SALINITY AND/OR SODICITY TOLERANT PLANTS

[0001] This application claims priority from Australian provisional patent application number 2019902392 filed on 5 July 2019, the content of which is to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to salinity and/or sodicity tolerant plants. Specifically, methods for the identification and generation of salinity and/or sodicity tolerant plants are provided, together with genetic markers associated with salinity and/or sodicity tolerance in plants (including wheat), as well as salinity and/or sodicity tolerant wheat germplasm and uses thereof.

BACKGROUND OF THE INVENTION

[0003] Global food requirements are expected to increase by approximately 90% by 2050, and as land degradation, urban spread and seawater intrusion are increasing, gains in agricultural productivity must come from marginal land, including saline soils. However, soil salinity and sodicity severely constrain crop production in Australia and globally. The total global area of saline and sodic soils is estimated to be around 830 million hectares, more than 6% of the world’s land and rising. Indeed, it is estimated that over 50% of global arable land will be salinized by 2050.

[0004] Although the actual cost from lost agricultural production is hard to quantify, and varies with crop species, timing, duration and severity of the stress, it is apparent that losses in yield and profit are significant. For example, with respect to wheat, yield reductions of 50% in durum wheat under dryland salinity, 88% in bread wheat under high irrigation salinity, and 70% under sodicity, have been reported. This highlights the scale of likely yield and profit losses on saline and sodic soils, and potential gain if the yield can be improved.

[0005] When cropping on saline and sodic soils, there are limited options to raise productivity, and they are complementary: (i) soil management; and (ii) plant breeding. The soil management option (i.e. leaching salts below the root zone and gypsum application) for dryland cropping systems is not always practical and rarely cost-effective. Furthermore, despite the potential of the plant breeding approach, progress in breeding cereal cultivars with salinity or sodicity tolerance has been slow. In this regard, at present there are a few salt-tolerant dryland cereal varieties developed via conventional breeding; however, no varieties have ever been released based on physiological mechanisms. There are several possible reasons for this apparent slow progress, such as genetic and physiological complexities of the salt tolerance trait, and lack of a reliable and rapid screening assay. Moreover, elite germplasm may not include genes able to confer worthwhile salt/sodicity tolerance, and introgression from wild wheat relatives and/or genetic engineering may be required for progress.

[0006] While salinity tolerance is complex and requires combined mechanisms in plants to achieve such tolerance (i.e. Na ⁺ exclusion, osmotic tolerance, and tolerance to high internal Na ⁺ concentration), it is acknowledged that osmotic stress is the critical component of salinity stress and most plants are naturally efficient Na ⁺ excluders. Indeed, Na ⁺ exclusion theory has for around two decades defined the salinity tolerance research paradigm.

[0007] For example, previous studies of a wild wheat relative examined the effect of introgression of the Na ⁺ exclusion genes Nax1 and Nax2 from the diploid bread wheat ancestor Triticum monococcum L. (C68-101) into durum wheat Tamaroi. Nax1 removes Na ⁺ from the xylem in roots and leaf sheaths, while Nax2 removes Na ⁺ from xylem in the roots only. Tamaroi possessing the Nax2 gene showed lower leaf Na ⁺ concentration and yielded, on average, 20% higher grain yield under salinity and sodicity. These two genes were also transferred from durum wheat into bread wheat cv. Westonia, and subsequently shown to reduce leaf 3 Na ⁺ concentration. A recent saline field trial with three Westonia- Nax2 and two Westonia-A/axi lines indicated, compared to Westonia, a 9% yield increase (average over two seasons) in one of the Westonia-/\/ax2 lines (Westonia-/\/ax2-5924). These results are encouraging, but not conclusive. Therefore, there is a need to verify the effects of these genes in bread wheat in controlled environment studies involving salinity and sodicity, especially as bread wheat has much greater Na ⁺ exclusion than durum wheat.

[0008] Sodicity, of which high Na ⁺ is the key component, affects greater land area than salinity, but there has been little specific research on sodicity tolerance and its mechanisms. This is unsurprising as screening for sodicity tolerance has been difficult in laboratory or glasshouse environments, which are needed to test large numbers of accessions in a relatively controlled manner. Problems with current screening methods include: (i) very high pH of sodic soils, hence difficulty of separating pH effects from those of Na ⁺ toxicity; (ii) inability to control soil composition when sourced from field sites; and (iii) months of waiting before pH stabilizes, and thereafter the possibility of toxicity from excess salt (sodium bicarbonate) not adsorbed at cation exchange sites. [0009] In efforts to develop genetic markers for breeding programs, numerous markers for the Na ⁺ exclusion trait in cereals have been identified by QTL mapping of bi-parental populations. However, most of these genetic markers have not been implemented in breeding programs. This is likely due to the presence of many markers with small effects and the lack of validation in other genetic backgrounds and in realistic environments (soil or field). An alternative to the aforementioned QTL mapping is association mapping or genome-wide association studies (GWAS). Two major advantages of association mapping over QTL mapping are: (i) a much larger and more representative gene pool can be surveyed; and (ii) it bypasses the time-consuming and expensive process of constructing bi-parental mapping populations. Although GWAS can be applied to a variety of plant species and conditions, only a few studies have reported on Na ⁺ exclusion and/or salt tolerance in rice, wheat, and barley.

[0010] In order to address the issues of sustaining adequate crop production under increasing salinized arable land, there is a clear need for the development of methods which can be used to generate and identify salinity and sodicity tolerant plants. Furthermore, new genetic markers associated with salinity and sodicity tolerance in plants (including wheat) are also required which can be deployed in breeding programs. Ultimately, elite germplasm from salinity and sodicity tolerant plants (including wheat) is needed from which next generation salinity and sodicity tolerant plants can be grown.

[0011] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[0012] The present invention arises from the identification of a new paradigm for the production of plants having salinity and/or sodicity tolerance. This new paradigm relies on the use of a plant with low sodium exclusion in breeding programs which runs counter intuitive to the prevailing opinion that low sodium (Na ⁺ ) in plants (i.e. high sodium exclusion) confers such tolerance.

[0013] Accordingly, in a first aspect, the present invention provides a method of producing a plant having salinity and/or sodicity tolerance, the method comprising: (i) crossing two parental plants; and

(ii) screening progeny plants produced from the cross for salinity and/or sodicity tolerance to identify a progeny plant having salinity and/or sodicity tolerance,

wherein at least one of the parental plants has low sodium exclusion.

[0014] In some embodiments, the parental plants are wheat plants. In some embodiments, at least one of the parental wheat plants is a hexaploid wheat plant. In one embodiment, the parental hexaploid wheat plant has low sodium exclusion. For example, the parental hexaploid wheat plant has a third leaf blade sodium concentration of at least 2,000 mg/kg DW when grown in the presence of 100 mM sodium chloride.

[0015] In some embodiments, the parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride. In some embodiments, the parental hexaploid wheat plant is of the species Triticum aestivum. For example, the parental hexaploid wheat plant is variety W4909 registered in the journal Crop Science as GP-730 (PI 631 164).

[0016] In some embodiments, the other parental wheat plant is a hexaploid wheat plant. In some embodiments, the other parental hexaploid wheat plant has high sodium exclusion. In some embodiments, the other parental hexaploid wheat plant has a penultimate leaf sodium concentration at heading of less than 500 mg/kg DW when grown in the presence of 100 mM sodium chloride.

[0017] In some embodiments, the other parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride. In some embodiments, the other parental hexaploid wheat plant is of the species Triticum aestivum. For example, the other parental hexaploid wheat plant is variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009.

[0018] In some embodiments, the parental plants are variety W4909 registered in the journal Crop Science as GP-730 (PI 631164) and variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009.

[0019] In some embodiments, the progeny plant has low sodium exclusion. [0020] In some embodiments, screening progeny plants for salinity tolerance comprises determining penultimate leaf sodium concentration at heading of the progeny plants when grown in the presence of 100 mM sodium chloride. In some embodiments, a progeny plant with salinity tolerance has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the progeny plant has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0021] In some embodiments, screening progeny plants for sodicity tolerance comprises determining penultimate leaf sodium concentration at heading of the progeny plants when grown in the presence of 8 g/kg sodium humate. In some embodiments, a progeny plant having sodicity tolerance has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, the progeny plant has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0022] In some embodiments, the progeny plant has a higher absolute grain yield when grown in the absence of sodium chloride when compared to the absolute grain yield of the progeny plant when grown in the presence of 100 mM sodium chloride or 8 g/kg sodium humate.

[0023] In some embodiments, screening progeny plants for salinity and/or sodicity tolerance comprises determining if DNA of the progeny plants comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0024] In some embodiments, a progeny plant having salinity and/or sodicity tolerance comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and (ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0025] In some embodiments, screening progeny plants for salinity and/or sodicity tolerance comprises determining if DNA of the progeny plants comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0026] In some embodiments, a progeny plant having salinity and/or sodicity tolerance comprises one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0027] In some embodiments, screening progeny plants for salinity and/or sodicity tolerance comprises determining if DNA of the progeny plants comprises one or more single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more SNPs include: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 1 1 ; (iv) a SNP at position 51 of SEQ I D NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ I D NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ I D NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ I D NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ I D NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ I D NO: 54; (xlvii) a SNP at position 51 of SEQ I D NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ I D NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0028] In some embodiments, a progeny plant having salinity and/or sodicity tolerance comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ I D NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0029] In some embodiments, screening progeny plants for salinity and/or sodicity tolerance comprises determining the expression level of one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein.

[0030] In some embodiments, a progeny plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; (iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0031] In some embodiments, the progeny plant having salinity and/or sodicity tolerance is a hexaploid wheat plant. In some embodiments, the progeny plant is of the species Triticum aestivum.

[0032] In some embodiments, the progeny plant is MW#293 representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0033] In some embodiments, the parental hexaploid wheat plant has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the parental hexaploid wheat plant has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0034] In some embodiments, the parental hexaploid wheat plant has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, the parental hexaploid wheat plant has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0035] In some embodiments, DNA of the parental hexaploid wheat plant comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and (ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0036] In some embodiments, the parental hexaploid wheat plant comprises:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0037] In some embodiments, DNA of the parental hexaploid wheat plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0038] In some embodiments, the parental hexaploid wheat plant comprises one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0039] In some embodiments, DNA of the parental hexaploid wheat plant comprises one or more single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more SNPs include: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 11 ; (iv) a SNP at position 51 of

SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of

SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of

SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ I D NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ I D NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xiv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ I D NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ I D NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0040] In some embodiments, the parental hexaploid wheat plant comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ I D NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ I D NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ I D NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ I D NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0041] In some embodiments, the parental hexaploid wheat plant comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; (iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0042] In some embodiments, the parental hexaploid wheat plant is of the species Triticum aestivum. In some embodiments, the parental hexaploid wheat plant is MW#293 representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0043] In some embodiments, the other parental wheat plant is a hexaploid wheat plant.

[0044] In a second aspect, the present invention provides a plant having salinity and/or sodicity tolerance produced by the method of the first aspect of the invention.

[0045] In a third aspect, the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining if DNA of the plant comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species. [0046] In some embodiments of the third aspect of the invention, a plant having salinity and/or sodicity tolerance comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0047] In some embodiments of the third aspect of the invention, the method further comprises determining if DNA of the plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0048] In some embodiments of the third aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0049] In some embodiments of the third aspect of the invention, the plant has sodicity tolerance.

[0050] In a fourth aspect, the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining if DNA of the plant comprises one or more single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more SNPs include: (i) a SNP at position 51 of SEQ I D NO: 9; (ii) a SNP at position 51 of SEQ I D NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 1 1 ; (iv) a SNP at position 51 of SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ I D NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ I D NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ I D NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ I D NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ I D NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ I D NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0051] In some embodiments of the fourth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ I D NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ I D NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ I D NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ ID NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ I D NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0052] In some embodiments of the fourth aspect of the invention, the plant has salinity tolerance.

[0053] In some embodiments of the third and fourth aspects of the invention, the method further comprises determining the expression level of one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein, in the plant. [0054] In some embodiments of the third and fourth aspects of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0055] In some embodiments of the third and fourth aspects of the invention, the plant is produced by a cross between two parental plants, wherein at least one of the parental plants has low sodium exclusion.

[0056] In some embodiments of the third and fourth aspects of the invention, the parental plants are wheat plants. In some embodiments, at least one of the parental wheat plants is a hexaploid wheat plant. In some embodiments, the parental hexaploid wheat plant has low sodium exclusion. In some embodiments, the parental hexaploid wheat plant has a third leaf blade sodium concentration of at least 2,000 mg/kg DW when grown in the presence of 100 mM sodium chloride.

[0057] In some embodiments of the third and fourth aspects of the invention, the parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride. In some embodiments, the parental hexaploid wheat plant is of the species Triticum aestivum. For example, the parental hexaploid wheat plant is variety W4909 registered in the journal Crop Science as GP-730 (PI 631 164). [0058] In some embodiments of the third and fourth aspects of the invention, the other parental wheat plant is a hexaploid wheat plant. In some embodiments, the other parental hexaploid wheat plant has high sodium exclusion. In some embodiments, the other parental hexaploid wheat plant has a penultimate leaf sodium concentration at heading of less than 500 mg/kg DW when grown in the presence of 100 mM sodium chloride.

[0059] In some embodiments of the third and fourth aspects of the invention, the other parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride. In some embodiments, the other parental hexaploid wheat plant is of the species Triticum aestivum. For example, the other parental hexaploid wheat plant is variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009.

[0060] In some embodiments of the third and fourth aspects of the invention, the parental plants are variety W4909 registered in the journal Crop Science as GP-730 (PI 631 164) and variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009.

[0061] In some embodiments of the third and fourth aspects of the invention, the plant having salinity and/or sodicity tolerance has low sodium exclusion.

[0062] In some embodiments of the third and fourth aspects of the invention, the plant having salinity tolerance has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the plant having salinity tolerance has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0063] In some embodiments of the third and fourth aspects of the invention, the plant having sodicity tolerance has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, the plant having sodicity tolerance has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0064] In some embodiments of the third and fourth aspects of the invention, the plant having salinity and/or sodicity tolerance has a higher absolute grain yield when grown in the absence of sodium chloride when compared to the absolute grain yield of the plant when grown in the presence of 100 mM sodium chloride or 8 g/kg sodium humate.

[0065] In some embodiments of the third and fourth aspects of the invention, the plant having salinity and/or sodicity tolerance is a wheat plant. In some embodiments, the wheat plant is a hexaploid wheat plant. In some embodiments, the hexaploid wheat plant is of the species Triticum aestivum.

[0066] In some embodiments of the third and fourth aspects of the invention, the wheat plant having salinity and/or sodicity tolerance is MW#293 representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0067] In a fifth aspect, the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining the expression level of one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TI F1-4, Putative high- affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein, in the plant.

[0068] In some embodiments of the fifth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and (vi) a decreased expression of AVP1-like protein when grown in the presence of

100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0069] In some embodiments of the fifth aspect of the invention, the method further comprises determining if DNA of the plant comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0070] In some embodiments of the fifth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0071] In some embodiments of the fifth aspect of the invention, the method further comprises determining if DNA of the plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0072] In some embodiments of the fifth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) a cytosine (C) residue at position

101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0073] In some embodiments of the fifth aspect of the invention, the method further comprises determining if DNA of the plant comprises one or more single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more SNPs include: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 11 ; (iv) a SNP at position 51 of SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ I D NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ I D NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a

SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ I D NO: 59; (Iii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0074] In some embodiments of the fifth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ I D NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ I D NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ I D NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ ID NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ I D NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0075] In some embodiments of the fifth aspect of the invention, the plant is produced by a cross between two parental plants, wherein at least one of the parental plants has low sodium exclusion. In some embodiments, the parental plants are wheat plants.

[0076] In some embodiments of the fifth aspect of the invention, at least one of the parental wheat plants is a hexaploid wheat plant. In some embodiments, the parental hexaploid wheat plant has low sodium exclusion.

[0077] In some embodiments of the fifth aspect of the invention, the parental hexaploid wheat plant has a third leaf blade sodium concentration of at least 2,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride.

[0078] In some embodiments of the fifth aspect of the invention, the parental hexaploid wheat plant is of the species Triticum aestivum. For example, the parental hexaploid wheat plant is variety W4909 registered in the journal Crop Science as GP-730 (PI 631164).

[0079] In some embodiments of the fifth aspect of the invention, the other parental wheat plant is a hexaploid wheat plant. In some embodiments, the other parental hexaploid wheat plant has high sodium exclusion.

[0080] In some embodiments of the fifth aspect of the invention, the other parental hexaploid wheat plant has a penultimate leaf sodium concentration at heading of less than 500 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the other parental hexaploid wheat plant has a salinity tolerance of <50% when grown in the presence of 100 mM sodium chloride.

[0081] In some embodiments of the fifth aspect of the invention, the other parental hexaploid wheat plant is of the species Triticum aestivum. For example, the other parental hexaploid wheat plant is variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009. [0082] In some embodiments of the fifth aspect of the invention, the parental plants are variety W4909 registered in the journal Crop Science as GP-730 (PI 631 164) and variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009.

[0083] In some embodiments of the fifth aspect of the invention, the plant having salinity and/or sodicity tolerance has low sodium exclusion.

[0084] In some embodiments of the fifth aspect of the invention, the plant having salinity tolerance has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the plant having salinity tolerance has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0085] In some embodiments of the fifth aspect of the invention, the plant having sodicity tolerance has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, the plant having sodicity tolerance has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0086] In some embodiments of the fifth aspect of the invention, the plant having salinity and/or sodicity tolerance has a higher absolute grain yield when grown in the absence of sodium chloride when compared to the absolute grain yield of the plant when grown in the presence of 100mM sodium chloride or 8g/kg sodium humate.

[0087] In some embodiments of the fifth aspect of the invention, the plant having salinity and/or sodicity tolerance is a wheat plant. In some embodiments, the wheat plant is a hexaploid wheat plant. In some embodiments, the hexaploid wheat plant is of the species Triticum aestivum.

[0088] In some embodiments of the fifth aspect of the invention, the wheat plant having salinity and/or sodicity tolerance is MW#293 representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422. [0089] In a sixth aspect, the present invention provides a plant having salinity and/or sodicity tolerance identified by the method of the third to fifth aspects of the invention.

[0090] In a seventh aspect, the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is a single nucleotide polymorphism (SNP), wherein the SNP is selected from one or more of the group consisting of:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0091] In some embodiments of the seventh aspect of the invention, the SNP is selected from one or more of the group consisting of:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0092] In some embodiments of the seventh aspect of the invention, the marker is a further SNP, wherein the further SNP is selected from one or more of the group consisting of: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ I D NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0093] In some embodiments of the seventh aspect of the invention, the further SNP is selected from one or more of the group consisting of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0094] In some embodiments of the seventh aspect of the invention, a plant having salinity and/or sodicity tolerance comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0095] In some embodiments of the seventh aspect of the invention, the plant having salinity and/or sodicity tolerance comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0096] In some embodiments of the seventh aspect of the invention, the marker is a marker of sodicity tolerance.

[0097] In an eighth aspect, the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is a single nucleotide polymorphism (SNP), wherein the SNP is selected from one or more of the group consisting of: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 11 ; (iv) a SNP at position 51 of SEQ I D NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ I D NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ I D NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ I D NO: 59; (lii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0098] In some embodiments of the eighth aspect of the invention, the SNP is selected from one or more of the group consisting of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ I D NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ I D NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ I D NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ I D NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ I D NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ I D NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ I D NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ I D NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ I D NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ ID NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ I D NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0099] In some embodiments of the eighth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more SNPs in homozygous form, wherein the one or more SNPs include: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ I D NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0100] In some embodiments of the eighth aspect of the invention, the marker is a marker of salinity tolerance.

[0101] In a ninth aspect, the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is selected from one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein.

[0102] In some embodiments of the ninth aspect of the invention, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0103] In a tenth aspect, the present invention provides a wheat plant designated MW#293, representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0104] In an eleventh aspect, the present invention provides seed of the wheat plant of the tenth aspect of the invention.

[0105] In a twelfth aspect, the present invention provides a tissue culture of cells of the wheat plant of the tenth aspect of the invention.

[0106] In some embodiments of the twelfth aspect of the invention, the tissue culture is generated from cells of a tissue selected from the group consisting of seeds, leaves, stems, pollens, roots, root tips, shoots, anthers, ovules, petals, flowers, embryos, fibers, and bolls. [0107] In a thirteenth aspect, the present invention provides an isolated wheat plant comprising at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 ; and

(ii) a thyime (T) residue at position 51 of SEQ ID NO: 2.

[0108] In some embodiments of the thirteenth aspect of the invention, the wheat plant comprises one or more further SNPs in homozygous form, and wherein the one or more further SNPs are selected from the group consisting of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7.

[0109] In some embodiments of the thirteenth aspect of the invention, the wheat plant has sodicity tolerance.

[0110] In some embodiments of the thirteenth aspect of the invention, the wheat plant comprises one or more further SNPs in homozygous form, and wherein the one or more further SNPs comprise: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55. In some embodiments, the wheat plant has salinity tolerance.

[0111] In some embodiments of the thirteenth aspect of the invention, the wheat plant has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DWwhen grown in the presence of 100 mM sodium chloride. In some embodiments, the wheat plant has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0112] In some embodiments of the thirteenth aspect of the invention, the wheat plant has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, the wheat plant has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0113] In some embodiments of the thirteenth aspect of the invention, the wheat plant is MW#293, representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0114] In a fourteenth aspect, the present invention provides a method of increasing salinity and/or sodicity tolerance of a plant, the method comprising one or more of:

(i) decreasing expression and/or activity of Na7H ⁺ antiporter NhaB in one or more cells of the plant; (ii) increasing expression and/or activity of Aquaporin-like protein TIF1-4 in one or more cells of the plant;

(iii) increasing expression and/or activity of Putative high-affinity potassium transporter in one or more cells of the plant;

(iv) increasing expression and/or activity of NHX1 in one or more cells of the plant;

(v) increasing expression and/or activity of NHX2 in one or more cells of the plant; and

(vi) decreasing expression and/or activity of AVP1-like protein in one or more cells of the plant.

[0115] In some embodiments of the fourteenth aspect of the invention, the expression and/or activity is increased or decreased by genetic modification of one or more cells of the plant.

[0116] In some embodiments of the fourteenth aspect of the invention, expression and/or activity of Na H ⁺ antiporter NhaB and AVP1-like protein is decreased by decreasing expression of nucleic acid encoding Na7H ⁺ antiporter NhaB and/or AVP1-like protein, and/or by decreasing expression of Na7H ⁺ antiporter NhaB protein and AVP1-like protein. In some embodiments, when expression of nucleic acid encoding Na7H ⁺ antiporter NhaB and AVP1-like protein, and/or expression of Na7H ⁺ antiporter NhaB protein and AVP1-like protein is decreased in one or more cells of the plant, the salinity and/or sodicity tolerance of the plant is increased.

[0117] In some embodiments of the fourteenth aspect of the invention, expression and/or activity of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 is increased by increasing expression of nucleic acid encoding Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2, and/or by increasing expression of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter protein, NHX1 protein, and NHX2 protein. In some embodiments, when expression of nucleic acid encoding Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2, and/or expression of Aquaporin-like protein TIF1- 4, Putative high-affinity potassium transporter protein, NHX1 protein, and NHX2 protein is increased in one or more cells of the plant, the salinity and/or sodicity tolerance of the plant is increased. [0118] In some embodiments of the fourteenth aspect of the invention, a plant with increased salinity and/or sodicity tolerance has a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, a plant with increased salinity tolerance has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0119] In some embodiments of the fourteenth aspect of the invention, a plant with increased sodicity tolerance has a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate. In some embodiments, a plant with increased sodicity tolerance has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0120] In some embodiments of the fourteenth aspect of the invention, a plant with increased salinity and/or sodicity tolerance has a higher absolute grain yield when grown in the absence of sodium chloride when compared to the absolute grain yield of the plant when grown in the presence of 100 mM sodium chloride or 8 g/kg sodium humate.

[0121] In some embodiments of the fourteenth aspect of the invention, the plant having increased salinity and/or sodicity tolerance is a wheat plant. In some embodiments, the wheat plant is a hexaploid wheat plant. In some embodiments, the hexaploid wheat plant is of the species Triticum aestivum.

[0122] In a fifteenth aspect, the present invention provides a genetically modified plant cell with increased salinity and/or sodicity tolerance compared to a wild-type form of the plant cell, wherein expression and/or activity of Na7H ⁺ antiporter NhaB and/or AVP1-like protein is decreased in the plant cell, and/or wherein expression and/or activity of one or more of Aquaporin-like protein, Putative high-affinity potassium transporter, NHX1 , and NHX2 is increased in the plant cell.

[0123] In some embodiments of the fifteenth aspect of the invention, expression of nucleic acid encoding Na7H ⁺ antiporter NhaB and/or AVP1-like protein, and/or expression of Na7H ⁺ antiporter NhaB protein and/or AVP1-like protein, is decreased in the plant cell.

[0124] In some embodiments of the fifteenth aspect of the invention, expression of nucleic acid encoding Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and/or NHX2, and/or expression of Aquaporin-like protein TIF1-4, Putative high- affinity potassium transporter protein, NHX1 protein, and/or NHX2 protein, is increased in the plant cell.

[0125] In some embodiments of the fifteenth aspect of the invention, the cell is a wheat cell. In some embodiments, the wheat cell is a hexaploid wheat cell. In some embodiments, the hexaploid wheat cell is a Triticum aestivum cell.

[0126] In a sixteenth aspect, the present invention provides a multicellular structure having salinity and/or sodicity tolerance, wherein the multicellular structure comprises one or more plant cells according to the thirteenth aspect of the invention.

[0127] In some embodiments of the sixteenth aspect of the invention, the multicellular structure comprises a whole plant, plant tissue, plant organ, plant part, plant reproductive material or cultured plant tissue.

BRIEF DESCRIPTION OF THE FIGURES

[0128] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying figures which illustrate certain embodiments of the present invention.

[0129] FIGURE 1 - Incremental water use (weekly) curves until heading in Westonia, Westonia-/Vax7, Westonia-/\/ax2 and Baart-46 under different levels of salinity (mM NaCI, left panels) and sodicity (g kg ^-1 Na ⁺ humate, right panels) in Experiment 1. Estimated slopes of the linear component of these curves are presented in Table 4.

[0130] FIGURE 2 - Graphs showing relative grain yield (%) (salinity or sodicity tolerance), and best linear unbiased estimates for leaf Na ⁺ and Cl concentrations in wheat cv. Westonia, Westonia-/Vax7, Westonia-/\/ax2 and Baart-46 under different levels of salinity

(left panels) and sodicity applied as Na ⁺ humate (right panels) in Experiment 1. The vertical bars indicate Least Significant Difference test value at P= 0.05 for variety x treatment interaction. See Table 6 for back-transformed Na ⁺ and Cl concentrations for comparisons with published data.

[0131] FIGURE 3 - Boxplots of leaf Na ⁺ (back-transformed), Cl , K ⁺ , Ca ²⁺ and Mg ²⁺ concentrations (mg kg ¹ DW) at heading in 100 bread wheats, 12 durum wheats and a barley cultivar grown under sodicity (8 g kg ¹ Na ⁺ -humate) in Experiment 2. See Table 8 for individual responses. The box represents the middle 50 % of the distribution (the median is drawn as the solid line within the box) with whiskers extending to the lowest/highest value within 1.5* IQR (Inter-Quartile Range). Values outside this range are plotted separately.

[0132] FIGURE 4 - Population structure based on 41 ,036 SNP markers (MAF>0.05). Each genotype is represented by a horizontal bar partitioned into k segments that represent the genotype’s estimated membership fractions. The most likely number of clusters in this population is k=2>.

[0133] FIGURE 5 - Manhattan plot showing the association signals for leaf Na ⁺ concentration using 100 bread wheat entries grown under sodicity (8 g kg ^-1 Na ⁺ -humate) in Experiment 2. The x-axis indicates the physical location of SNP markers along each wheat chromosome; the y-axis shows the P-value of SNP markers for the association test on a log scale. The horizontal red line indicates the significance threshold (P-value= 8.91 e- 5). The marker names of SNPs above the threshold are shown.

[0134] FIGURE 6 - Charts showing the allelic effects on leaf Na ⁺ concentration of SNP markers; seven mapped with a physical location and two unmapped. The x-axis is the allelic class and the y-axis is leaf Na ⁺ concentration. The two entries MW#293 and MW#451 with extremely high Na ⁺ concentration are indicated in each panel. For SNP markers tplb0049b24_1152 (2D) and tplb0058o04_1071 (2A), MW#293 and MW#451 could not be classified into either“AA” or“BB” type due to missing scores.

[0135] FIGURE 7 - Incremental water use curves until heading in 20 bread wheats ( Triticum aestivum L), three durum wheats ( Triticum turgidum subsp durum cv. Tamaroi, Tamaroi- Nax2 and Yawa) and one barley ( Hordeum vulgare L. cv. Clipper) under control, salinity (100 mM NaCI) and sodicity (8 g kg ¹ Na ⁺ -humate) in Experiment 3. Estimated slopes of the linear component of these curves are presented in Table 10.

[0136] FIGURE 8 - Charts showing the relationships of Na ⁺ , Cl , K ⁺ , Ca ²⁺ and Mg ²⁺ concentrations (mg kg ¹ DW), and grain yield (g plant ¹ ) under control, salinity (100 mM NaCI) and sodicity (8 g kg ¹ Na ⁺ -humate) in 20 bread wheats in Experiment 3 (r=0.561 ,d/=18, P<0.01). MW#293 is shown as an empty circle.

[0137] FIGURE 9 - Graphs showing best linear unbiased estimates for grain yield (A), and tolerance (grain yield under sodicity or salinity as a percentage of grain yield under control) (B) of 20 bread wheats ( Triticum aestivum L), three durum wheats ( Triticum turgidum subsp durum cv. Tamaroi, Jamard\-Nax2 and Yawa) and one barley ( Hordeum vulgare L. cv. Clipper) in Experiment 3. The vertical bars indicate Least Significant Difference test value at P= 0.05 for variety x treatment interaction. Entries are ordered in ascending order of leaf Na ⁺ concentration.

[0138] FIGURE 10 - Graphs showing best linear unbiased estimates for grain yield (A), and leaf Na ⁺ (B) and Cl (C) concentrations at heading in bread wheat cv. Mace, and two low- Na ⁺ (MW#28 and MW#491) and two-high Na ⁺ (MW#293 and MW#451) doubled-haploid lines selected from a cross between Mace and high-Na ⁺ germplasm W4909. Sodium concentration data were transformed to natural logarithms. The vertical bars indicate Least Significant Difference test value at P= 0.05 for variety x treatment interaction. Wheat lines are ordered in ascending order of salinity tolerance (ratio of grain yield under salinity to grain yield under control, expressed as percent).

[0139] FIGURE 11 - Photographs of representative pots of bread wheat ( Triticum aestivum) cv. Mace (A) and doubled-haploid line MW#293 (B) grown under control and salinity (100 mM NaCI) in Experiment 4.

[0140] FIGURE 12 - Graphs showing best linear unbiased estimates for leaf K ⁺ (A), Ca ²⁺ (B) and Mg ²⁺ (C) concentrations at heading in bread wheat ( Triticum aestivum L.) cv. Mace, and two low-Na ⁺ (MW#28 and MW#491) and two high-Na ⁺ (MW#293 and MW#451) doubled-haploid lines selected from a cross between Mace and high-Na ⁺ germplasm W4909. The vertical bars indicate Least Significant Difference test values at P= 0.05 for variety x treatment interactions. Wheat lines are ordered in ascending order of salinity tolerance (ratio of grain yield under salinity to grain yield under control, expressed as percent).

[0141] FIGURE 13 - Graphs showing best linear unbiased estimates (BLUEs) for candidate genes (copy number ng ^_1 mRNA) identified by GWAS in Experiment 2, and known genes (NHX and A VP) in penultimate leaves in low-Na ⁺ bread wheat ( Triticum aestivum L.) cv. Mace, and two low-Na ⁺ (MW#28 and MW#491) and two high-Na ⁺ (MW#293 and MW#451) doubled-haploid lines selected from a cross between wheat cv. Mace and a high-Na ⁺ line W4909 under control and salinity (100 mM NaCI) in Experiment 4. The vertical bars indicate Least Significant Difference test value at P= 0.05 for variety x treatment interaction. [0142] FIGURE 14 - Photographs of the growth room used for the study in Example 2 (A), bread wheats Longreach Dart (B) and MW#293 (C). Plants were grown in University of California potting mix supplied with (right pot in each image) or without (left pot in each image) 100 mM NaCI.

[0143] FIGURE 15 - A graph showing shoot dry weights of 100 bread wheats (first group), 12 durum wheats (second group) and barley cultivar Clipper under control and salinity. The arrow indicates bread wheat germplasm MW#293. All entries were harvested at the time when awns were visible, and ranked in ascending order of shoot dry weight under control. For a complete list of names, see Table 12.

[0144] FIGURE 16 - Graphs showing the relationships between growth under control and salinity in bread wheat (A) and durum wheat (B). Shoot DW; g p ; n=100 and 12 for bread wheat and durum wheat, respectively.

[0145] FIGURE 17 - Graphs showing the relationships between leaf sodium concentration (A and B), and chloride concentration (C and D) with salinity tolerance measured as relative shoot dry weight (A and C) and shoot dry weight under salinity (B and D) in bread wheat. Empty circle represents bread wheat germplasm MW#293.

DETAILED DESCRIPTION OF THE INVENTION

[0146] Nucleotide sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing has also been provided at the time of filing this application.

TABLE 1

Summary of Sequence Identifiers

[0147] As set out above, the present invention is predicated, in part, on the identification of a new paradigm for the production of plants having salinity and/or sodicity tolerance. Despite the prevailing opinion that low sodium (Na ⁺ ) in plants (i.e. high sodium exclusion) confers such tolerance, the results presented herein show otherwise. That is, the new paradigm relies on the use of a plant with low sodium exclusion in breeding programs.

[0148] Accordingly, certain disclosed embodiments provide methods, markers, and compositions, that have one or more advantages. For example, some of the advantages of some embodiments disclosed herein include one or more of the following: improved methods for producing plants having salinity and/or sodicity tolerance; improved methods for identifying plants having salinity and/or sodicity tolerance; plants having salinity and/or sodicity tolerance produced or identified using such methods; new genetic markers of salinity and/or sodicity tolerance in plants; plants and/or germplasm thereof having salinity and/or sodicity tolerance, and use of said plants and/or germplasm for the production of further salinity and/or sodicity tolerance plants; methods for increasing the salinity and/or sodicity tolerance of plants; genetically modified plant cells with increased salinity and/or sodicity tolerance; or the provision of a commercial alternative to existing methods, markers and compositions. Other advantages of some embodiments of the present disclosure are provided herein.

[0149] In a first aspect the present invention provides a method of producing a plant having salinity and/or sodicity tolerance, the method comprising:

(i) crossing two parental plants; and

(ii) screening progeny plants produced from the cross for salinity and/or sodicity tolerance to identify a progeny plant having salinity and/or sodicity tolerance,

wherein at least one of the parental plants has low sodium exclusion.

[0150] Reference herein to a“parental plant” or“plant” may include seed plant species such as monocotyledonous angiosperm plants (“monocots”), dicotyledonous angiosperm plants (“dicots”) and/or gymnosperm plants.

[0151] In some embodiments, the plant is a monocot plant. In some embodiments, the plant is a cereal crop plant. As used herein, the term“cereal crop plant” may include a member of the Poaceae (grass) family that produces grain. Examples of Poaceae cereal crop plants include wheat, rice, barley, maize, millets, sorghum, rye, triticale, oats, teff, wild rice, spelt, and the like. The term cereal crop plant should also be understood to include a number of non-Poaceae plant species that also produce edible grain, which are known as the pseudocereals and include, for example, amaranth, buckwheat and quinoa.

[0152] In some embodiments, the plant is a wheat plant. As referred to herein,“wheat” should be understood as a plant of the genus Triticum. Thus, the term“wheat” encompasses hexaploid wheat, tetraploid wheat, and diploid wheat. Hexapioid wheat may include T. aestivum, T. speita, T. macha, T. compactum, T sphaerococcum, T. vaviiovii, and interspecies crosses thereof. Tetraploid wheat may include T. durum, T. dicoccoides, T. dicoccum, T. ispahanicum, T. karamyschevii, T. turgidum, T. polonicum, T. turanicum, T carthiicum, and interspecies cross thereof. Diploid wheat may include T boeoticum, T. urartu, T . monococcum, and T. tauschii (also known as Aegiiops squarrosa or Aegilops tauschn). In some embodiments, the wheat plant is of the species Triticum aestivum.

[0153] In some embodiments, the plant is a rice plant. As referred to herein,“rice” should be understood to include several members of the genus Oryza, including the species Oryza sativa and Oryza glaberrima. The term“rice” thus encompasses rice cultivars such as japonica or sinica varieties, indica varieties and javonica varieties. In some embodiments, the term“rice” refers to rice of the species Oryza sativa.

[0154] In some embodiments, the plant is a barley plant. As referred to herein,“barley” includes several members of the genus Hordeum. The term “barley” encompasses cultivated barley including two-row barley ( Hordeum distichum ), four-row barley ( Hordeum tetrastichum) and six-row barley ( Hordeum vulgare). In some embodiments, barley may also refer to wild barley ( Hordeum spontaneum ). In some embodiments, the term“barley” refers to barley of the species Hordeum vulgare.

[0155] According to the first and other aspects of the invention, the parental plants may be of the same species or of a different species, provided that at least one of the parental plants has low sodium exclusion. For example, one parent plant may be Trilicum sp. used in a sexual cross with a no n-Triiicum species (such as rye - Seca/e cereaie). The combination of parent plant species is not limited by the present invention; however, it is recognised that natural barriers exist that may preclude the formation of progeny between certain plants, whether they are of a different species, or in some instances of the same species. Natural barriers will Include asynchronous flowering between and within species, gametic or zygotic incompatibility, and reduced hybrid fitness or hybrid sterility. However, the crossing compatibility of various plants would be well understood by those skilled In the art.

[0156] In some embodiments, the parental plants are of the same species. For example, both parental plants may be wheat plants. In this regard, the cross may be between hexaploid/hexaploid, tetraploid/tetraploid, or diploid/diploid wheat plants. Alternatively, the cross may be between a hexaploid wheat plant and a tetraploid or diploid wheat plant, or between a tetraploid wheat plant and a diploid wheat plant.

[0157] As indicated above, at least one of the parental plants has low sodium exclusion. A parental plant with“low sodium exclusion” is taken to mean that the plant retains sodium in its tissues (such that it has a high rate of sodium accumulation), when the plant is grown in saline and/or sodic soils.

[0158] A saline soil is defined as a soil having a concentration of soluble salts high enough to affect plant growth. Salt concentration in a soil is measured in terms of its electrical conductivity. Accordingly, as used herein a "saline soil" has an EC _e of at least 1 dS/m, at least 2 dS/m, at least 3 dS/m, or at least 4 dS/m. EC _e is the electrical conductivity of the solution extracted from a soil sample after being mixed with sufficient water to produce a saturated paste (a“saturated paste extract”). Typically, a plant grown in the presence of at least 100 mM sodium chloride will be considered as being grown in a saline soil.

[0159] Sodic soils have a low concentration of soluble salts, but a high percent of exchangeable sodium; that is, sodium forms a high percent of ail cations bound to the negative charges on the clay particles that make up the soil complex. Sodicity is defined in terms of the threshold ESP (exchangeable sodium percentage) that causes degradation of soil structure. As used herein a "sodic soil" has an ESP of at least 5. Typically, a plant grown in the presence of at least 4 g/kg of sodium humate will be considered as being grown in a sodic soil.

[0160] The level of sodium exclusion of a plant is typically determined by measuring the level of sodium in tissues of the plant. Such tissues may include leaves, seeds, roots, shoots, etc. In some embodiments, the level of sodium in the plant is measured in the third leaf blade of the plant as it matures, or in the penultimate leaf (i.e. the leaf immediately below the flag leaf) at heading.

[0161] In some embodiments, when measuring the level of sodium in the third leaf blade of the plant a low sodium excluder may have a sodium concentration in the range of at least 2,000 mg/kg to 10,000 mg/kg dry weight (DW) when grown in the presence of 100 mM sodium chloride. For example, the sodium concentration may be 2,000 mg/kg to 9,000 mg/kg DW, 2,000 mg/kg to 8,000 mg/kg DW, 2,000 mg/kg to 7,000 mg/kg DW, 2,000 mg/kg to 6,000 mg/kg DW, 2,000 mg/kg to 5,000 mg/kg DW, 2,000 mg/kg to 4,000 mg/kg DW, 2,000 mg/kg to 3,000 mg/kg DW, 3,000 mg/kg to 10,000 mg/kg DW, 3,000 mg/kg to 9,000 mg/kg DW, 3,000 mg/kg to 8,000 mg/kg DW, 3,000 mg/kg to 7,000 mg/kg DW, 3,000 mg/kg to 6,000 mg/kg DW, 3,000 mg/kg to 5,000 mg/kg DW, 3,000 mg/kg to 4,000 mg/kg DW, 4,000 mg/kg to 10,000 mg/kg DW, 4,000 mg/kg to 9,000 mg/kg DW, 4,000 mg/kg to 8,000 mg/kg DW, 4,000 mg/kg to 7,000 mg/kg DW, 4,000 mg/kg to 6,000 mg/kg DW, 4,000 mg/kg to 5,000 mg/kg DW, 5,000 mg/kg to 10,000 mg/kg DW, 5,000 mg/kg to 9,000 mg/kg DW, 5,000 mg/kg to 8,000 mg/kg DW, 5,000 mg/kg to 7,000 mg/kg DW, 5,000 mg/kg to 6,000 mg/kg DW, 6,000 mg/kg to 10,000 mg/kg DW, 6,000 mg/kg to 9,000 mg/kg DW, 6,000 mg/kg to 8,000 mg/kg DW, 6,000 mg/kg to 7,000 mg/kg DW, 7,000 mg/kg to 10,000 mg/kg DW, 7,000 mg/kg to 9,000 mg/kg DW, 7,000 mg/kg to 8,000 mg/kg DW, 8,000 mg/kg to 10,000 mg/kg DW, 8,000 mg/kg to 9,000 mg/kg DW, or 9,000 mg/kg to 10,000 mg/kg DW.

[0162] In some embodiments of the first aspect of the invention, and other aspects of the invention as described below, at least one of the parental plants is a wheat plant. In some embodiments, the wheat plant is a hexaploid wheat plant which has low sodium exclusion. In this regard, a parental hexaploid wheat plant having low sodium exclusion will have a third leaf blade sodium concentration of at least 2,000 mg/kg dry weight (DW) when grown in the presence of 100 mM sodium chloride. In some embodiments, the ranges listed above are contemplated.

[0163] In some embodiments, the parental hexaploid wheat plant having low sodium exclusion is variety W4909 registered in the journal Crop Science as GP-730 (PI 631 164)(see Wang RR-C et al., 2003, Crop Science, 43: 746, March-April).

[0164] In some embodiments of the first aspect of the invention, and other aspects of the invention as described below, the other parental plant has high sodium exclusion. A parental plant with“high sodium exclusion” is taken to mean that the plant excludes sodium from its tissues (such that it has a low rate of sodium accumulation), when the plant is grown in saline and/or sodic soils, as described herein.

[0165] In some embodiments, when measuring the level of sodium in the penultimate leaf blade of the plant a high sodium excluder may have a sodium concentration in the range of less than 500 mg/kg to 1 mg/kg dry weight (DW) when grown in the presence of 100 mM sodium chloride. For example, the sodium concentration may be 500 mg/kg to 10 mg/kg DW, 500 mg/kg to 50 mg/kg DW, 500 mg/kg to 100 mg/kg DW, 500 mg/kg to 150 mg/kg DW, 500 mg/kg to 200 mg/kg DW, 500 mg/kg to 250 mg/kg DW, 500 mg/kg to 300 mg/kg DW, 500 mg/kg to 350 mg/kg DW, 500 mg/kg to 400 mg/kg DW, 500 mg/kg to 450 mg/kg DW, 450 mg/kg to 1 mg/kg DW, 450 mg/kg to 10 mg/kg DW, 450 mg/kg to 50 mg/kg DW, 450 mg/kg to 100 mg/kg DW, 450 mg/kg to 150 mg/kg DW, 450 mg/kg to 200 mg/kg DW, 450 mg/kg to 250 mg/kg DW, 450 mg/kg to 300 mg/kg DW, 450 mg/kg to 350 mg/kg DW, 450 mg/kg to 400 mg/kg DW, 400 mg/kg to 1 mg/kg DW, 400 mg/kg to 10 mg/kg DW, 400 mg/kg to 50 mg/kg DW, 400 mg/kg to 100 mg/kg DW, 400 mg/kg to 150 mg/kg DW, 400 mg/kg to 200 mg/kg DW, 400 mg/kg to 250 mg/kg DW, 400 mg/kg to 300 mg/kg DW, 400 mg/kg to 350 mg/kg DW, 350 mg/kg to 1 mg/kg DW, 350 mg/kg to 10 mg/kg DW, 350 mg/kg to 50 mg/kg DW, 350 mg/kg to 100 mg/kg DW, 350 mg/kg to 150 mg/kg DW, 350 mg/kg to 200 mg/kg DW, 350 mg/kg to 250 mg/kg DW, 350 mg/kg to 300 mg/kg DW, 300 mg/kg to 1 mg/kg DW, 300 mg/kg to 10 mg/kg DW, 300 mg/kg to 50 mg/kg DW, 300 mg/kg to 100 mg/kg DW, 300 mg/kg to 150 mg/kg DW, 300 mg/kg to 200 mg/kg DW, 300 mg/kg to 250 mg/kg DW, 250 mg/kg to 1 mg/kg DW, 250 mg/kg to 10 mg/kg DW, 250 mg/kg to 50 mg/kg DW, 250 mg/kg to 100 mg/kg DW, 250 mg/kg to 150 mg/kg DW, 250 mg/kg to 200 mg/kg DW, 200 mg/kg to 1 mg/kg DW, 200 mg/kg to 10 mg/kg DW, 200 mg/kg to 50 mg/kg DW, 200 mg/kg to 100 mg/kg DW, 200 mg/kg to 150 mg/kg DW, 150 mg/kg to 1 mg/kg DW, 150 mg/kg to 10 mg/kg DW, 150 mg/kg to 50 mg/kg DW, 150 mg/kg to 100 mg/kg DW, 100 mg/kg to 1 mg/kg DW, 100 mg/kg to 10 mg/kg DW, 100 mg/kg to 50 mg/kg DW, 50 mg/kg to 1 mg/kg DW, 50 mg/kg to 10 mg/kg DW, or 10 mg/kg to 1 mg/kg DW.

[0166] In some embodiments, the other parental plant is a wheat plant. In some embodiments, the other parental wheat plant is a hexaploid wheat plant which has high sodium exclusion. In some embodiments, a parental hexaploid wheat plant having high sodium exclusion will have a penultimate leaf sodium concentration at heading of less than 500 mg/kg DW when grown in the presence of 100 mM sodium chloride. In some embodiments, the ranges listed above are contemplated.

[0167] In some embodiments, the other parental hexaploid wheat plant having high sodium exclusion is variety cv. Mace having Australian Plant Breeders Rights Certificate Number 3895 granted on 28 September 2009 (see http://pericles.ipaustralia.gov.au/pbr_db/plant_detail.cfm7A I D=27781374). A deposit of this variety is held with the Australian Winter Cereals Collection (Tamworth, NSW, Australia).

[0168] The methods by which the two parental plants can be crossed would be well understood by a person skilled in the art. Exemplar methods are set out in standard text books in the art such as: Allard RW, 1999, Principles of Plant Breeding, second edition, John Wily & Sons, ISBN 0471023094; Ram M, 2014, Plant Breeding Methods, PHI Learning Private Limited, ISBN 9788120348509; Brown J and Caligari P, 2008, An Introduction to Plant Breeding, Blackwell Publishing Limited, ISBN 9781405133449; Poehlman JM, 1959, Breeding Field Crops, Holt, Rinehart and Winston Pub., ISBN 9780030065804; and Poehlman JM and Sleper D, 1999, Breeding Field Crops, Fourth Edition, Wiley- Blackwell Publ., ISBN 9780813824277.

[0169] In one particular example, with respect to the crossing of wheat plants, classical/conventional plant breeding methods may be used which rely on choosing a suitable spike on the female parent in which pollen is not yet ripened. For convenience, two outer flowers of the spikelets may be used for crossing. The remaining spikelets are cut off close to the rachis with dissecting scissors. Individual spikelets are clipped just above the anthers. Unripe greenish anthers (pollen factories) from the female plants are then removed by a pair of forceps with fine points (emasculation). The spikes of the emasculated plants are covered by paper bags to ensure they are not pollinated by random pollens. In the next few days, with the help of a pair of fine forceps, the pollens from the male plant are transferred onto the stigmas of female plants. The resulting seed is an F1 hybrid (also known as filial 1 hybrid) (for details see Florell VH, 1934, A method of making wheat crosses, Journal of Heredity 25: 157-161). F1 progeny are then used to generate populations via methods such as the wheat-maize hybridization method or single seed descend method to study inheritance of the trait. Wheat-maize hybridization includes six steps; emasculation of wheat flower, pollination of emasculated flower with maize pollen, hormone treatment, embryo rescue, haploid plant regeneration in tissue culture medium, and chromosome doubling (Broughton S et ai, 2014, In vitro Culture for Doubled Haploids: Tools for Molecular Breeding, Methods Mol. Biol., 1 145: 167-189; Santra M et a!., 2017, Wheat Biotechnology pp 235-249). With this method, it is possible to achieve 100% homozygosity at all loci in a single generation, while achieving homozygosity in the case of the single seed descent method takes several generations.

[0170] The term“salinity” as used herein generally refers to the level of all soluble salts in the growing environment of a plant. These salts are free to move in the soil water and can be readily taken up by plants. The term“sodicity” as used herein generally refers to the level of sodium held in a soil that a plant is growing in. Sodium is a cation that is held loosely on clay particles in soil. Therefore, in some embodiments, the terms“salinity tolerance” and “sodicity tolerance” relate to the capacity of a plant cell or plant to survive, grow, and/or reproduce at a particular environmental salt concentration.

[0171] However, the most relevant salt for a majority of cropping systems is sodium chloride. Therefore, in some embodiments, the term “salinity tolerance” or “sodicity tolerance” refers to the capacity of a plant to survive, grow, and/or reproduce at a particular environmental sodium concentration. In some embodiments, salinity tolerance or sodicity tolerance also refers to the ability of a plant to maintain a suitable sodium concentration in one or more tissues of the plant at a particular environmental sodium concentration.

[0172] Accordingly, a progeny plant having“salinity tolerance” and/or“sodicity tolerance” refers to a plant that is capable of surviving, growing, and/or reproducing in saline and/or sodic soil conditions. Typically, a plant having salinity tolerance and/or sodicity tolerance will have a yield less than that of the plant when grown in normal (non-saline and non-sodic) soil conditions. However, the yield of a plant having salinity tolerance and/or sodicity tolerance will be greater than that of a plant of the same or similar species which does not have salinity and/or sodicity tolerance.

[0173] Screening progeny plants for salinity tolerance and/or sodicity tolerance can be achieved a number of ways as would be known in the art. In some embodiments, salinity and/or sodicity tolerance can be measured by observation alone as indicated above - namely the ability to survive and grow in saline and/or sodic soil conditions. Alternatively, salinity and/or sodicity tolerance can be assessed through measuring other phenotypic aspects of the plant, including grain yield, germination percentage, shoot dry weight (shoot biomass), and seedling mass of the plant. For example, relative grain yield can be determined which is a measure of the ratio of grain yield of the plant at a particular salinity and/or sodicity stress level to grain yield of the plant under no salinity and/or sodicity stress.

[0174] When relative grain yield is used as the screening parameter, in some embodiments of the first and other aspects of the invention, the progeny plant has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0175] The sodicity tolerance of a progeny plant can also be determined by growth of the progeny plant in a soil containing sodium humate, as indicated above. In this regard, the use of sodium humate allows the study of ionic effects of sodium toxicity without interference from chloride. The use of sodium humate in this manner is described in Gene Y et al. , 2016, New Phytologist, 201 : 145-156. Accordingly, in some embodiments the progeny plant has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0176] In some embodiments of the first and other aspects of the invention, one or both of the parental plants have a salinity tolerance of <50% when grown in saline and/or sodic soils, as defined herein (for example when grown in the presence of 100 mM sodium chloride). Accordingly, one or both of the parental plants may be considered to lack salinity tolerance.

[0177] Progeny plants can also be screened for salinity tolerance and/or sodicity tolerance by determining the level of sodium in tissues of the plant - which is also a measure of the sodium exclusion capabilities of the parental plants as described above. For example, the penultimate leaf sodium concentration at heading of the progeny plant when grown in the presence of sodium chloride or when grown in the presence of sodium humate can be determined.

[0178] In some embodiments, a progeny plant that is salinity tolerant will have a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg dry weight (DW) when grown in the presence of 100 mM sodium chloride. In this regard, said progeny plant has a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride.

[0179] In some embodiments, a progeny plant that is sodicity tolerant will have a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg dry weight (DW) when grown in the presence of 8 g/kg sodium humate. In this regard, said progeny plant has a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0180] Progeny plants produced by the method of the first aspect of the invention include Triticum aestivum cultivars MW#293 and MW#451. These plants were produced from a cross between wheat varieties W4909 and cv. Mace the details of which have been described above. Seed from progeny plant MW#293 has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0181] When progeny wheat cultivars MW#293 and MW#452 are grown under conditions of sodicity, DNA of these cultivars share in common at least two single nucleotide polymorphisms (SNPs) in homozygous form, which are considered to be markers for the sodicity (and/or salinity) tolerant phenotype. These two SNPs are represented by a polymorphism at position 51 of SEQ ID NOs: 1 and 2. Accordingly, these SNPs can be used to screen progeny wheat plants, including progeny Triticum aestivum plants, for salinity and/or sodicity tolerance. Furthermore, the same SNPs in equivalent nucleotide sequences from other species of plants are useful for screening purposes in those plants.

[0182] Accordingly, in some embodiments of the first aspect of the invention, and other aspects of the invention as described below, screening progeny plants for salinity and/or sodicity tolerance comprises determining if DNA of the progeny plants comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0183] With respect to the progeny wheat cultivars MW#293 and MW#452, the SNPs are represented by a guanine (G) residue at position 51 of SEQ ID NO: 1 , and a thymine (T) residue at position 51 of SEQ ID NO: 2.

[0184] Accordingly, in some embodiments of the first and other aspects of the invention, a progeny plant having salinity and/or sodicity tolerance comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0185] The DNA from each of progeny wheat cultivars MW#293 and MW#452 (when grown under conditions of sodicity) also has one or more further SNPs in common. These further SNPs are represented by a polymorphism at position 101 of SEQ ID NO: 3, and a polymorphism at position 51 of SEQ ID NOs: 4 to 9. Accordingly, these SNPs can also be used to screen progeny wheat plants, including progeny Triticum aestivum plants, for salinity and/or sodicity tolerance when used in combination with the SNPs at position 51 of SEQ ID NOs: 1 and 2. Furthermore, the same SNPs in equivalent nucleotide sequences from other species of plants are useful for screening purposes in those plants.

[0186] Accordingly, in some embodiments of the first aspect of the invention, and other aspects of the invention as described below, screening progeny plants for salinity and/or sodicity tolerance comprises determining if DNA of the progeny plants comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ I D NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0187] With respect to the progeny wheat cultivars MW#293 and MW#452, these further SNPs comprise one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0188] Accordingly, in some embodiments of the first and other aspects of the invention, a progeny plant having salinity and/or sodicity tolerance comprises one or more further SNPs comprising one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0189] When progeny wheat cultivars MW#293 and MW#452 are grown under conditions of salinity, DNA of of these cultivars share in common a number of additional single nucleotide polymorphisms (SNPs) in homozygous form, which are considered to be markers for the salinity (and/or sodicity) tolerant phenotype. These additional SN Ps include: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 1 1 ; (iv) a SNP at position 51 of SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a

SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a

SNP at position 51 of SEQ I D NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a

SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a

SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ I D NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ I D NO: 54; (xlvii) a SNP at position 51 of SEQ I D NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ I D NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species..

[0190] Accordingly, in some embodiments of the first aspect of the invention, and other aspects of the invention as described below, a progeny plant having salinity and/or sodicity tolerance comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ I D NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ I D NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ I D NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ I D NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ I D NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ I D NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ I D NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ I D NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ I D NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ ID NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ I D NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0191] The presence of the aforementioned SNPs can be determined using standard assays known in the art. Such assays may require the isolation of DNA from a sample of the progeny plant to be tested, or from the whole plant. A sample of the progeny plant may be a tissue sample, such as a leaf, stem, shoot, root, grain, etc. DNA may be isolated from the sample according to any methods well known to those of skill in the art, for example see methods disclosed in Gelvin SB et ai, Plant Molecular Biology Manual, Springer, 1989 (ISBN 978-94-010-6918-2).

[0192] Determining the presence of SNPs can be achieved using allele-specific primers and probes, for example PCR-based approaches that use oligonucleotide primers which specifically bind to a SNP being tested for. Such oligonucleotides which detect single nucleotide variations in target sequences may be referred to by such terms as "allele- specific probes", or "allele-specific primers". The design and use of allele-specific probes for detecting known sequence variations is described in, for example, Mutation Detection A Practical Approach, ed. Cotton et at. Oxford University Press, 1998; Saiki et ai, 1986 ( Nature , 324: 163-166); EP235726; and WO 89/11548. In one example, a probe or primer may be designed to hybridize to a segment of target DNA that includes the SNP such that the actual SNP aligns with either the 5' most end or the 3' most end of the probe or primer. In some assays, the amplification may include a labeled primer, thereby allowing detection of the amplification product of that primer. In one example, the amplification may include a multiplicity of labeled primers; typically, such primers are distinguishably labeled, allowing the simultaneous detection of multiple amplification products.

[0193] In one type of PCR-based assay, an allele-specific primer hybridizes to a segment of target DNA that overlaps with the SNP site and only primes amplification of an allelic form to which the primer exhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res. 17:2427-2448). Typically, the primer's 3'-most nucleotide is aligned with and complementary to the actual SNP being tested for. This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers, producing a detectable product that indicates which allelic form is present in the test sample. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the SNP site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification or substantially reduces amplification efficiency, so that either no detectable product is formed, or it is formed in lower amounts or at a slower pace. The method generally works most effectively when the mismatch is at the 3'-most position of the oligonucleotide (i.e. the 3'-most position of the oligonucleotide aligns with the SNP position) because this position is most destabilizing to elongation from the primer (see for example WO 93/22456). A person skilled in the art would readily be able to design allele-specific primer sequences for detecting the SNPs referred to above based on the disclosure of SEQ ID NOs: 1 to 62, and with reference to Table 1 and the accompanying sequence listing which is incorporated herein.

[0194] In one example, a primer contains a sequence substantially complementary to a segment of DNA containing the SNP except that the primer has a mismatched nucleotide in one of the three nucleotide positions at the 3'-most end of the primer, such that the mismatched nucleotide does not base pair with a particular allele at the SNP site. The mismatched nucleotide in the primer can be the first, second or the third nucleotide from the last nucleotide at the 3'-most position of the primer. In some examples, primers and/or probes are labeled with detectable labels. [0195] In an alternative approach, tagged allele specific primer pairs can be used to detect one of the aforementioned SNPs (Strom et ai, 2005, Genet. Med. 7:633-63). In one example, two tagged allele-specific primers overlap the SNP site in the target DNA; however, only the correctly hybridized primer(s) will be extended to generate a labeled product(s). A non-complementary primer will not be extended or labeled due to the 3' mismatched base. The labeled extended product can be detected based on the detectable label. The tagged extended primers can also be captured on a solid support such as beads that are coupled to anti-tag sequences. The immobilized extended primer product can be detected by commercially available means such as Luminex 100 LabMAP™ (Luminex Corporation, Austin TX).

[0196] The most common approach to detect the aforementioned SNPs is amplification of a segment of DNA comprising the SNP using PCR, and then isolating and sequencing the PCR fragment that has been amplified. Primers for the amplification procedure can be designed based on the disclosure of SEQ ID NOs: 1 to 62 herein. Procedures for designing primers, conducting PCR, isolating amplified fragments, and sequencing said fragments are known in the art and provided in Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0197] As indicated above, the Triticum aestivum sequences disclosed herein (SEQ ID NOs: 1 to 62) which contain the SNPs representative of salinity and sodicity tolerant Triticum aestivum plants, will have equivalent sequences (and corresponding SNPs) in other plant species. The nature of those equivalent sequences can be determined a number of ways as would be understood in the art. For example, a BLAST sequence query with any one of SEQ ID NOs: 1 to 62 could be conducted on the ENSEMBL plants database (https://plants.ensembl.org/index.html).

[0198] The SNPs identified herein represent markers which may be linked to one or more genes responsible for the salinity and/or sodicity tolerant phenotype. In this regard, the location of a particular SNP on the relevant plant genome can point to genes in close physical proximity which can be examined for a role in said phenotypes. For example, in the present study seven of the aforementioned SNPs identified could be physically mapped to the wheat genome and therefore genes located nearby were analysed. In this instance, four candidate genes with putative functions in regulating Na ⁺ concentration were identified in close physical location to these seven SNPs and the level of expression of three of these genes (calcium-transporting ATPase (TraesCS4D01G343200.1), Na(+)/H(+) antiporter NhaB (TraesCS4D01G344200.1), and Aquaporin-like protein TIF1-4 (TraesCS4D01 G344300.1)), was determined in various wheat plants under control and salinity/sodicity conditions to determine if the level of expression and/or activity of each gene could be correlated to a particular phenotype.

[0199] The term“gene” as used herein is to be understood to mean a region of genomic nucleotide sequence (nuclear or mitochondrial) which includes a coding region that is transcribed and translated into protein. A "gene" in the context of the present invention can therefore include regulatory regions (e.g. promoter regions), transcribed regions, exons, introns, untranslated regions and other functional and/or non-functional sequence regions associated with the gene.

[0200] As would be understood by a person skilled in the art, the term“expression” with respect to a gene includes: (1) transcription of the gene into a messenger RNA (mRNA) molecule; and/or (2) translation of the mRNA into the corresponding protein. In effect, the expression of a gene may be decreased or increased at the RNA and/or protein stages of expression. With respect to the term“activity” in relation to a gene, this should be taken to mean the expected function of the translated protein.

[0201] The expression level of a gene may be increased or decreased in a plant with salinity and/or sodicity tolerance compared to the expression level of the gene in a control plant (e.g. a plant lacking salinity and/or sodicity tolerance).

[0202] Reference herein to“decreased” with respect to the expression level of a gene in a progeny plant having salinity and/or sodicity tolerance, whether at the transcriptional (mRNA) or translational (protein) stage, is intended to mean, for example, at least a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.1 -fold, 1.2-fold, 1.3-fold,

1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4- fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold,

3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100- fold or greater reduction in the level of mRNA or protein of the gene in the progeny plant compared to the level in a control plant.

[0203] Reference herein to“decreased” with respect to the level of activity of a gene in a progeny plant having salinity and/or sodicity tolerance is intended to mean a reduction in the function of the protein encoded by the gene in the plant. In some embodiments, the level of activity of a gene may be reduced by at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9- fold, 4-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100-fold, or greater, in the progeny plant compared to the level of activity in a control plant.

[0204] Similarly, reference herein to“increased” with respect to the expression level of a gene in a progeny plant having salinity and/or sodicity tolerance, whether at the transcriptional (mRNA) or translational (protein) stage, is intended to mean, for example, at least a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.1-fold, 1.2- fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3- fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 5-fold, 10-fold, 20 fold, 50- fold, or 100-fold or greater increase in the level of mRNA or protein of the gene in the progeny plant compared to the level in a control plant.

[0205] Furthermore, reference herein to“increased” with respect to the level activity of a gene in a progeny plant having salinity and/or sodicity tolerance is intended to mean an increase in the function of the protein encoded by the gene in the plant. In some embodiments, the level activity of a gene may be increased by at least 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,

1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6- fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold,

3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100-fold, or greater, in the progeny plant compared to the level of activity in a control plant.

[0206] In some embodiments of the first aspect of the invention, and other aspects of the invention as described below, screening progeny plants for salinity and/or sodicity tolerance comprises determining the expression level of genes encoding one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein.

[0207] The Na7H ⁺ antiporter NhaB gene is fully functionally characterised in the bacterial species Escherichia coli. It acts to extrude Na ⁺ in exchange for external protons and is important for regulation of intracellular pH in alkaline environments. In higher plants, SOS1 in Arabidopsis thaliana (At2G01980) is the archetypal NhaB member. Expression of this gene is localised to cell plasma membrane and is concentrated around xylem vessels, and root tip cells where vacuoles are either very small or absent and vacuolar sequestration of Na+ is not possible. A NhaB-like gene in Triticum aestivum (Na7H ⁺ antiporter NhaB gene) encodes putative mRNA and amino acid sequences set out in SEQ ID NOs: 63 and 64, respectively, and is represented by the High Confidence Gene Model TraesCS4D01G344200.1 (IWGSC v1.1). The gene is located on chromosome 4DL. Further details regarding the nucleotide and amino acid sequence for the NhaB-like gene in wheat can be accessed from the International Wheat Genome Sequencing Consortium (https://wheat-urgi.versailles.inra.fr) and the UniProt database (www.uniprot.org) wherein the UniProt ID for this sequence is A0A3B6JRJ7. The contents of the IWGSC and UniProt records are incorporated herein by reference.

[0208] The Aquaporin-like protein TIF1-4 gene is located on Chr4D in Triticum aestivum and is a member of the Major Intrinsic Protein (MIP) family found across bacteria, archae and eukaryotes. MIPs are membrane-spanning channels that facilitate and regulate the transport of non-charged molecules such as water and small non-polar solutes. In plants, they are classified into five sub-families. The Aquaporin-like protein TIF1-4 Chr4D gene in Triticum aestivum is classified as belonging to the Tonoplast Intrinsic Protein (TIP) sub family. The Aquaporin-like protein TIF1-4 Chr4D gene encodes the mRNA and amino acid sequences set out in SEQ ID NOs: 65 and 66, respectively, and is represented by the High Confidence Gene Model TraesCS4D01G344300.1 (IWGSC v1.1). The gene is located on chromosome 4DL. Further details regarding the nucleotide and amino acid sequence for the Aquaporin-like protein TIF1-4 Chr4D gene in wheat and other species can be accessed from the International Wheat Genome Sequencing Consortium (https://wheat- urgi.versailles.inra.fr) and the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the gene is represented by a Triticum aestivum (cv. Chinese Spring) cDNA clone having GenBank Accession number AK331084. Further details regarding the amino acid sequence for the Aquaporin-like protein TIF1-4 Chr4D gene can be accessed from the UniProt database (www.uniprot.org) wherein the UniProt ID for a fragment representing this sequence is A0A090A0K0. The contents of the IWGSC, GenBank and UniProt records are incorporated herein by reference.

[0209] The Putative high-affinity potassium transporter gene sequence in Triticum aestivum encodes the mRNA and amino acid sequences set out in SEQ ID NOs: 67 and 68, respectively, and is represented by the GenBank Accession number AK455305 and the high-confidence gene model TraesCS2A02G256100 (IWGSC v1.1). The gene is located on chromosome 2A. The gene and its encoded protein are classified as a putative high-affinity potassium transporter on the basis of functional domain analyses and comparisons with characterised high-affinity potassium transporter proteins in other species. Further details of the putative high-affinity potassium transporter gene in Triticum aestivum and other plant species can be accessed from the International Wheat Genome Sequencing Consortium (https://wheat-urgi.versailles.inra.fr) and the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). Further details regarding the amino acid sequence for the putative high-affinity potassium transporter gene can be accessed from the UniProt database (www.uniprot.org) wherein the UniProt I D for this gene is A0A3B6AZ09. The contents of the IWGSC, GenBank and UniProt records are incorporated herein by reference.

[0210] The Arabidopsis vacuolar pyrophosatase gene (AVP-1 like protein) encodes an enzyme that breaks down inorganic pyrophosphate and transports protons, and has been shown to play a role in the sequestration of excess Na ⁺ ions. Regulation of pyrophosphate levels in plant cells assists in the regulation of growth and developmental processes. The AVP-like gene sequence in Triticum aestivum encodes the mRNA and amino acid sequences set out in SEQ ID NOs: 69 and 70, respectively, and is represented by GenBank Accession numbers MH376297 and QBC16611.1 , respectively, and the high-confidence gene model TraesCS7A02G517700 (IWGSC v1.1). The gene is located on chromosome 7 A and there are homeologous sequences on chromosomes 7B and 7D. The AVP-like gene and its encoded protein are classified on the basis of functional domain analyses. Further details of the AVP-like gene in Triticum aestivum and other plant species can be accessed from the International Wheat Genome Sequencing Consortium (https://wheat- urgi.versailles.inra.fr) and the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the Gene I D number for Arabidopsis thaliana AVP1 is 838138. Further details regarding the amino acid sequence for the putative high-affinity potassium transporter gene can be accessed from the UniProt database (UniProt ID A0A3B6RN65). The contents of the IWGSC, GenBank and UniProt records are incorporated herein by reference.

[0211] The NHX1 gene is a Na7H ⁺ antiporter that acts in the low affinity electroneutrai exchange of protons for cations such as Na ⁺ or K ⁺ across membranes. The gene is involved in vacuolar ion compartmentalizafion necessary for ceil volume regulation and cytoplasmic Na ⁺ detoxification in Triticum aestivum, the gene encodes the mRNA and amino acid sequences set out in SEQ ID NOs: 71 and 72, respectively, and represented by GenBank Accession Numbers AY296910 and AAK76737.1 , respectively. Further details of the NHX1 gene in Triticum aestivum and other plant species can be accessed from the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the Gene ID number for Triticum aestivum NHX1 is 543443, for Arabidopsis thaliana is 832773, for Glycine max is 732573, for Solanum lycopersicum is 543771 , for Oryza sativa is 4344217, for Gossypium hirsutum is 107891968, and for Cicer arietinum is 101515538. Further details regarding the nucleotide and amino acid sequence for NHX1 in wheat and other plant species can be accessed from the UniProt database (www.uniprot.org) wherein the UniProt ID for Triticum aestivum NHX1 is Q94BM4. The contents of the GenBank and UniProt records are incorporated herein by reference.

[0212] The NHX2 gene encodes a vacuolar K+/H+ exchanger essential for active K+ uptake at the tonoplast and is involved in regulating stomatal closure. In Triticum aestivum, the gene encodes the mRNA and amino acid sequences set out in SEQ ID NOs: 73 and 74, respectively, and represented by GenBank Accession Numbers AY040246 and AAK76738.2, respectively. Further details of the NHX2 gene in Triticum aestivum and other plant species can be accessed from the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the Gene ID number for Triticum aestivum NHX2 is 543060, for Arabidopsis thaliana is 819665, for Solanum lycopersicum is 544178, for Oryza sativa is 433781 1 , for Hordeum vulgare is 548178, for Populus euphratica is 105124949, for Ricinus communis is 8262382, for Helianthus annuus is 110930025, and for Brassica napus is 111200868. Further details regarding the nucleotide and amino acid sequence for NHX2 in wheat and other plant species can be accessed from the UniProt data3ase (www.uniprot.org) wherein the UniProt ID for Triticum aestivum NHX2 is Q94BM4. The contents of the GenBank and UniProt records are incorporated herein by reference.

[0213] It is to be made clear that reference to the specific genes described above, includes a reference to naturally-occurring variants of these genes. In this regard, a“variant” of a gene may exhibit an amino acid or nucleic acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the native gene. In some embodiments, a variant of a native gene may retain native biological activity or a substantial equivalent thereof. In some embodiments, a variant may have no substantial biological activity, such as those variants which are precursors for the biologically active gene.

[0214] Methods which can be used to measure the level of expression of a gene are known in the art. With respect to measuring an increase or decrease in the transcription of a gene into mRNA, levels of mRNA may be measured by techniques which include, but are not limited to, RNAseq next generation sequencing (Metzker ML, 2010, Nat. Rev. Genet., 11 (1): 31-46; Mardis ER, 2008, Ann. Rev. Genomics Hum. Genet., 9: 387-402), Northern blotting, RNA in situ hybridisation, reverse-transcriptase PCR (RT-PCR), real-time (quantitative) RT- PCR, microarrays, or“tag based” technologies such as SAGE (serial analysis of gene expression). Microarrays and SAGE may be used to simultaneously quantitate the level of expression of more than one gene. Primers or probes may be designed based on the nucleotide sequence of the gene or transcripts thereof. Methodology similar to that disclosed in Paik et ai, 2004 ( NEJM , 351 (27): 2817-2826), or Anderson et ai, 2010 ( Journal of Molecular Diagnostics, 12(5): 566-575) may be used to measure the level of expression of a gene. Many methods are also disclosed in standard molecular biology text books such as Gelvin SB et ai, 1989, supra, or Green MR and Sambrook J, 2012, supra.

[0215] With respect to RT-PCR, the first step is typically the isolation of total RNA from a sample obtained from the plant under investigation. A typical sample in this instance would be a tissue sample (such as a leaf sample), although other sample sources are contemplated. Messenger RNA (mRNA) may be subsequently purified from the total RNA sample. The total RNA sample (or purified mRNA) is then reverse transcribed into cDNA using a suitable reverse transcriptase. The reverse transcription step is typically primed using oligo-dT primers, random hexamers, or primers specific for the gene, depending on the RNA template. The cDNA derived from the reverse transcription reaction then serves as a template for a typical PCR reaction. In this regard, two oligonucleotide PCR primers specific for the gene are used to generate a PCR product. A third oligonucleotide, or probe, designed to detect a nucleotide sequence located between the other two PCR primers is also used in the PCR reaction. The probe is non-extendible by the Taq DNA polymerase enzyme used in the PCR reaction and is labelled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together, as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is freed from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0216] In real-time RT-PCR the amount of product formed, and the timing at which the product is formed, in the PCR reaction correlates with the amount of starting template. RT- PCR product will accumulate quicker in a sample having an increased level of mRNA compared to a relevant control sample. Real-time RT-PCR measures either the fluorescence of DNA intercalating dyes such as Sybr Green into the synthesized PCR product, or can measure PCR product accumulation through a dual-labelled fluorigenic probe (i.e. , TaqMan probe). The progression of the RT-PCR reaction can be monitored using a PCR machine or a LightCycler which measure product accumulation in real-time. Real-time RT-PCR is compatible both with quantitative competitive PCR and with quantitative comparative PCR. The former uses an internal competitor for the target sequence for normalization, while the latter uses a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.

[0217] The production and application of microarrays for measuring the level of expression of a gene at the transcriptional level are well known in the art. In general, in a microarray, a nucleotide sequence (for example an oligonucleotide, a cDNA, or genomic DNA) representing a portion, or all, of the gene occupies a known location on a substrate. A nucleic acid target sample (for example total RNA or mRNA) obtained from a plant of interest is then hybridized to the microarray and the amount of target nucleic acid hybridized to each probe on the array is quantified and compared to the hybridisation which occurs to a relevant control sample. Fluorescently labelled cDNA probes may also represent the relevant nucleic acid target sample. Such probes can be generated through incorporation of fluorescent nucleotides during reverse transcription of total RNA or mRNA extracted from a sample of the plant to be tested. Labelled cDNA probes applied to the microarray will hybridize with specificity to the equivalent spot of DNA on the array. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance in the sample compared to the abundance observed in a relevant control sample. With dual colour fluorescence, separately labelled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization using microarray analysis affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels. A number of plant microarrays are also commercially available. Sources include Agilent Technologies (https://www.agilent.com/en/product/gene-expression-microarr ay-platform/gene- expression-exon-microarrays/plant-microarrays#5).

[0218] Methods which can be used to measure a level of expression of a gene at the translational level (protein level) are also known in the art. For example, the level of protein encoded by the gene may be measured by techniques which include, but are not limited to, antibody-based testing (including Western blotting, immunoblotting, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation and dissociation-enhanced lanthanide fluoro immuno assay (DELFIA)), proteomics techniques, surface plasmon resonance (SPR), versatile fibre-based SPR, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemistry, immunofluorescence, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), as described in WO 2009/004576 (including surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS), especially surface-enhanced affinity capture (SEAC), protein microarrays, surface-enhanced need desorption (SEND) or surface-enhanced photo label attachment and release (SEPAR)), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.

[0219] With respect to antibody-based testing methods such as immunohistochemistry and immunoblotting, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for the relevant protein are used to detect protein abundance in the sample. The antibodies can be detected by direct labelling of the antibodies themselves, for example with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody may be used in conjunction with a labelled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available. Antibodies can be produced by methods well known in the art, for example, by immunizing animals with the protein under investigation. Further detailed description is provided below. [0220] Also contemplated are traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. Nephelometry is an assay performed in liquid phase, in which antibodies are in solution. Binding of the protein to be detected to the antibody results in changes in absorbance, which are measured. In the SELDI-based immunoassay, a biospecific capture reagent for the protein is attached to the surface of an MS probe, such as a pre-activated ProteinChip array (see below). The protein is then specifically captured on the biochip through this reagent, and the captured protein is detected by mass spectrometry.

[0221] Proteomics can also be used to analyse the level of expression of a protein present in a sample at a certain point of time. In particular, proteomic techniques can be used to assess the global changes of protein expression in a sample (also referred to as expression proteomics). Proteomic analysis typically includes: (i) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (ii) identification of the individual polypeptides recovered from the gel, for example by mass spectrometry or N-terminal sequencing; and (iii) analysis of the data using bioinformatics.

[0222] As indicated above, the level of a protein can also be measured by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The mass spectrometer may be a laser desorption/ionization mass spectrometer. In laser desorption/ionization mass spectrometry, the protein to be detected is placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present the protein to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.

[0223] The analysis of protein by LDI can take the form of MALDI or of SELDI, as described below. The SELDI method is described, for example, in U.S. Patents Nos. 5,719,060 and 6,225,047, and relates to a method of desorption/ionization gas phase ion spectrometry (e.g. mass spectrometry) in which an analyte (in this instance the protein to be detected) is captured on the surface of a SELDI mass spectrometry probe. MALDI is a traditional method of laser desorption/ionization. In one MALDI method, the sample to be tested is mixed with matrix and deposited directly on a MALDI chip. Depending on the sample being tested, the protein being tested is preferably first captured with biospecific (e.g. an antibody) or chromatographic materials coupled to a solid support such as a resin (e.g. in a spin column). Specific affinity materials that may bind the protein being detected are described above. After purification on the affinity material, the protein is eluted and then detected by MALDI.

[0224] In a further approach, the protein can be captured with a solid-phase bound immuno- adsorbent that has antibodies that specifically bind to the protein. After washing the adsorbent to remove unbound material, the protein is eluted from the solid phase and is detected by applying it to a biochip that binds the protein.

[0225] Protein which is bound to the substrates are detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer. The protein is ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of protein typically will involve detection of signal intensity. Thus, both the quantity and mass of the protein can be determined.

[0226] Analysis of proteins by time-of-flight mass spectrometry generates a time-of-flight spectrum. The time-of-flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of- flight data is then subject to data processing using specialized software. Data processing typically includes TOF-to-M/Z transformation to generate a mass spectrum, baseline subtraction to eliminate instrument offsets and high frequency noise filtering to reduce high frequency noise.

[0227] Data generated by desorption and detection of protein can be analyzed with the use of a programmable digital computer. Data analysis can include steps of determining signal strength of the protein and removing data deviating from a predetermined statistical distribution. For example, the observed peak can be normalized, by calculating the height of the peak relative to a reference. The computer can transform the resulting data into various formats for display. The standard spectrum can be displayed, but in one useful format only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling proteins with nearly identical molecular weights to be more easily seen. In another useful format, two or more spectra are compared, conveniently highlighting protein that has varying expression levels between samples. Using any of these formats, one can readily determine whether the protein under investigation is present in a sample and to what level.

[0228] Other methods which may be employed to determine if the level of a protein under investigation has decreased or increased in a sample include assays which rely on known protein/protein interactions.

[0229] In some embodiments of the first aspect of the invention, and other aspects of the invention as described below, a progeny plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0230] In some embodiments, progeny plants produced by the method of the first aspect of the invention may be used in further crosses to improve the salinity and/or sodicity tolerance of further generation plants. For example, a progeny plant produced by the method of the first aspect of the invention includes the Triticum aestivum cultivar MW#293. Therefore, in some embodiments this cultivar may subsequently be used as a parent plant in further crosses using methods which are described in detail above.

[0231] Accordingly, in some embodiments of the first aspect of the invention, one of the parental plants in such crosses will have one or more of the following features: low sodium exclusion; a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride; a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride; a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate; and a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0232] Furthermore, in some embodiments, DNA of the parental plant comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0233] In some embodiments, the parental plant comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0234] In some embodiments, DNA of the parental plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ I D NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0235] In some embodiments, the parental plant comprises one or more of: (i) a cytosine (C) residue at position 101 of SEQ I D NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0236] In some embodiments, DNA of the parental plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 11 ; (iv) a SNP at position 51 of SEQ I D NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ I D NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ I D NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ I D NO: 59; (lii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0237] In some embodiments, the parental plant comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ I D NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ I D NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ I D NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ I D NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ ID NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0238] Furthermore, in some embodiments the parental plant comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0239] In a further aspect, the present invention provides a plant having salinity and/or sodicity tolerance produced by a method of the first aspect of the invention.

[0240] The methods of the first aspect of the invention have enabled the identification of markers (biomarkers) for salinity and/or sodicity tolerance in plants. In the context of the present invention, a biomarker is effectively an organic biomolecule which is present or absent, or differentially present, in a sample taken from a plant of one phenotypic status (e.g. having salinity tolerance) as compared with another phenotypic status (e.g. not having salinity tolerance). Therefore, biomarkers, alone or in combination, provide an indication that a plant belongs to one phenotypic status or another. [0241] In some embodiments, the biomarker will be a DNA marker, such as a polymorphism in DNA, which alone or in combination with other biomarkers, will be indicative of a salinity tolerant and/or sodicity tolerant phenotype in the plant. Genomic variability at the DNA level can be present in many forms including: single nucleotide polymorphisms (SNPs), variable number of tandem repeats (VNTRs)(e.g., mini- and micro-satellites), transposable elements (e.g., Alu repeats), structural alterations, and copy number variations.

[0242] In some embodiments of the present invention, the biomarker is a single nucleotide polymorphism (SNP). Methods for identifying SNPs have been described in detail above. SNPs represent high-density natural sequence variations in the plant genome. SNPs are mostly formed when errors occur (substitution, insertion and deletion) during DNA replication. SNPs are prominent sources of variation in the plant genome and therefore represent ideal genetic markers. Some regions of the genome are richer in SNPs than others. SNPs may occur within gene sequences or in intergenic sequences. SNPs are typically located in non-coding regions of the genome predominantly due to the abundance of non-coding regions compared to regions of the genome which encode proteins. While SNPs in non-coding regions mostly have no direct known impact on the phenotype of plants, SNPs which are present in genes may well have a consequence at the phenotypic level.

[0243] Accordingly, in a further aspect the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is a single nucleotide polymorphism (SNP) in homozygous form, wherein the SNP is selected from one or more of the group consisting of:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0244] In some embodiments, the SNP is selected from one or more of the group consisting of:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and (ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0245] In some embodiments, the marker is a further SNP, wherein the further SNP is selected from one or more of the group consisting of: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ ID NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ ID NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0246] In some embodiments, the further SNP is selected from one or more of the group consisting of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0247] In some embodiments, the marker is a marker of sodicity tolerance.

[0248] In a further aspect the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is a single nucleotide polymorphism (SNP) in homozygous form, wherein the SNP is selected from one or more of the group consisting of: (i) a SNP at position 51 of SEQ ID NO: 9; (ii) a SNP at position 51 of SEQ ID NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 11 ; (iv) a SNP at position 51 of SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ ID NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ I D NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ ID NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ I D NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ ID NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ ID NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xlv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ I D NO: 54; (xlvii) a SNP at position 51 of SEQ I D NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ ID NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ I D NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ ID NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0249] In some embodiments, the SNP is selected from one or more of the group consisting of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ I D NO: 11 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ I D NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ ID NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ I D NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ I D NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0250] In some embodiments, the marker is a marker of salinity tolerance.

[0251] While SNPs in non-coding regions are not likely to have an impact on the phenotype of plants, they may be in close physical proximity to a gene which does play a direct role in the phenotype. Such SNPs can be stated as being“linked” to the phenotype-causing gene as they are co-inherited with the causative gene. Therefore, detecting the presence of such SNPs by default can detect the presence of the phenotype.

[0252] The specific SNPs referred to above, are indeed in close physical proximity to a number of genes mapped to the wheat genome. Accordingly, in a further aspect the present invention provides a marker for salinity and/or sodicity tolerance in a plant, wherein the marker is selected from one or more of Na7H ⁺ antiporter NhaB, Aquaporin-like protein TIF1- 4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein.

[0253] As discussed above, a biomarker (such as a gene encoding a protein referred to above) may be differentially present between different phenotypic status groups. A biomarker (gene) is differentially present if the mean or median expression level of the biomarker (gene) is calculated to be different (i.e. higher or lower) between the groups. Methods for measuring differential expression of a gene are described in detail above.

[0254] In some embodiments, a plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0255] As markers for the salinity and/or sodicity tolerance of plants, the SNPs referred to above may be used in methods for identifying a plant having said phenotype. Accordingly, in a further aspect the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining if DNA of the plant comprises at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a SNP at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a SNP at position 51 of SEQ ID NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species. [0256] In some embodiments, a plant having salinity and/or sodicity tolerance comprises:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species; and

(ii) a thyime (T) residue at position 51 of SEQ I D NO: 2 with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0257] In some embodiments, the method of identifying a plant having salinity and/or sodicity tolerance further comprises determining if DNA of the plant comprises one or more further SNPs in homozygous form, wherein the one or more further SNPs include: (i) a SNP at position 101 of SEQ ID NO: 3; (ii) a SNP at position 51 of SEQ I D NO: 4; (iii) a SNP at position 51 of SEQ ID NO: 5; (iv) a SNP at position 51 of SEQ I D NO: 6; (v) a SNP at position 51 of SEQ ID NO: 7; (vi) a SNP at position 51 of SEQ ID NO: 8; and (vii) a SNP at position 51 of SEQ ID NO: 9, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0258] In some embodiments, a plant having salinity and/or sodicity tolerance comprises one or more of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0259] In some embodiments, the plant has sodicity tolerance.

[0260] In a further aspect the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining if DNA of the plant comprises one or more single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more SNPs include: (i) a SNP at position 51 of SEQ I D NO: 9; (ii) a SNP at position 51 of SEQ I D NO: 10; (iii) a SNP at position 51 of SEQ ID NO: 1 1 ; (iv) a SNP at position 51 of SEQ ID NO: 12; (v) a SNP at position 101 of SEQ ID NO: 13; (vi) a SNP at position 51 of SEQ ID NO: 14; (vii) a SNP at position 51 of SEQ ID NO: 15; (viii) a SNP at position 101 of SEQ I D NO: 16; (ix) a SNP at position 51 of SEQ ID NO: 17; (x) a SNP at position 101 of SEQ ID NO: 18; (xi) a SNP at position 51 of SEQ ID NO: 19; (xii) a SNP at position 51 of SEQ ID NO: 20; (xiii) a SNP at position 51 of SEQ I D NO: 21 ; (xiv) a SNP at position 51 of SEQ ID NO: 22; (xv) a SNP at position 101 of SEQ ID NO: 23; (xvi) a SNP at position 51 of SEQ ID NO: 24; (xvii) a SNP at position 51 of SEQ ID NO: 25; (xviii) a SNP at position 51 of SEQ ID NO: 26; (xix) a SNP at position 51 of SEQ ID NO: 27; (xx) a SNP at position 101 of SEQ ID NO: 28; (xxi) a SNP at position 101 of SEQ ID NO: 29; (xxii) a SNP at position 101 of SEQ ID NO: 30; (xxiii) a SNP at position 51 of SEQ ID NO: 31 ; (xxiv) a SNP at position 51 of SEQ I D NO: 32; (xxv) a SNP at position 51 of SEQ ID NO: 33; (xxvi) a SNP at position 51 of SEQ ID NO: 34; (xxvii) a SNP at position 51 of SEQ ID NO: 35; (xxviii) a SNP at position 51 of SEQ ID NO: 36; (xxix) a SNP at position 51 of SEQ ID NO: 37; (xxx) a SNP at position 51 of SEQ ID NO: 38; (xxxi) a SNP at position 51 of SEQ ID NO: 39; (xxxii) a SNP at position 51 of SEQ ID NO: 40; (xxxiii) a SNP at position 101 of SEQ ID NO: 41 ; (xxxiv) a SNP at position 51 of SEQ ID NO: 42; (xxxv) a SNP at position 51 of SEQ ID NO: 43; (xxxvi) a SNP at position 51 of SEQ ID NO: 44; (xxxvii) a SNP at position 51 of SEQ ID NO: 45; (xxxviii) a SNP at position 51 of SEQ ID NO: 46; (xxxix) a SNP at position 51 of SEQ ID NO: 47; (xl) a SNP at position 51 of SEQ ID NO: 48; (xli) a SNP at position 101 of SEQ ID NO: 49; (xlii) a SNP at position 51 of SEQ I D NO: 50; (xliii) a SNP at position 51 of SEQ ID NO: 51 ; (xliv) a SNP at position 51 of SEQ ID NO: 52; (xiv) a SNP at position 51 of SEQ ID NO: 53; (xlvi) a SNP at position 51 of SEQ ID NO: 54; (xlvii) a SNP at position 51 of SEQ ID NO: 55; (xlviii) a SNP at position 51 of SEQ ID NO: 56; (xlix) a SNP at position 51 of SEQ I D NO: 57; (I) a SNP at position 51 of SEQ ID NO: 58; (li) a SNP at position 51 of SEQ ID NO: 59; (lii) a SNP at position 51 of SEQ ID NO: 60; (liii) a SNP at position 51 of SEQ ID NO: 61 ; and (liv) a SNP at position 51 of SEQ I D NO: 62, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0261] In some embodiments a plant having salinity and/or sodicity tolerance comprises one or more of: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ I D NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ I D NO: 55, with respect to wheat plants of the species Triticum aestivum, or the same nucleotide substitution in an equivalent sequence with respect to other plant species.

[0262] In some embodiments the plant has salinity tolerance.

[0263] As markers for the salinity and/or sodicity tolerance of plants, the genes referred to above may also be used in methods for identifying a plant having said phenotype. Accordingly, in a further aspect the present invention provides a method of identifying a plant having salinity and/or sodicity tolerance, the method comprising determining the expression level of one or more of Na H ⁺ antiporter NhaB, Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , NHX2, and AVP1-like protein, in the plant. [0264] In some embodiments, a plant having salinity and/or sodicity tolerance comprises one or more of:

(i) a decreased expression of Na7H ⁺ antiporter NhaB when grown in the presence of 100 mM sodium chloride, compared to expression of Na7H ⁺ antiporter NhaB in a plant with high sodium exclusion grown under the same conditions;

(ii) an increased expression of Aquaporin-like protein TIF1-4 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iii) an increased expression of Putative high-affinity potassium transporter when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(iv) an increased expression of NHX1 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride;

(v) an increased expression of NHX2 when grown in the presence of 100 mM sodium chloride, compared to expression when grown in the absence of sodium chloride; and

(vi) a decreased expression of AVP1-like protein when grown in the presence of 100 mM sodium chloride, compared to expression of AVP1-like protein in a plant with high sodium exclusion grown under the same conditions.

[0265] Methods for determining the expression level of genes has been described in detail above.

[0266] In a further aspect, the present invention provides a plant having salinity and/or sodicity tolerance identified by the aforementioned methods.

[0267] As described above, in some embodiments of the method of the first aspect of the invention a cross between wheat varieties W4909 and cv. Mace produced progeny plant Triticum aestivum cultivar MW#293. Accordingly, in a still further aspect, the present invention provides a wheat plant designated MW#293, representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0268] In a further aspect, the present invention provides seed of the wheat plant designated MW#293. Seed may be produced according to methods well known in the art. [0269] In a further aspect, the present invention provides a tissue culture of cells of the wheat plant designated MW#293. As used herein the term "tissue culture" refers to plant cells or plant parts from which wheat plants can be generated, including plant protoplasts, plant cali, plant clumps, and plant cells that are intact in plants, or part of plants, such as seeds, leaves, stems, pollens, roots, root tips, anthers, ovules, petals, flowers, embryos, fibers and bolls.

[0270] Techniques of generating plant tissue culture and regenerating plants from tissue culture are well known in the art. For example, such techniques are set forth in: Vasil, 1984, Cell Culture and Somatic Cell Genetics of Plants, Vol I, II, III Laboratory Procedures and Their Applications Academic Press, New York; Green et ai, 1987, Plant Tissue and Cell Culture, Academic Press, New York; Weissbach and Weissbach, 1989, Methods for Plant Molecular Biology, Academic Press; Gelvin et ai, 1990, Plant Molecular Biology Manual, Kluwer Academic Publishers; Evans et ai, 1983, Handbook of Plant Cell Culture, MacMillian Publishing Company, New York; and Klee et ai, 1987, Ann. Rev. of Plant Phys. 38:467-486.

[0271] In a further aspect, the present invention provides an isolated wheat plant comprising at least two single nucleotide polymorphisms (SNPs) in homozygous form, wherein the at least two SNPs include:

(i) a guanine (G) residue at position 51 of SEQ ID NO: 1 ; and

(ii) a thyime (T) residue at position 51 of SEQ ID NO: 2.

[0272] In some embodiments, the isolated wheat plant further comprises one or more further SNPs in homozygous form, and wherein the one or more further SNPs are selected from the group consisting of: (i) a cytosine (C) residue at position 101 of SEQ ID NO: 3; (ii) a guanine (G) residue at position 51 of SEQ ID NO: 4; (iii) a guanine (G) residue at position 51 of SEQ ID NO: 5; (iv) a thymine (T) residue at position 51 of SEQ ID NO: 6; and (v) an adenine (A) residue at position 51 of SEQ ID NO: 7.

[0273] In some embodiments, the isolated wheat plant has sodicity tolerance.

[0274] In some embodiments, the isolated wheat plant comprises one or more further single nucleotide polymorphisms (SNPs) in homozygous form, wherein the one or more further SNPs include: (i) an adenine (A) residue at position 51 of SEQ ID NO: 10; (ii) a cytosine (C) residue at position 51 of SEQ ID NO: 1 1 ; (iii) a thymine (T) residue at position 51 of SEQ ID NO: 12; (iv) an adenine (A) residue at position 101 of SEQ ID NO: 13; (v) a guanine (G) residue at position 51 of SEQ ID NO: 14; (vi) an adenine (A) residue at position 51 of SEQ ID NO: 15; (vii) a cytosine (C) residue at position 101 of SEQ ID NO: 16; (viii) a guanine (G) residue at position 51 of SEQ ID NO: 17; (ix) a guanine (G) residue at position 101 of SEQ ID NO: 18; (x) an adenine (A) residue at position 51 of SEQ ID NO: 19; (xi) a cytosine (C) residue at position 51 of SEQ ID NO: 20; (xii) a guanine (G) residue at position 51 of SEQ ID NO: 21 ; (xiii) a thymine (T) residue at position 51 of SEQ ID NO: 22; (xiv) an adenine (A) residue at position 101 of SEQ ID NO: 23; (xv) a thymine (T) residue at position 51 of SEQ ID NO: 24; (xvi) a cytosine (C) residue at position 51 of SEQ ID NO: 25; (xvii) a thymine (T) residue at position 51 of SEQ ID NO: 26; (xviii) a guanine (G) residue at position 51 of SEQ ID NO: 27; (xix) a guanine (G) residue at position 101 of SEQ ID NO: 28; (xx) a guanine (G) residue at position 101 of SEQ I D NO: 29; (xxi) a guanine (G) residue at position 101 of SEQ ID NO: 30; (xxii) an adenine (A) residue at position 51 of SEQ ID NO: 31 ; (xxiii) an adenine (A) residue at position 51 of SEQ ID NO: 32; (xxiv) an adenine (A) residue at position 51 of SEQ ID NO: 33; (xxv) a guanine (G) residue at position 51 of SEQ ID NO: 34; (xxvi) a cytosine (C) residue at position 51 of SEQ ID NO: 35; (xxvii) a guanine (G) residue at position 51 of SEQ ID NO: 36; (xxviii) an adenine (A) residue at position 51 of SEQ ID NO: 37; (xxix) a cytosine (C) residue at position 51 of SEQ ID NO: 38; (xxx) an adenine (A) residue at position 51 of SEQ ID NO: 39; (xxxi) a guanine (G) residue at position 51 of SEQ ID NO: 40; (xxxii) a thymine (T) residue at position 101 of SEQ ID NO: 41 ; (xxxiii) an adenine (A) residue at position 51 of SEQ ID NO: 42; (xxxiv) a thymine (T) residue at position 51 of SEQ ID NO: 43; (xxxv) a cytosine (C) residue at position 51 of SEQ ID NO: 44; (xxxvi) a guanine (G) residue at position 51 of SEQ ID NO: 45; (xxxvii) an adenine (A) residue at position 51 of SEQ I D NO: 46; (xxxviii) an adenine (A) residue at position 51 of SEQ ID NO: 47; (xxxix) a thymine (T) residue at position 51 of SEQ ID NO: 48; (xl) an adenine (A) residue at position 101 of SEQ ID NO: 49; (xli) a thymine (T) residue at position 51 of SEQ ID NO: 50; (xlii) a cytosine (C) residue at position 51 of SEQ ID NO: 51 ; (xliii) an adenine (A) residue at position 51 of SEQ ID NO: 52; (xliv) a thymine (T) residue at position 51 of SEQ ID NO: 53; (xlv) a thymine (T) residue at position 51 of SEQ ID NO: 54; and (xlvi) an adenine (A) residue at position 51 of SEQ ID NO: 55.

[0275] In some embodiments, the isolated wheat plant has salinity tolerance.

[0276] In some embodiments, the isolated wheat plant has one or more of the following features: a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride; a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride; a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate; and a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0277] In some embodiments, the isolated wheat plant is MW#293, representative seed of which has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB - Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on 14 June 2019 under accession number NCIMB 43422.

[0278] As described above, the inventors have identified a number of genes that are differentially expressed in plants that have salinity and/or sodicity tolerance. Accordingly, in a further aspect the present invention provides a method of increasing salinity and/or sodicity tolerance of a plant, the method comprising one or more of:

(i) decreasing expression and/or activity of Na7H ⁺ antiporter NhaB in one or more cells of the plant cell;

(ii) increasing expression and/or activity of Aquaporin-like protein TIF1-4 in one or more cells of the plant cell;

(iii) increasing expression and/or activity of Putative high-affinity potassium transporter in one or more cells of the plant cell;

(iv) increasing expression and/or activity of NHX1 in one or more cells of the plant cell;

(v) increasing expression and/or activity of NHX2 in one or more cells of the plant cell; and

(vi) decreasing expression and/or activity of AVP1-like protein in one or more cells of the plant cell.

[0279] As indicated above, the expression and/or activity of the aforementioned genes may be increased or decreased at the RNA and/or protein stages of expression. In some embodiments, expression and/or activity of the relevant gene is increased or decreased by genetic modification of one or more cells of the plant.

[0280] In some embodiments of this aspect of the invention, expression and/or activity of Na7H ⁺ antiporter NhaB and AVP1-like protein is decreased by decreasing expression and/or activity of nucleic acid encoding Na7H ⁺ antiporter NhaB and/or AVP1 -like protein. In some embodiments, expression and/or activity of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 is increased by increasing expression and/or activity of nucleic acid encoding Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2.

[0281]“Decreasing” in this context is intended to mean, for example, a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or greater, or 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater, reduction in transcription of nucleic acid encoding the relevant gene. Decreasing also includes the substantially complete inhibition (e.g. knockout) of expression of nucleic acid encoding the relevant gene in one or more cells of the plant that normally have such activity.

[0282]“Increasing” in this context is intended to mean, for example, a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, or 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater, increase in transcription of nucleic acid encoding the relevant gene. Increasing also includes introducing expression of nucleic acid encoding the relevant gene into one or more cells of the plant that do not normally express the relevant gene.

[0283] Any means by which the expression of nucleic acid encoding the relevant gene may be decreased or increased is contemplated. Methods for modulating the expression of nucleic acid include, for example: genetic modification of one or more cells of the plant to decrease or increase endogenous nucleic acid expression; genetic modification by transformation with a nucleic acid; genetic modification to increase the copy number of a nucleic acid in a cell; administration of a nucleic acid molecule to a cell which modulates expression acid in a cell of an endogenous nucleic encoding the relevant gene; and the like.

[0284] In some embodiments, the expression of nucleic acid encoding the relevant gene is modulated by genetic modification of a cell. The term“genetically modified”, as used herein, should be understood to include any genetic modification that effects an alteration in the expression of a nucleic acid in the genetically modified cell relative to a non-genetically modified form of the cell. Exemplary types of genetic modification include: random mutagenesis such as transposon, chemical, UV and phage mutagenesis together with selection of mutants which overexpress or underexpress an endogenous nucleic acid encoding the relevant gene; transient or stable introduction of one or more nucleic acid molecules into a cell which direct the expression and/or overexpression in the cell of nucleic acid encoding the relevant gene; modulation of an endogenous protein encoded by the relevant gene by site-directed mutagenesis of an endogenous nucleic acid encoding the relevant gene; introduction of one or more nucleic acid molecules which inhibit the expression of an endogenous nucleic acid encoding the relevant gene in the cell, e.g. a cosuppression construct, an RNAi construct or a miRNA construct; and the like.

[0285] In some embodiments, increasing expression and/or activity of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 in a cell is achieved by introducing Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 nucleic acid into the cell, upregulating the expression of Aquaporin-like protein, Putative high-affinity potassium transporter, NHX1 , and NHX2 nucleic acid in the cell, and/or increasing the copy number of Aquaporin-like protein TIF1- 4, Putative high-affinity potassium transporter, NHX1 , and NHX2 nucleic acid in the cell.

[0286] Methods for transformation and expression of an introduced nucleotide sequence in various cell types are well known in the art, and the present invention contemplates the use of any suitable method.

[0287] However, by way of example with regard to the transformation of plant cells, reference is made to Zhao et al. ( Mol Breeding DOI 10.1007/s 1 1032-006-9005-6, 2006), Katsuhara et al. ( Plant Cell Physiol 44(12): 1378-1383, 2003), Ohta et al. ( FEBS Letters 532: 279-282, 2002) and Wu et al. ( Plant Science 169: 65-73, 2005). Further suitable methods for introduction of a nucleic acid into plant cells include, for example: Agrobacterium-med atedi transformation, other bacterially-mediated transformation (see Broothaerts et al., 2005, supra) microprojectile bombardment-based transformation methods and direct DNA uptake-based methods. Roa-Rodriguez et al. (Agrobacterium- mediated transformation of plants, 3 ^rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003) review a wide array of suitable Agrobacterium-medi atedi plant transformation methods for a wide range of plant species. Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, and such methods are reviewed by Casas et al. ( Plant Breeding Rev. 13: 235-264, 1995). Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995). In addition to the methods mentioned above, a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849- 858, 2002). A range of other plant transformation methods may also be evident to those of skill in the art.

[0288] In some embodiments, decreasing expression of nucleic acid encoding Na7H ⁺ antiporter NhaB and/or AVP1-like protein in a cell can be facilitated by methods such as knockout, knockdown or downregulation of a Na7H ⁺ antiporter NhaB and/or AVP1-like protein nucleic acid in a cell using methods including, for example:

insertional mutagenesis including knockout or knockdown of a nucleic acid in a cell by homologous recombination with a knockout construct (for an example of targeted gene disruption see Terada et ai, Nat. Biotechnol. 20: 1030-1034, 2002);

post-transcriptional gene silencing (PTGS) or RNAi of a nucleic acid in a cell (for review of PTGS and RNAi see Sharp, Genes Dev. 15(5): 485-490, 2001 ; and Hannon, Nature 418: 244-51 , 2002);

transformation of a cell with an antisense construct directed against a nucleic acid (for examples of antisense suppression see van der Krol et a!., Nature 333: 866-869; van der Krol et ai, BioTechniques 6: 958-967; and van der Krol et ai, Gen. Genet. 220: 204- 212);

transformation of a cell with a co-suppression construct directed against a nucleic acid (for an example of co-suppression see van der Krol et ai, Plant Cell 2(4): 291-299); transformation of a cell with a construct encoding a double stranded RNA directed against a nucleic acid (for an example of dsRNA mediated gene silencing see Waterhouse et ai, Proc. Natl. Acad. Sci. USA 95: 13959-13964, 1998);

transformation of a cell with a construct encoding an siRNA or hairpin RNA directed against a nucleic acid (for an example of siRNA or hairpin RNA mediated gene silencing see Lu et ai, Nuci Acids Res. 32(21): e171 ; doi:10.1093/nar/gnh170, 2004);

insertion of a miRNA target sequence such that it is in operable connection with a nucleic acid (for an example of miRNA mediated gene silencing see Brown et ai, Blood 110(13): 4144-4152, 2007);

the use of synthetic oligonucleotides, for example, siRNAs or miRNAs directed against nucleic acid (for examples of synthetic siRNA mediated silencing see Caplen et ai, Proc. Natl. Acad. Sci USA 98: 9742-9747, 2001 ; Elbashir et ai, Genes Dev. 15: 188-200, 2001 ; Elbashir et ai, Nature 411 : 494-498, 2001 ; Elbashir et ai, EMBO J. 20: 6877-6888, 2001 ; and Elbashir et ai, Methods 26: 199-213, 2002); and

gene editing technologies such as mutagenesis and gene knock-down using zinc- finger nucleases (see Carroll D, 2011 , Genetics 188: 773-782; Sander JD et ai, 2011 , Nat. Methods 8: 67-69; and Miller JC et ai, 2007, Nat. Biotechnol., 25: 778-785), mutagenesis and gene knock-down using transcription activator- 1 ike effector nuclease (TALEN) systems (see Bogdanove AJ and Voytas DF, 201 1 , Science 333: 1843-1846; Streubel J et ai, 2012, Nat. Biotechnol., 30: 593-595; Cermak T et al., 2011 , Nucl. Acids Res., 39: e82; Chen K and Gao C, 2013, J. Genet. Genomics 40: 271-279; Voytas DF, 2013, Ann. Rev. Plant Biol., 64: 327-350; and Wang Y et ai., 2014, Nat. Biotechnol., 32: 947-951), and mutagenesis and gene knock-down using the CRISPR/Cas9 system or equivalently adapted systems (see Belhaj K et al., 2015, Current Opinion in Biotechnology, 32: 76-84; Shan Q et al., 2014, Nature Protocols, 9: 2395-2410; and Wang Y et ai., 2014, Nat. Biotechnol., 32: 947-951).

[0289] These knockout, knockdown or downregulation technologies are merely representative and are not limiting to other mechanisms that may be employed.

[0290] In addition to the examples above, a nucleic acid may be introduced into the cell wherein said nucleic acid comprises a nucleotide sequence which is not directly related to a nucleic acid encoding the relevant gene but, nonetheless, may directly or indirectly modulate the expression of a nucleic acid encoding the relevant gene in the cell. Examples include nucleic acid molecules that encode transcription factors or other proteins which promote or suppress the expression of an endogenous nucleic acid molecule in a cell; and other non-translated RNAs which directly or indirectly promote or suppress endogenous protein expression and the like.

[0291] In order to effect expression of an introduced nucleic acid in a cell, where appropriate, the introduced nucleic acid may be operably connected to one or more transcriptional control sequences and/or promoters.

[0292] The term“transcriptional control sequence” should be understood to include any nucleic acid sequence which effects the transcription of an operably connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activating sequence, and transcription terminator. Typically, a transcriptional control sequence at least includes a promoter. The term“promoter” as used herein, describes any nucleic acid which confers, activates or enhances expression of a nucleic acid molecule in a cell.

[0293] In some embodiments, at least one transcriptional control sequence is operably connected to a nucleic acid encoding the relevant gene. For the purposes of the present specification, a transcriptional control sequence is regarded as“operably connected” to a given gene or other nucleotide sequence when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the gene or other nucleotide sequence.

[0294] A promoter may regulate the expression of an operably connected nucleotide sequence constitutively, or differentially, with respect to the cell, tissue, organ or developmental stage at which expression occurs, in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others, or in response to one or more transcriptional activators. As such, the promoter used in accordance with the present invention may include, for example, a constitutive promoter, an inducible promoter, a tissue- specific promoter, or an activatable promoter.

[0295] Plant constitutive promoters typically direct expression in nearly all tissues of a plant and are largely independent of environmental and developmental factors. Examples of constitutive promoters that may be used in accordance with the present invention include plant viral derived promoters such as the Cauliflower Mosaic Virus 35S and 19S (CaMV 35S and CaMV 19S) promoters; bacterial plant pathogen derived promoters such as opine promoters derived from Agrobacterium spp., eg. the Agrobacterium- derived nopaline synthase (nos) promoter; and plant-derived promoters such as the rubisco small subunit gene (rbcS) promoter, the plant ubiquitin promoter (Pubi) and the rice actin promoter (Pact).

[0296] In some embodiments, a constitutive transcriptional control sequence may be used. In some embodiments, the constitutive transcriptional control sequence comprises one or more repeats of a CaMV 35S promoter. In some embodiments the transcriptional control sequence comprises two repeats of the CaMV 35S promoter.

[0297]“Inducible” promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: alcohol regulated promoters (eg. see European Patent 637 339); tetracycline regulated promoters (eg. see US Patent 5,851 ,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (eg. see US Patent 5,512,483), estrogen receptor promoters (eg. see European Patent Application 1 232 273), ecdysone receptor promoters (eg. see US Patent 6,379,945) and the like; metal-responsive promoters such as metallothionein promoters (eg. see US Patent 4,940,661 , US Patent 4,579,821 and US 4,601 ,978); and pathogenesis related promoters such as chitinase or lysozyme promoters (eg. see US Patent 5,654,414) or PR protein promoters (eg. see US Patent 5,689,044, US Patent 5,789,214, Australian Patent 708850, US Patent 6,429,362).

[0298] In some embodiments, a salt or sodium inducible promoter may be used. Examples of such promoters include the AtGRP9 promoter (Chen et al., Journal of Plant Research 120: 337-343, 2007; accession number At2g05440) and the VHAc3 promoter (accession number At4g38920).

[0299] An inducible promoter may also be a physically regulated promoter which is regulated by non-chemical environmental factors such as temperature (both heat and cold), light and the like. Examples of physically regulated promoters include heat shock promoters (eg. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097); cold inducible promoters (eg. see US Patent 6,479,260, US Patent 6, 184,443 and US Patent 5,847, 102); light inducible promoters (eg. see US Patent 5,750,385 and Canadian Patent 132 1563); light repressible promoters (eg. see New Zealand Patent 508103 and US Patent 5,639,952).

[0300]“Tissue specific promoters” include promoters which are preferentially or specifically expressed in one or more specific cells, tissues or organs in an organism and/or one or more developmental stages of the organism. It should be understood that a tissue specific promoter can also be constitutive or inducible.

[0301] Examples of plant tissue specific promoters include: root specific promoters such as those described in US Patent Application 2001047525; fruit specific promoters including ovary specific and receptacle tissue specific promoters such as those described in European Patent 316 441 , US Patent 5,753,475 and European Patent Application 973 922; and seed specific promoters such as those described in Australian Patent 612326 and European Patent application 0 781 849 and Australian Patent 746032.

[0302] In some embodiments, a promoter which preferentially or specifically directs expression in a root, or one or more parts thereof, may be used. Examples of root-specific or preferential promoters that may be used include the promoter of the root stelar gene AtGRP9 (At2g05440) as described by Chen et al. ( J . Plant Res. 120: 337-343, 2007) and the root cortex promoter from tobacco as described in US patent 5,837,876. [0303] The promoter may also be a promoter that is activatable by one or more transcriptional activators, referred to herein as an“activatable promoter”. For example, the activatable promoter may comprise a minimal promoter operably connected to an Upstream Activating Sequence (UAS), which comprises, inter alia, a DNA binding site for one or more transcriptional activators.

[0304] As referred to herein the term“minimal promoter” should be understood to include any promoter that incorporates at least an RNA polymerase binding site and, optionally a TATA box and transcription initiation site and/or one or more CAAT boxes. In some embodiments, the minimal promoter may be derived from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. The CaMV 35S derived minimal promoter may comprise, for example, a sequence that substantially corresponds to positions -90 to +1 (the transcription initiation site) of the CaMV 35S promoter (also referred to as a -90 CaMV 35S minimal promoter), -60 to +1 of the CaMV 35S promoter (also referred to as a -60 CaMV 35S minimal promoter) or -45 to +1 of the CaMV 35S promoter (also referred to as a -45 CaMV 35S minimal promoter).

[0305] As set out above, the activatable promoter may comprise a minimal promoter fused to an Upstream Activating Sequence (UAS). The UAS may be any sequence that can bind a transcriptional activator to activate the minimal promoter. Exemplary transcriptional activators include, for example: yeast derived transcription activators such as Gal4, Pdr1 , Gcn4 and Ace1 ; the viral derived transcription activator, VP16; Hap1 (Hach et ai, J Biol Chem 278: 248-254, 2000); Gaf1 (Hoe et ai., Gene 215(2): 319-328, 1998); E2F (Albani et ai, J Biol Chem 275: 19258-19267, 2000); HAND2 (Dai and Cserjesi, J Biol Chem 277: 12604-12612, 2002); NRF-1 and EWG (Herzig et ai., J Cell Sci 113: 4263-4273, 2000); P/CAF (Itoh et ai, Nucl Acids Res 28: 4291 - 4298, 2000); MafA (Kataoka et ai, J Biol Chem 277: 49903-49910, 2002); human activating transcription factor 4 (Liang and Hai, J Biol Chem 272: 24088 - 24095, 1997); Bel 10 (Liu et ai., Biochem Biophys Res Comm 320(1): 1-6, 2004); CREB-H (Omori et ai., Nucl Acids Res 29: 2154 - 2162, 2001); ARR1 and ARR2 (Sakai et ai, Plant J 24(6): 703-711 , 2000); Fos (Szuts and Bienz, Proc Natl Acad Sci USA 97: 5351-5356, 2000); HSF4 (Tanabe et ai, J Biol Chem 274: 27845 - 27856, 1999); MAML1 (Wu et ai., Nat Genet 26: 484-489, 2000).

[0306] In some embodiments, the UAS comprises a nucleotide sequence that is able to bind to at least the DNA-binding domain of the GAL4 transcriptional activator. [0307] An example of an activatable promoter includes the enhancer trap system for Arabidopsis and rice as described by Johnson et al. (Plant J. 41 : 779-789, 2005), and Moller et al. (Plant Cell 21 : 2163-2178, 2009).

[0308] In some embodiments, expression and/or activity of Na7H ⁺ antiporter NhaB and AVP1-like protein is decreased by decreasing expression and/or activity of Na7H ⁺ antiporter NhaB protein and/or AVP1 -like protein. In some embodiments, expression and/or activity of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 is increased by increasing expression and/or activity of Aquaporin-like protein TIF1- 4, Putative high-affinity potassium transporter protein, NHX1 protein, and NHX2 protein.

[0309] Decreasing or increasing expression of the relevant protein should be understood to include a decrease or increase in the level or amount of the relevant protein in a cell or a particular part of a cell. Similarly, decreasing or increasing the“activity” of relevant protein should be understood to include a decrease or increase in, for example, the total activity, specific activity, half-life and/or stability of a relevant protein in the cell.

[0310]“Decreasing” in this context is intended to mean, for example, a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or greater, or 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater, reduction in the level or activity of a relevant protein in the cell.“Increasing” in this context is intended to mean, for example, a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, or 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100-fold, or greater, increase in the level of activity of a relevant protein in the cell.

[0311]“Increasing” should also be understood to include introducing a relevant protein into a cell which does not normally express the introduced protein, and“decreasing” should also be understood to include the substantially complete inhibition of a relevant protein activity in a cell that normally expresses such a protein.

[0312] Any means by which the expression of a relevant protein in a cell is modulated is contemplated. This includes, for example, methods such as the application of agents which modulate protein activity in a cell, including the application of agonists or antagonists to the relevant protein; the application of agents which mimic activity of the relevant protein in the cell; modulating the expression of a nucleic acid which encodes the relevant protein in the cell (see discussion above); effecting the expression of an altered or mutated nucleic acid in a cell such that the relevant protein with an increased or decreased specific activity, half- life and/or stability is expressed by the cell (see discussion above); or modulating the expression level, pattern and/or targeting of a relevant protein in a cell, for example via modification of a transcriptional control sequence and/or signal polypeptide associated with the relevant protein.

[0313] In the method of increasing salinity and/or sodicity tolerance of a plant as described above, a plant with increased salinity and/or sodicity tolerance has one or more of the following features: a penultimate leaf sodium concentration at heading of at least 1 ,000 mg/kg DW when grown in the presence of 100 mM sodium chloride; a salinity tolerance of >50% when grown in the presence of 100 mM sodium chloride; a penultimate leaf sodium concentration at heading of at least 2,000 mg/kg DW when grown in the presence of 8 g/kg sodium humate; and a sodicity tolerance of >50% when grown in the presence of 8 g/kg sodium humate.

[0314] In some embodiments of this method, the plant having increased salinity and/or sodicity tolerance is a wheat plant. In some embodiments, the wheat plant is a hexaploid wheat plant, such as a plant of the species Triticum aestivum.

[0315] In a further aspect, the present invention provides a genetically modified plant cell with increased salinity and/or sodicity tolerance compared to a wild-type form of the plant cell, wherein expression and/or activity of Na7H ⁺ antiporter NhaB and/or AVP1-like protein is decreased in the plant cell, and/or wherein expression and/or activity of one or more of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and NHX2 is increased in the plant cell.

[0316] As referred to herein, a“genetically modified plant cell” comprises a cell that is genetically modified with respect to the wild type of the cell. As such, a genetically modified plant cell may be a cell which has itself been genetically modified and/or the progeny of such a cell.

[0317] In some embodiments, expression of nucleic acid encoding Na7H ⁺ antiporter NhaB and/or AVP1 -like protein, and/or expression of Na7H ⁺ antiporter NhaB protein and/or AVP1- like protein, is decreased in the plant cell. In some embodiments, expression of nucleic acid encoding Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter, NHX1 , and/or NHX2, and/or expression of Aquaporin-like protein TIF1-4, Putative high-affinity potassium transporter protein, NHX1 protein, and/or NHX2 protein, is increased in the plant cell. The cell may be of a type as described in detail above.

[0318] In a further aspect, the present invention provides a multicellular structure having salinity and/or sodicity tolerance, wherein the multicellular structure comprises one or more plant cells according to the aforementioned aspect of the invention.

[0319] As referred to herein, a“multicellular structure” includes any aggregation of one or more plant cells as hereinbefore described. As such, a multicellular structure specifically encompasses tissues, organs, whole plant and parts thereof. Furthermore, a multicellular structure should also be understood to encompass multicellular aggregations of cultured cells such as colonies, plant calli, liquid or suspension cultures and the like.

[0320] In light of the above, the term “multicellular structure” should be understood to include a whole plant, plant tissue, plant organ, plant part, plant reproductive material or cultured plant tissue (e.g. callus or suspension culture).

[0321] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0322] The term“about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1% or less, or +/- 0.1 % or less of and from the numerical value or range recited or claimed.

[0323] As used herein, the singular forms“a,”“an,” and“the” may refer to plural articles unless specifically stated otherwise.

[0324] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. [0325] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0326] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0327] Furthermore, the description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0328] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0329] The invention is further illustrated in the following examples. The examples are for the purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description. It will be appreciated by those skilled in the art that the disclosure may be embodied in many other forms.

EXAMPLE 1

Generation and Characterisation of Salinity and/or Sodicity Tolerant Wheat and

Genetic Markers Thereof

[0330] This Example encompasses four experiments. These experiments were conducted to: (1) determine the effects of the Na ⁺ exclusion genes Nax1 and Nax2 under salinity and sodicity in bread wheat ( Triticum aestivum L.) cv. Westonia background; (2) identify genetic markers for Na ⁺ exclusion/accumulation using genome-wide association studies; (3) quantify effects of a wide range of Na ⁺ exclusion under salinity and sodicity; and (4) develop/characterize high-Na ⁺ and sodicity/salinity tolerant wheat germplasm carrying alien introgression.

Materials and Methods

Plant Material

[0331] In Experiment 1 , there were four bread wheat ( Triticum aestivum L.) entries (Westonia, Westonia with Na ⁺ exclusion gene Nax1, Westonia with Na ⁺ exclusion gene Nax2 and Baart-46). The Nax1 ( TmHKT1;4-A2 ) and Nax2 ( TmHKT1;5-A ) genes have been previously characterized (James et ai, 2006, Plant Physiology 142: 1537-1547; Byrt et at., 2007, Plant Physiology 143: 1918-1928). As bread wheat cv. Westonia represents low Na ⁺ wheat genotypes, a bread wheat genotype representing high-Na ⁺ wheat genotypes, cv. Baart-46 (Gene et ai, 2007, Plant Cell and Environment 30: 1486-1498), was also included for comparison.

[0332] In Experiment 2, 100 bread wheat entries were included (see Table 2) forming a diversity panel based on differential growth, yield and Na ⁺ exclusion (Richards et ai, 1987, Field Crops Research 15: 277-287; Shavrukov et ai, 2006, “Screening for sodium exclusion in wheat and barley”. Proceedings of 13 ^th Agronomy Conference, 10-14 September 2006, Perth, Western Australia; Gene et ai, 2007, supra James et ai, 201 1 , Journal of Experimental Botany 62: 2939-2947; and Setter et ai, 2016, Field Crops Research 194: 31-42). These were released cultivars, breeding lines and germplasms (MW#28, MW#293, MW#451 and MW#491) developed in-house from a cross between low sodium cv. Mace and high sodium wheat germplasm line W4909 (Wang et ai, 2003, Crop Science 43: 745-746)(see breeding methodology below). Twelve durum wheats ( Triticum turgidum subsp durum) and an Australian barley ( Hordeum vulgare L) cv. Clipper were also included as checks. As durum wheats generally accumulate higher Na ⁺ levels than bread wheats, two unique durum lines were included each having low Na ⁺ concentration (cv. Tamaroi with the Nax2 and breeding line WID902 with both Nax1 and Nax2).

[0333] In Experiment 3, there were 20 bread wheat entries (representing the range in leaf Na ⁺ concentration in Experiment 2), four durum wheat entries and a barley entry Clipper as checks.

[0334] In Experiment 4, cv. Mace and wheat germplasm lines with low leaf Na ⁺ concentration (MW#28 and MW#491) and high leaf Na ⁺ concentration (MW#293 and MW#451) were used. TABLE 2

Name, year of registration, origin, species, pedigree and the source of entries used in the present study

st wheat pedigree data were compiled from http://www.wheatpedigree.net/. Commercial cultivars grown in South Australia in 2015 are given in bold tps://grdc.com.au/ _ data/assets/pdf_file/0020/109055/sa-sowing-guide-2015-pdf.pd f.pdf). SARDI: South Australian Research and Development Institute (P search Centre, Waite Campus, 2b Hartley Grove, Urrbrae, South Australia, 5064, Australia). AGG: Australian Grains Genebank (Horsham, Victoria, 3400, Austr : University of Adelaide (Waite Campus, Waite Road, Urrbrae, South Australia, 5064, Australia). LRPB: LongReach Plant Breeders (Park Drive, Bundoora, Vict 83, Australia). CSIRO: Commonwealth Scientific and Industrial Research Organisation (Canberra, ACT, 2061 , Australia).

Crosses between low sodium cv. Mace and high sodium wheat germplasm line W4909

[0335] A classical/conventional plant breeding method was used to obtain bread wheat germplasm lines MW#28, MW#293, MW#451 and MW#491. Briefly, this is a method of crossing closely or distantly related species to create new crops with desirable characteristics. A suitable spike is chosen on the female parent in which pollen is not yet ripened. For convenience, two outer flowers of the spikelets are used for crossing. The remaining spikelets are cut off close to the rachis with dissecting scissors. Individual spikelets are clipped just above the anthers. Unripe greenish anthers (pollen factories) from the female plants (cv,.Mace) are then removed by a pair of forceps with fine points (emasculation). The spikes of the emasculated plants are covered by paper bags to ensure they are not pollinated by random pollens. In the next few days, with the help of a pair of fine forceps, the pollens from the male plant (W4909) are transferred onto the stigmas of female plants. The resulting seed is an F1 hybrid (also known as filial 1 hybrid).

F1 progeny were then used to generate populations via the wheat-maize hybridization method to study inheritance of the trait. MW#28, MW#293, MW#451 and MW#491 were four of over 200 doubled-haploid lines developed using the wheat-maize method. Wheat- maize hybridization includes six steps; emasculation of wheat flower, pollination of emasculated flower with maize pollen, hormone treatment, embryo rescue, haploid plant regeneration in tissue culture medium, and chromosome doubling (Broughton et ai, 2014, supra ; Santra et ai, 2017, supra).

Growth medium, treatments, seedling establishment and growth conditions

[0336] All four experiments used University of California potting mix, described previously (Gene et ai., 2016, New Phytoiogist 2M^\ 145-156). In Experiment 1 , there were five salinity (0, 50, 100, 150 and 200 mM NaCI) and four sodicity (2, 4, 8 and 16 g kg ¹ Na ⁺ -humate) levels which were replicated four times. As plants were grown to maturity, 4 kg capacity pots were used as described in Gene et ai, 2016, supra. There were three plants per pot.

[0337] In Experiment 2, as plants were grown to heading to determine Na ⁺ concentration in leaves at a single level of sodicity (8 g kg ^-1 Na ⁺ -humate), four plants per pot were grown in 3 kg capacity pots to enable testing of more wheat entries. There were four replications.

[0338] In Experiment 3, as plants were grown to maturity, 4 kg capacity pots were used. There were four replications and three plants per pot, and grown under control, sodicity (8 g kg ^-1 Na ⁺ -humate) and salinity (100 mM NaCI). [0339] In Experiment 4, plants were grown to heading or maturity under control and salinity in 4 kg capacity pots. There were five (nutrient analysis) or nine (gene expression) replications, and three plants per pot. At heading, penultimate leaves were sampled for gene expression, while in the other set penultimate leaves were sampled for elemental analysis and plants were grown to maturity.

Plant traits measured

[0340] In all experiments the pots were weighed daily and watered to field capacity (7.4% w/w) till heading and 10% thereafter with milli-Q water. Weekly incremental water uptake was used to establish growth curves in Experiments 1 and 3 (Gene et ai, 2016, supra). At heading (main culm fully emerged), penultimate leaves were sampled for analysis of Na ⁺ , potassium (K ⁺ ), Ca ²⁺ , magnesium (Mg ²⁺ ) and Cl (Gene et ai, 2016, supra). Handling of leaf samples and analytical methods used in nutrient analyses were described earlier (Gene et ai, 2016, supra). At maturity, grain yield per plant was determined. For across and within species comparisons, relative grain yield (the ratio of yield at an individual stress level to that under nil stress and expressed as percent) was also calculated.

Candidate gene selection, primer design, and gene expression in penultimate leaves under control and salinity

[0341] Candidate genes and previously reported genes were selected based on published literature and findings of the present study (see Table 3). Primers for qPCR reactions were sourced from previous studies or designed against relevant cDNA sequences from NCBI. Primers were designed using NCBI/Primer-BLAST, targeting amplicon sizes between 60 and 170 bases. Specific qPCR amplification for each candidate gene was confirmed by obtaining a single, distinct peak in melt curve analysis, and by sequencing the qPCR products.

[0342] Total RNA was isolated from penultimate leaves using Spectrum Plant Total RNA kit (Sigma) with an on-column DNase treatment. The concentration and integrity of the RNA was determined using a NanoDrop 8000 (Thermo Scientific) spectrophotometer. Superscript III Reverse Transcriptase kit (Life Technologies) was used to synthesize the cDNA. The reaction contained 500 ng purified RNA from each sample in a final reaction volume of 20 pi, performed according to manufacturer’s instructions. The cDNA samples were diluted 10 times in sterile milli Q water before being used as a template in qPCR reactions. The qPCR assays were prepared according to manufacturer’s instructions using PrecisionFAST qPCR mix (Primer Design Ltd). TABLE 3

Accession number/probe set, annotation/gene function, primer sequence, amplicon of candidate genes, previously published genes and housekeeping genes investigated in this study

Mott & Wang (2007): Mott IW and Wang RR-C, 2007, Plant Science, 173: 327-339. Tounsi et al. (2016): Tounsi S et ai, 2016, Plant and Cell Physiology, 57: 20 2057.

Amplifications were performed in a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher) with 3 min of 95°C followed by 40 cycles of 3s at 95°C, 20s at 60°C, and fluorescent acquisition at 60°C, followed by melt curve analysis. Three wheat genes, encoding actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and elongation factor 1-alpha (EF1 a) were used together for normalization of target gene expression. Purified PCR products of target genes, covering six orders of magnitude were used to construct a standard curve of copy number in relation to the cycle threshold (Ct) value from which the actual copy number of RNA was obtained.

Experimental design and statistical analysis

[0343] For Experiments 1 and 4, the variety by treatment combinations were allocated to pots in a single growth room using a randomized complete block design (RCBD) with four replicates of each combination. In Experiments 2 and 3 the number of pots required exceeded the size of a single growth room and consequently two growth rooms with identical settings were used and variety by treatment combinations were allocated to pots within each growth room using an RCBD with two replicates. To overcome the problem of variance heterogeneity, all leaf Na ⁺ and some of Cl data were log-transformed prior to model fitting.

[0344] For each experiment, analysis of measured elemental and grain yield related traits was conducted using linear mixed models that appropriately captured sources of treatment and variety variation as well as environmental variation associated with the experiments. In each model the fixed component contained term accounting for variety and treatment main effects as well as variety by treatment interaction effects. To appropriately account for extraneous variation, physical design constraints such as multiple growth rooms and replicates within growth rooms were accounted for using random effects. For any given trait, perceived observational outliers were down-weighted using a simple indicator covariate random effect term (Gumedze et al., 2010, Computational Statistics and Data Analysis 54: 2128-2144).

[0345] From each of the fitted models best linear unbiased estimates (BLUEs) and standard errors for the variety by treatment interaction means were extracted for summary. For traits analysed from Experiments 1 , 3 and 4, where there was a reduced number of variety by treatment combinations, a Least Significant Difference (LSD) at P= 0.05 was calculated and used to compare variety by treatment means. For traits analysed from Experiment 2, the Honest Significant Difference (HSD) at P= 0.05 was used to control the familywise error rate when comparing between means.

[0346] Similar to Gene et al., 2016, supra, plant water uptake in Experiments 1 and 3 was statistically assessed using weekly incremental water use from 15 days of transplanting to heading of each variety. To determine differences in the rate of water use between the levels of salinity or sodicity treatments across varieties, a longitudinal regression analysis was conducted using a linear mixed model. In this model, the fixed component contained terms to model the intercept and linear slope of the water use over time for each of the variety by treatment combinations. Additional non-linearity was modelled using a random cubic smoothing spline term (Verbyla et al., 1999). For each model the estimated variety by treatment linear coefficients were extracted and an LSD at P= 0.05 was calculated to provide a comparison between estimates. Model based prediction curves of incremental water use were also calculated for graphical summary.

[0347] All linear mixed modelling of grain yield, elemental and water use traits was computationally conducted using the flexible ASReml-R software (Butler et al., 2009, ASReml-R Reference Manual (Version 3). Queensland Department of Primary Industries), available as a package in the R statistical computing environment (R Core Team, 2018, https://www. R-project. org/) .

Genome-wide association study and identification of candidate genes

[0348] DNA of the 100 bread wheats was extracted from leaf tissue using the phenol/chloroform extraction method and subsequently genotyped with the 90K wheat SNP array (Wang et al., 2014, Plant Biotechnology Journal 12: 787-796). Population structure was estimated using ADMIXTURE (v1.23) software (Alexander and Lange, 2011 , BMC Bioinformatics 12: 246) which uses a model-based algorithm to estimate the ancestry of individuals. Cross-validation was used to determine the most likely number of clusters to be used in the subsequent modelling.

[0349] For the 100 bread wheats, the BLUEs of Na ⁺ extracted from the fitted model of Experiment 2 were used in genome-wide association mapping, based on 41 ,035 SNP markers with minor allele frequency (MAF)> 0.05 and <50% missing call rate. For each of the SNPs, a mixed-linear model (MLM) was fitted where the fixed component of the model contained a numerical version of the SNP as well as a covariate to adjust for the confounding effects of population structure. The MLM also contained a random effect for the lines with an assumed variance structure equivalent to the kinship matrix centred using the IBS method and then compressed to optimum groups. This then allowed the P3D (population parameters previously determined) compressed MLM method to be used to speed up computation time (Zhang et al., 2010, Nature Genetics 42: 355-360). From each of the fitted models, SNP effects were assessed using a significant p-value threshold set at P= 8.91 e-5 equivalent to a level of 0.05 after Bonferroni correction using the simple/W method (Gao X et al., 2008, Genet. Epidemiol., 32: 361-369). Bonferroni correction assumes that the hypothesis tests are independent which is not true due to linkage disequilibrium among the SNP in GWAS study. Briefly, the simple/W method calculated the effective number of independent test using principal component analysis based on the SNP data. Subsequently, the number of test in the Bonferroni correction formula was replaced by the effective number of independent test. All genome wide association mapping and assessment was computationally conducted using TASSEL software (Bradbury et al., 2007, Bioinformatics 23: 2633-2635). Based on the physical position of the markers and high confidence gene content in the Chinese Spring Reference Genome (IWGSC RefSeq v1.0 - https://wheat-urgi.versailles.inra.fr/Seq-Repository/Annotat ions), candidate genes located within a 1 Mb region flanking the significant SNP were reported. The IWGSC RefSeq v1.0 annotation is publicaly available for download

(https://urgi. Versailles. inra.fr/download/iwgsc/IWGSC_RefSeq_Annotations/v1.0/).

Results

Experiment 1. Growth responses of Westonia, Westonia-Nax1, Westonia-Nax2 and Baart- 46 under a range of salinity and sodicity

[0350] A regression analysis of weekly incremental water use indicated that all wheat lines had reduced water uptake and consequently reduced growth rates under increasing salinity and sodicity (Figure 1 ; Table 4), and reductions were greater under salinity than sodicity. Under salinity, there were no differences amongst the Westonia lines, while Baart-46 had higher growth rates than the Westonia lines until 100 mM NaCI was reached. Under sodicity, Westonia-/Vax7 had higher growth rate than Westonia-/\/ax2 at 8 g kg ^-1 soil Na ⁺ - humate, but there were no other significant differences amongst Westonia lines at other levels. Baart-46 had higher growth rates than Westonia lines at all levels of sodicity (Table 4). TABLE 4

Slopes of incremental water use over time (surrogate for growth rate)

LSD refers to Least Significant Difference at P= 0.05 for species x treatment interaction

[0351] Penultimate leaf Na ⁺ concentrations were much higher under sodicity than salinity (Figure 2; Tables 5 and 6). Baart-46 maintained much higher Na ⁺ concentrations than Westonia and Nax lines under both stresses (Figure 2; Tables 5 and 6). As compared to Westonia, the presence of Nax1 and Nax2 genes was associated with reduced Na ⁺ concentrations (Figure 2; Tables 5 and 6). Reductions were similar for both genes and became more pronounced at higher rates of salinity and sodicity, reaching maxima of 72- 82% and 32-34% reductions at 8 g kg ¹ Na ⁺ -humate and 100 mM NaCI, respectively. Chloride concentrations were much higher under salinity than sodicity, and higher in Baart- 46 than Westonia and Nax lines, the latter group being similar to each (Figure 2; Tables 5 and 6).

[0352] For other cations, Ca ²⁺ concentrations were lower under sodicity than salinity, Mg ²⁺ concentrations were similarly low under both salinity and sodicity, while K ⁺ concentrations remained unaffected, either by salinity or sodicity (Table 5). The most notable genetic differences were higher K ⁺ , and lower Ca ²⁺ and Mg ²⁺ in Baart-46 than the other three lines under salinity and sodicity. Reduced Na ⁺ concentrations in the Westonia Nax lines were not accompanied by higher grain yields, with small grain yield increases observed only under moderate salinity and low sodicity (6-9% at 50 mM NaCI and 5-10% at 2 g kg ^-1 Na ⁺ -humate) (Table 7). At these salinity and sodicity rates, despite much TABLE 5

Best linear unbiased estimates for leaf Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ and Cl concentrations (mg kg ¹ DW) in bread wheat ( Triticum aestivum L.) cv. Westonia, Weston \a-Nax1, Westonia-A/ax2 and Baart-46 under salinity and sodicity in Experiment 1

_ LogNa ^* * _ _ j _ _ Ca ^2* _ _ Mg ^2* LogCI *alinity Westonia West- West- Baart-46 Westonia West- West- Baart-46 Westonia West- West- Baart-46 Westonia West- West- Baart-46 Westonia West- W M NaCI) Nax1 Nax2 Nax1 Nax2 Nax1 Nax2 Nax1 Nax2 Nax1 N

0 2.722 2.525 2.656 5.430 40287 39061 39879 34218 5756 6540 7704 6226 1952 2307 2182 2462 8.260 8.310

50 2.861 2.967 2.860 6.304 39132 36755 40141 34746 7691 7090 8176 7059 1723 1738 1769 2100 9.556 9.500

100 3.293 2.928 2.957 6.466 40792 37667 40929 33610 7619 6807 7932 7139 1674 1596 1618 1790 9.913 9.800

150 3.468 3.152 3.1 12 6.584 40565 42681 40238 36637 7378 7263 7769 7708 1427 1490 1558 1554 10.051 10.081

200 3.444 3.293 3.272 6.539 38349 40453 39440 36447 7128 7645 7318 6915 1329 1408 1386 1349 10.077 10.065

LSD** 0.305 2320 862 135 0.110

LogNa ^* *

odicity Westonia West- West- Baart-46

kg ^'1 soil Nax1 Nax2

a ^* -humate)

0 2.722 2.525 2.656 5.430 40287 39061 39879 34218 5756 6540 7704 6226 1952 2307 2182 2462 8.260 8.310

2 2.738 2.685 2.690 6.669 40861 41077 43036 36832 5041 5115 6172 4825 1730 2032 1924 2333 8.524 8.516

4 3.896 2.838 2.846 7.107 42188 42140 43703 38079 4120 4163 4958 3543 1738 1997 1780 1982 8.676 8.741

8 4.921 3.691 3.214 7.227 43877 42081 43708 37564 3624 3598 4160 3016 1683 1694 1712 1708 8.997 8.999

16 6.301 5.186 5.479 7.777 41599 41985 42496 36909 2932 3477 2894 2774 1502 1690 1507 1524 8.969 9.141

LSD** 0.338 2526 654 178 0.118 ⁺ and Cl· concentration data were transformed to natural logarithms. **LSD refers to Least Significant Difference at P= 0.05 for species x treatment interaction. Althtrol treatment appeared once for both treatments in the experimental design, the control values are presented for both treatments separately to assist comparisons wh treatment. However, in statistical analysis, all data were analysed together with the control treatment appearing once.

TABLE 6

Back-transformed Na ⁺ and Cl concentrations in bread wheat ( Triticum aestivum L.) cv. Westonia, Westonia-A/ax·/, Westonia-A/ax2 and Baart-46 under different levels of salinity and sodicity in Experiment 1

Although control treatment appeared once for both treatments in the experimental design, the control values were presented for both treatments separately to assist comparisons within each treatment. However, in statistical analysis, all data were analysed together with the control treatment appearing once.

TABLE 7

Best linear unbiased estimates for grain yield in bread wheat Triticum aestivum L·.) cv. Westonia, Westonia-A/ax·/, Westonia-A/ax2 and Baart-46 under different levels of salinity and sodicity in Experiment 1

*LSD refers to Least Significant Difference at P= 0.05 for species x treatment interaction. higher Na ⁺ concentrations than Westonia and Nax lines, cv. Baart-46 was similar or higher for grain yield (Table 7; Figure 2). There were no other notable differences in salinity or sodicity tolerance (relative grain yield %) amongst the four bread wheat lines and salinity/sodicity rates (Table 7).

Experiment 2. Genome-wide association mapping of Na ⁺ accumulation in 100 bread wheat entries

[0353] Given the benefits of Na ⁺ exclusion under sodicity but not salinity observed in our initial study (Gene et al., 2016, supra), here we screened a bread wheat diversity set under sodicity to determine genetic variation for Na ⁺ exclusion. Figure 3 and Table 8 show that there is genetic variation in Na ⁺ exclusion (P<0.001), but almost all elite bread wheat entries had high Na ⁺ exclusion (<2,000 mg kg ^-1 DW), compared to typical durum wheat entries (15,000-30,000 mg Na kg ^-1 DW) and barley entry Clipper (14,300 mg kg ^-1 DW). Leaf Na ⁺ concentrations in bread wheats varied from 50 mg kg ^-1 DW in Westonia-/\/ax2 to 2,800 mg kg ^-1 DW in cv. Olympic. The only exceptions to this were two bread wheat germplasm lines (MW#451 and MW#293; >15,000 mg kg ^-1 DW) which grouped with the durum wheats and barley. The presence of Na ⁺ exclusion genes Nax1 and Nax2 in durum wheat ( Nax1 and Nax2 in WID902; Nax2 in Tamaroi) was associated with much lower Na ⁺ concentrations (600-4,000 mg kg ^-1 DW) than in durum wheats lacking these genes (Figure 3; Table 8).

[0354] Calcium, K ⁺ and Mg ²⁺ concentrations varied significantly amongst the bread wheat entries (850-5,240, 24,820-48,400 and 940-2,460 mg kg ^-1 DW, respectively), but variations were much lower than those observed for Na ⁺ concentration, and values were lower in durum wheats (Figure 3; Table 8).

Single nucleotide polymorphism (SNP) markers significantly associated with Na ⁺ concentration in 100 bread wheat entries

[0355] Genome-wide association mapping was performed with 41 ,035 SNP markers (MAF > 5%) in the 100 bread wheat accessions taking population structure effect (K=3) into account (Figure 4). We identified nine SNPs significantly (P-value<8.91 e- 5) associated with leaf Na ⁺ concentration (log-transformed)(Table 9; Figure 5). Using the IWGSC RefSeq v1.0, seven SNPs were mapped to chromosomes 2A, 2B, 2D, 4B, 4D, 5B, and 7A. We examined the high confidence (HC) genes located within 1 Mb left and right of each significant SNP, and identified four candidate genes with potential functions in Na ⁺ accumulation/exclusion. TABLE 8

Best linear unbiased estimates (BLUEs) for element concentrations (mg kg ^~1 DW) at heading in 100 bread wheats ( Triticum aestivum L·.), 12 durum wheats Triticum turgidum subsp durum) and a barley cultivar ( Hordeum vulgare L.) grown under sodicity (8 g kg ¹ Na ⁺ -humate) in Experiment 3

HSD 0.847 3502 6694 1143 367

Na ⁺ concentration data were transformed to natural logarithms for analysis

These were calcium-transporting ATPase (TraesCS4D01 G343200.1), Na ⁽⁺⁾ /H ⁽⁺⁾ antiporter NhaB (T raesCS4D01 G344200.1), Aquaporin-like protein TIF1-4

(T raesCS4D01 G344300.1 ) , and Aquaporin PIP2 (TraesCS4B01G362300.1). It is notable that MW#451 and MW#293 contain the rare alleles that gave rise to extremely high Na ⁺ concentrations (allele effects for SNPs shown in Figure 6). We did not identify any mapped significant SNPs in proximity to the Na ⁺ exclusion genes Nax1 (2A) and Kna 1 (4D; TaHKT1;5-D) which is homologous to Nax2 in the diploid bread wheat ancestor T. monococcum.

Experiment 3. Effects of a wide range of Na ⁺ exclusion on salinity and sodicity tolerance in 20 bread wheats, three durum wheats and a barley entry

[0356] Regression analyses of incremental water use up to heading stage indicated that growth rates were reduced significantly by salinity and sodicity in all bread wheats, durum wheats and barley cv. Clipper, with a much lower reduction occurring in bread wheat germplasm line MW#293 which had the highest growth rate under salinity and sodicity (Table 10; Figure 7). The Nax2 gene in durum wheat Tamaroi was associated with a non significant increase in growth rate under sodicity, while there was no benefit under salinity. Barley cv. Clipper had generally higher growth rates than the averages of bread wheats under salinity and sodicity. There were also differences in growth rates under control.

TABLE 9

SNPs significantly associated with leaf Na ⁺ concentration and their flanking candidate genes identified in Experiment 2

Candidates genes selected for expression analyses are presented in bold. ^a SNP positions according to IWGSC RefSeq v1 .0 (https://wheat- urgi.versailles.inra.fr/Seq-Repository/Annotations).

TABLE 10

Slopes of incremental water use over time (surrogate for growth rate) derived from linear regressions up to heading stage in 20 bread wheats ( Triticum aestivum L.), three durum wheats (T ticum turgidum subsp durum cv. Tamaroi , Tamaroi-A/ax2, and Yawa) and one barley cultivar ( Hordeum vulgare L. cv. Clipper) under control, salinity (100 mM NaCI) and sodicity (8 g kg ^-1 Na ⁺ - humate) in Experiment 3

Control Sodicity Salinity

AGT Katana 2.8 1 .1 1.1

Axe 2.1 0.9 0.7

Krichauff 2.7 1 .0 1.2

Longreach Cobra 3.0 1 .0 1.3

Baart-46 4.5 2.2 2.4

Beckom 3.2 1 .8 1.4

Mace 3.3 1.5 1.4

MW#293 4.7 3.9 3.7

Correll 3.2 1.3 1.4

Condor 4.5 2.6 2.4

MW#451 3.3 1.4 1.5

Pitic-62 4.3 2.3 2.1

Drysdale 3.0 1 .0 1.2

Federation 6.0 3.4 2.7

Halberd 4.6 1 .8 1.8

Hartog 2.9 1 .0 1.3

Kharchia-65 4.9 3.0 2.4

Westonia 4.0 1.5 1.6

Wyalkatchem 2.4 1 .1 1.1

Yitpi 3.7 1.5 1.6

Tamaroi 4.6 1 .8 2.0

Tamaroi -Nax2 4.5 2.4 1.7

Yawa 5.1 3.0 2.9

Clipper 5.6 2.4 2.8

LSD cont vs sodicity 0.8

LSD cont vs salinity 0.8

*LSD refers to Least Significant Difference test value at P= 0.05 for species x treatment interaction. [0357] Salinity and sodicity increased leaf Na ⁺ concentrations in all entries, and concentrations were higher under sodicity than salinity (Table 11). Amongst the commercial wheats, older cultivars such as Federation and Baart-46 had higher Na ⁺ concentrations (430-460 and 1 ,700-1 ,800 mg kg ¹ DW under salinity and sodicity) than modern cultivars (<400 and 1 ,200 mg kg ¹ DW under salinity and sodicity) (Table 11). However, none of the cultivars had Na ⁺ concentrations as high as the two novel germplasm lines (MW#451 and MW#293) derived from wild relatives of bread wheat (back-transformed averages of two lines; 5,600 and 13,000 mg kg ¹ DW under salinity and sodicity, respectively) (Table 1 1).

[0358] There was a significant correlation between Na ⁺ concentration under salinity and sodicity (r=0.984, df= 18, P<0.01) (Figure 8). Barley entry Clipper had Na ⁺ concentrations (7,000 and 17,000 mg kg ¹ DW under salinity and sodicity) as high as those in high- Na ⁺ wheat germplasm lines MW#293 and MW#451 , while durum wheats Yawa and Tamaroi had overall the highest Na ⁺ concentrations (6,700 and 1 1 ,400 under salinity; 20,600 and 32,700 mg kg ¹ DW under sodicity, respectively). As expected, concentrations of Cl rose significantly under salinity in all entries, and the increases were similar for the three species (Table 1 1).

[0359] The other cations measured were K ⁺ , Ca ²⁺ and Mg ²⁺ . Reduced concentrations of K ⁺ and Ca ²⁺ under sodicity and salinity were moderate, while Mg ²⁺ was significantly reduced, especially under sodicity (Table 11). Durum wheats Yawa and Tamaroi and barley entry Clipper exhibited mild Mg ²⁺ deficiency symptoms under sodicity. There were significant correlations between salinity and sodicity for concentrations of K ⁺ , Ca ²⁺ and Mg ²⁺ (Table 1 1) .

[0360] Bread wheat entries varied in grain yield under control, sodicity and salinity (3-, 4- and 5-fold, respectively; Figure 9). Axe produced the lowest, while germplasm line MW#293 produced the highest grain yield under all conditions and doubled the grain yield of almost all other entries under salinity and sodicity (Figure 9). There was a close correlation between grain yield produced under control and either salinity or sodicity (r=0.867 and r=0.927 when comparing control vs salinity or control vs sodicity) (Figure 8). The correlation was even greater when grain yields were compared between salinity and sodicity (r=0.961 , df= 18, P<0.01) (Figure 8). Depending on wheat entries, tolerance (relative grain yield %) was higher, lower or similar between salinity and sodicity (Figure 9). TABLE 11

Best linear unbiased estimates for leaf Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ and Cl concentrations (mg kg ¹ DW) in 20 bread wheats ( Triticum aestivum L.), three durum wheats ( Triticum turgidum subsp durum cv. Tamaroi , Tamaroi-A/ax2, and Yawa) and one barley cultivar ( Hordeum

vulgare L.cv. Clipper) under control, salinity (100 mM NaCI) and sodicity (8 g kg ^-1 Na ⁺ -humate) in Experiment 3

logNa logCI iC Ca^ M£ heat control sodicity salinity control sodicity salinity control sodicity salinity control_ sodicity salinity control sodicity salini ch Cobra 3.0 5.1 3.4 8.6 9.0 9.9 36195 40803 35571 7256 3348 8420 2841 1897 227 ia 2.8 5.4 3.4 8.5 8.8 9.7 35116 42279 33991 8171 3785 7174 2527 1622 188 ff 2.8 5.8 4.2 8.6 8.7 9.9 37910 43897 39750 6638 2628 5842 2458 1292 161

3.2 5.9 4.1 8.7 9.1 9.9 41062 45004 41691 5393 2319 5246 2597 1498 169 chem 3.2 6.1 4.6 8.7 9.1 10.1 36371 43430 39166 8055 3192 8515 2881 1607 195

3.8 6.4 4.9 8.7 8.8 9.9 42832 45870 45646 6302 2579 6241 2156 1181 150 2.7 6.4 4.8 8.8 8.9 10.1 42081 41028 38207 6809 2290 7873 2479 1270 199 3.0 6.4 4.7 9.1 9.2 10.2 44062 48719 44317 5976 2793 7558 2775 1612 217 3.1 6.4 5.2 8.4 8.6 9.9 34783 39531 33095 5961 1755 7784 2570 1313 202 2.6 6.5 4.7 8.9 9.2 10.2 36922 42016 35453 8415 3061 9240 2987 1658 237 a-65 4.0 6.6 5.1 8.8 8.9 10.2 30194 34456 24958 7983 4563 9971 2851 1931 293 tana 2.9 6.6 4.8 8.6 8.9 9.9 38951 43964 37232 9212 4056 8290 2744 1676 196 e 5.1 6.7 5.8 8.5 8.7 9.7 35883 36594 35793 5687 3017 6953 2475 1605 180

3.1 6.9 5.9 9.2 9.3 10.4 31681 36493 32557 7995 3735 9150 2949 1798 253 3.7 7.0 5.0 8.9 9.1 10.1 38771 41952 37019 6890 2468 7346 2742 1387 193 5.6 7.1 5.9 8.9 8.9 9.9 35614 37094 34640 9058 3827 8337 2710 1881 213 ion 5.2 7.4 6.1 8.6 8.9 10.0 32973 34989 29931 7853 2905 8472 2930 1769 245 6 5.6 7.4 6.0 8.8 9.1 10.0 33777 37723 32390 7643 3756 7014 2509 1606 191 3 6.8 9.5 8.7 8.6 8.6 10.0 36460 28490 31981 4549 1343 5094 2229 1135 153 1 7.1 9.5 8.6 8.8 8.9 9.9 38135 27516 34032 5615 1759 5588 2421 1419 183 i -Nax2 5.8 8.3 7.5 8.4 8.6 10.0 39468 39393 42829 8042 4102 7634 1784 1188 144

120

7.6 9.9 8.7 8.6 8.3 10.2 37015 21706 32653 7556 2383 8613 1764 762 154i 7.8 10.3 9.2 8.5 8.6 10.2 38756 16836 31415 7780 2189 7512 1803 665 134

8.0 9.6 8.7 9.3 9.4 10.0 30070 18646 19045 10572 2960 10977 1844 724 147ntrol vs

0.4 0.2 3092 908 200ntrol vs

0.4 0.1 2916 1381 232 and Ch concentration data were transformed to natural logarithms. **LSD refers to Least Significant Difference at P= 0.05 for species x treatment interaction

[0361] Similar to water use, the most noteworthy effects were the higher sodicity tolerance in Tamaroi -Nax2 compared to Tamaroi, and the highest salinity and sodicity tolerance in MW#293 (Figure 9). There was a modest positive correlation between leaf Na ⁺ concentration and salinity or sodicity tolerance (Figure 8; df= 18, P< 0.05; r=0.475 and r=0.463 for sodicity and salinity respectively). However, this relationship was greatly influenced by the inclusion of MW#293. When MW#293 was omitted from the analyses, there were no correlations.

Experiment 4. Grain yield, leaf element concentration, and expression profiling of candidate genes identified in the present study, and of previously reported genes associated with high leaf Na ⁺

[0362] The five wheat lines varied significantly in grain yield under control and salinity; higher yielding lines under control were also higher yielding under salinity (Figure 10). Mace was the lowest yielding, while MW#293 the highest. Salinity tolerance measured as relative grain yield (%) ranged from 47% in Mace to 81 % in MW#293. Salinity increased leaf Na ⁺ concentrations significantly but mainly in MW#451 and MW#293 (7,770 and 8,400 mg kg ^-1 DW), almost two orders of magnitude higher than for Mace, MW#28 and MW#491 (35, 122 and 124 mg kg ^-1 DW, respectively) (Figure 10). In agreement with Experiment 3, Cl concentrations increased significantly under salinity (Figure 10). Representative pots of bread wheat ( Triticum aestivum) cv. Mace and doubled-haploid line MW#293 grown under control and salinity (100 mM NaCI) are shown in Figure 1 1.

[0363] Despite differences across wheat lines and treatments, leaf Ca ²⁺ , K ⁺ and Mg ²⁺ concentrations were all above the critical levels for deficiency thus indicating adequate nutrition (Figure 12; Reuter and Robinson, 1997, Plant Analysis. An Interpretation Manual. 2nd edn. CSIRO, Australia).

[0364] Three candidate genes identified by GWAS in Experiment 2, and five previously published genes that were differentially expressed under control and salt, and between the parental line of high-Na ⁺ wheat germplasm MW#293 (high Na ⁺ -W4909) and cv. Chinese Spring (low-Na ⁺ bread wheat) (Mott and Wang, 2007, Plant Science, 173: 327-339), were selected for analysis of gene expression (Table 3). One of the three candidate genes (calcium-transporting ATPase) and the Nax1 gene had very low levels of expression (data not shown), while two genes ( Na ⁺ /H ⁺ antiporter NhaB and Aquaporin-like protein TIF1-4) were differentially expressed between wheat lines and treatments. Na ⁺ /H ⁺ antiporter NhaB was highly expressed in low-Na ⁺ wheat lines Mace, MW#28 and MW#491 , while very low expression levels were observed in high-Na ⁺ wheat lines MW#293 and MW#451 (Figure 13). Wheat lines showed similar expression levels under control and salinity, with the notable exception of MW#491 showing higher expression under salinity (Figure 13). Aquaporin-like protein TIF1-4 levels were higher in wheat line Mace than the other four wheat lines, and levels varied depending on treatments; lower (Mace and MW#451), higher (MW#293) or no change (MW#28 and MW#451) under salinity (Figure 13).

[0365] Of the five previously reported genes, four ( Na ⁺ /H ⁺ antiporters NHX1 and NHX2, putative high affinity potassium transporter, and vacuolar pyrophosphatase similar to A VP1 ) were differentially expressed between wheat lines and treatments (Figure 13). The main effects were (i) higher expression of putative high-affinity potassium transporter and NHX2 in MW#293 and (ii) higher expression of NHX1 in MW#451 under salinity, and (iii) lower expression of AVP1 like gene in MW#451 and MW#293 under both control and salinity (Figure 13). Moreover, the probe set Ta.22954.1.S1_at showed very low levels of expression in the present study (data not shown); in contrast to an 8-fold higher expression in high-Na ⁺ bread wheat germplasm line W4909 (parental line of MW#293) compared with low-Na ⁺ Chinese Spring bread wheat under salinity (see Table S2 in Mott and Wang, 2007, supra).

Discussion

Sodium exclusion in bread wheat and its relationship with salinity and sodicity tolerance as measured by a novel screening method

[0366] Our results demonstrate that whilst there is genetic variation for Na ⁺ concentration in modern bread wheat (n=98; Table 8; Figure 3), most wheats contain relatively low Na ⁺ concentrations. We found no correlation between leaf Na ⁺ concentration and either salinity or sodicity tolerance based on grain yield (n=18; Experiment 3). In fact, wheat germplasm MW#293 achieved the highest salinity tolerance despite having a 14-fold higher Na ⁺ concentration (6,044 mg kg ^-1 DW) than the highest of the naturally occurring bread wheats (cv. Federation, 425 mg kg ^-1 DW) (Table 8). Similarly, under sodicity MW#293 had a 7-fold higher Na ⁺ concentration (12,939 mg kg ^-1 DW) than the second highest bread wheat cv. Federation (1 ,651 mg kg ^-1 DW), and still had the highest sodicity tolerance (Figure 9; Table 1 1) .

[0367] Despite the prevailing opinion that low Na ⁺ confers tolerance, the results in Experiment 3 and other studies in wheat, barley and maize show that low Na ⁺ concentration is not necessarily associated with salinity tolerance. This suggests that additional mechanisms (tissue tolerance/osmotic adjustment) need to be considered in order to breed salinity tolerant bread wheat.

Effects of Nax1 and Nax2 on salinity and sodicity tolerance in low-Na ⁺ bread wheat cv. Westonia

[0368] Our results confirm that Westonia-A/axi and Westonia-/\/ax2 lines were lower in Na ⁺ concentration compared to Westonia, and showed slightly higher but non-significant grain yield increase at moderate salinity (50 mM NaCI) and low sodicity (2 g kg ^-1 Na ⁺ -humate) (Figure 2; Tables 5 and 6). However, compared to high-Na ⁺ bread wheat Baart-46, Na ⁺ concentrations of Westonia and Nax lines were low, and hence small differences in Na ⁺ concentration between Westonia and the Nax lines are unlikely to make a difference to grain yield (Figure 2; Tables 5 and 6). This supposition is supported by two lines of evidence: In Experiment 1 , Baart-46 had much higher Na ⁺ concentration but yielded higher than the three Westonia lines at all levels of salinity and sodicity. Secondly, in a saline field trial (Setter et ai, 2016), only one Westonia-/\/ax2 line (5924) yielded higher (1 1 %) than Westonia, while the other four Westonia-Nax lines were, on average, no different to Westonia. The results indicate that transferring Nax1 and Nax2 genes into an already efficient Na ⁺ excluding bread wheat confers little, if any, improvement in overall salinity tolerance.

[0369] Unlike low-Na ⁺ bread wheat, when the Nax2 gene was introduced into high-Na ⁺ durum wheat cv. Tamaroi, a significant yield increase was reported under salinity in the field (Munns et ai, 2012, Nature Biotechnology 30: 360-364) and under sodicity in the growth room (Gene et ai, 2016, supra). The differences between the Na ⁺ excluding abilities of bread and durum wheats are attributed to modern bread wheats possessing homologs of the Na ⁺ exclusion genes Nax1 and Nax2 and/or other Na ⁺ exclusion genes (Byrt et ai, 2007, Plant Physiology 143: 1918-1928; cf. Gene et ai, 2010, Plant and Soil 327: 331-345; Husain et ai, 2017, Scientific Reports 7: 15662; Oyiga et ai, 2018, Plant, Cell and Environment 41 : 919-935), while durum wheats are thought to lack such genes. Hence, the introduction of Nax type genes is more useful in durum wheat backgrounds.

SNPs and candidate genes for Na ⁺ accumulation, and their relationships with salinity tolerance

[0370] Of the 9 SNPs significantly associated with leaf Na ⁺ concentration, seven were mapped to chromosomes 2A, 2B, 2D, 4B, 4D, 5B, and 7A, while the rest could not be assigned to a particular chromosome (Table 9), hence there is limited discussion with published studies. Using the latest available Chinese Spring Reference Genome (IWGSC RefSeq v1.0), we compared the physical position of previously reported QTL (Gene et al., 2013, Molecular Breeding 32:39-59; Oyiga et al., 2018, Plant, Cell and Environment 41 : 919-935) to the seven mapped SNPs identified in the present study. It appears that all seven SNPs are novel. Four candidate genes with putative functions in regulating Na ⁺ concentration were identified in close physical location to these significant SNPs, of which two were significantly different between treatments and wheat lines in the gene expression study (Table 3; Figure 13).

[0371] Some of the genes described in this study ( NHX1 and NHX2, AVP1, putative high- affinity potassium transporter) have been previously reported (Mott and Wang, 2007, supra), while others were identified for the first time (Table 9). Contrary to no differences in Mott and Wang, 2007, supra, NHX1 and NHX2 expression levels varied between treatments and wheat lines (Figure 13). We found no correlation between expression levels of NHX genes and grain yield. However, other studies have found that increased expression of NHX genes can enhance growth under saline conditions (Apse et al., 1999, Science 285: 1256-1258; Zhang et al., 2001 , Proceedings of the National Academy of Sciences of the United States of America 98: 12832-12836; Bayat et al., 2011 , Australian Journal of Crop Science 5: 428-432).

[0372] Non-significant expression levels observed for vacuolar pyrophosphatase gene were in contrast to findings of Mott and Wang, 2007, supra, who reported higher expression in high-Na ⁺ germplasm line W4909 than low-Na ⁺ Chinese Spring bread wheat under control and salinity, but much greater expression levels under control. In barley, AVP expression was correlated with shoot biomass and grain yield (Schilling et al., 2014, Plant Biotechnology Journal 12:378-86), while in our study there was no evidence for this.

[0373] Putative high-affinity transporter, a candidate gene for osmoregulation (Mott and Wang 2007, supra), had higher expression under salinity in MW#293 only (Figure 13), and this was in contrast to higher expression levels in low Na ⁺ Chinese Spring bread wheat than in high Na ⁺ germplasm W4909 in Mott and Wang, 2007, supra. In addition, candidate genes were identified that showed similarity to an Na+/H+ antiporter NhaB, Aquaporin-like protein TIF1-4, and Aquaporin PIP2. Aquaporin (involved in water and nutrient uptake) gene expression levels did not correlate with leaf Na ⁺ or grain yield, while Na ⁺ /H ⁺ antiporter NhaB (which remains to be characterized in plants) expression was much lower or not expressed in high Na ⁺ than in low Na ⁺ germplasm lines, although there was no correlation between its expression and grain yield. The differences in gene expressions between the present study and previous studies may be due to different germplasm and experimental conditions. There is also a need to perform a time-point analysis of these differentially expressed genes across different plant tissues and demonstrate if they are responsible for improved salinity tolerance.

Potential of tissue tolerance/osmotic adjustment for further improvement of salinity tolerance in bread wheat

[0374] Tissue tolerance (the ability of an organ to maintain function in the presence of elevated tissue Na ⁺ and Cl concentrations) and osmotic adjustment (maintaining turgor by accumulating inorganic ions (mainly Na ⁺ , K ⁺ , Ca ²⁺ and Cl ), organic acids, carbohydrates, and amino acids), are regarded as two of the three main mechanisms of salinity tolerance in plants (Munns and Tester, 2008, Ann. Rev. Plant Biol., 59: 651-681). However, there has been little focus on their physiological and genetic aspects as compared to Na ⁺ exclusion. In the present study, GWAS identified alleles from MW#451 and MW#293 associated with high leaf Na ⁺ in several candidate genes (Figure 6). This, together with gene expression data here (Figure 13) and elsewhere (Mott and Wang, 2007, supra) indicate that these genes may be potentially involved in tissue tolerance of salinity tolerant wheat germplasm MW#293. Higher relative growth rates in MW#293 under salinity also points to better osmotic adjustment in this line. Taken together, tissue tolerance and osmotic adjustment are likely to contribute to higher salinity of MW#293.

A novel wheat germplasm (MW#293) for development of next generation salt tolerant bread wheat

[0375] MW#293 was derived from an earlier bread wheat germplasm line (W4909) developed by Richard Wang and his colleagues (Wang et ai, 2003, supra). W4909 is a product of three species [Triticum aestivum cv. Chinese Spring), Aegilops speltoides and Thinopyrum junceum (sea wheatgrass)], and its ability to accumulate very high Na ⁺ sodium has been demonstrated in independent studies (Gene et al., 2007, supra ; Wang et ai, 2003, supra ; Mott and Wang, 2007, supra). However, its salt tolerance is debatable as studies so far have produced variable results (Gene et al., 2007, supra ; Wang et al., 2003, supra ; Mott and Wang, 2007, supra). In addition, the potential of high Na ⁺ as a source of osmotic adjustment/ tissue tolerance in a widely adapted and high yielding bread wheat has not been realized.

[0376] To introduce salt tolerance gene(s) of W4909 into a commercial bread wheat, we made a cross between a popular Australian bread wheat cv. Mace and W4909, and developed a doubled-haploid population (over 200 lines). As the population segregated for maturity and height markedly, a sub-selection of this population (n=18), agronomically similar to commercial lines, was grown under control and salinity using the soil assay described in Gene et al., 2016, supra. Of these 18 lines, MW#293 had the highest grain yield under both control and salinity, and doubled the grain yield of Mace under salinity despite having an 86-fold higher leaf Na ⁺ concentration (Gene et al., unpublished). When tested with 18 commercial wheats in Experiment 3, MW#293 produced the highest grain yield under control, salinity and sodicity, and its grain yield under salinity was three times higher despite 35-100-fold higher leaf Na ⁺ concentrations (under sodicity and salinity) than cv. Mace (Table 11). In Experiment 4, MW#293 recorded 200-fold higher leaf Na ⁺ concentration and 2-fold higher grain yield than cv. Mace (Figures 10 and 1 1). MW#293 also had the highest growth rates under salinity and sodicity (Table 10). These data suggest that MW#293 may have the ability to efficiently assimilate and sequester Na ⁺ levels that can support high growth rates. To our knowledge, such high Na ⁺ accumulation together with high grain yield/growth rate in bread wheat has not been previously reported. This represents a new paradigm in breeding for salinity tolerance.

Conclusions

[0377] Despite a 10- to 14-fold variation in leaf Na ⁺ concentration in modern bread wheats, there were no correlations between leaf Na ⁺ concentration and either salinity or sodicity tolerance, thus demonstrating the limits of using leaf Na ⁺ concentration alone as a selection parameter for salinity/sodicity tolerance. As modern bread wheats have an excellent Na ⁺ exclusion ability, further investment in the Na ⁺ exclusion mechanism is unlikely to improve sodicity/salinity tolerance significantly. Future efforts should focus on osmotic adjustment/tissue tolerance mechanisms.

[0378] Despite much higher Na ⁺ concentration, bread wheat germplasm MW#293 yielded 208% and 157% higher under salinity and sodicity, respectively, compared with the average of the other bread wheats. Development of MW#293 paves the way for next generation salinity/sodicity tolerant bread wheat.

EXAMPLE 2

Further Characterisation of Salinity and/or Sodicity Tolerant Wheat and Genetic

Markers Thereof

[0379] The 100 bread wheat entries of Experiment 2 were analysed for growth under control and salinity conditions. As with Experiment 2, twelve durum wheats ( Triticum turgidum subsp durum) and an Australian barley ( Hordeum vulgare L) cv. Clipper were also included as checks.

[0380] Plants were grown to awn-visible stage under control (0 mM NaCI) and salinity (100 mM NaCI). There were three plants per 3-kg capacity pots. Plants were grown in University of California potting mix (as described above) supplied with or without NaCI. The experiment was conducted in three growth rooms with identical settings (20/15°C day/night temperature, 14 h photoperiod; light intensity approx. 350-500 pmol nr ² s ¹ ). As the time of harvest depended on the appearance of awns, a few awnless wheats may have grown longer than wheats with awns. In contrast to other experiments, a decision was made to harvest plants earlier than heading to prevent nutrient deficiencies due to greater than expected growth especially in the control treatment under increased light intensity which was trialled for the first time in this experiment.

[0381] At the awn-visible stage, penultimate leaves were sampled for analysis of sodium (Na ⁺ ), potassium (K ⁺ ), Calcium (Ca ²⁺ ), magnesium (Mg ²⁺ ) and Cl (Gene et al., 2016, supra). Handling of leaf samples and analytical methods used in nutrient analyses were described earlier (Gene et ai, 2016, supra). Shoot dry weight (DW) for each plant (g plant ^-1 ) was determined under control and salinity (salt) conditions and was used as a measure of plant yield. Salinity tolerance (ST) was calculated based on shoot DW under salinity/shoot DW under control, expressed as percent. To determine salinity tolerance, we used an index based on the genetic response of the varieties under saline conditions compared to their control equivalents. To calculate this index, we fitted an initial linear mixed model (LMM) for shoot dry weight where the model contained a fixed term consisting of a T reatment (Control, Salt) by variety interaction. Additionally, non-genetic random effects were also required in the model to ensure variation arising from design constraints, such as different growth rooms, were accounted for. From this fitted LMM, the best linear unbiased estimators (BLUEs) of the varieties for both treatments were extracted and the response index was calculated as the residuals from the regression of the variety Salt BLUEs on the variety Control BLUEs. The resulting index is the centred around zero and becomes a proxy for salinity tolerance. Varieties with the highest ranked control BLUEs and a positive response index can be viewed, on average, as performing well under control conditions as well as exhibiting greater tolerance to salt. In contrast, varieties with the lowest ranked control BLUEs and negative response index are the poorer performing varieties in both control and saline conditions. [0382] As shown in Table 12, bread and durum wheat entries differed significantly in shoot growth under control and salinity (see also Figure 14). Of bread wheat entries, Olympic, MW#293, Federation, Egret and Kukri were top performers under control, while MW#293, Olympic, Egret, MW#451 and Federation were highest performers under salinity. International salt-tolerant bread wheat Kharchia-65 was a moderate performer. A number of Australian varieties outperformed this international check (Figure 15; Table 12).

[0383] Salinity tolerance ranged from 38% in Longreach Impala to 89% in MW#293 (Table 12). Reliance on ST% may not always be useful as in the case of low yield under control resulting in higher ST%. Therefore, we calculated the response index (residuals extracted from linear relationship) and regressed against shoot dry weight under control. The idea is to identify wheat varieties with a higher response index and higher growth under control. Based on this calculation, MW#293, can be considered the most salinity tolerant, while Kukri, is the least salinity tolerant (Table 13).

[0384] Durum wheats, WID802, Saintly, Kalka, WID902 and DBA-Aurora had the highest growth under control, while Kalka, Saintly, Tjilkuri, DBA-Aurora and WID902 had the highest growth under salinity. Salinity tolerance varied from 44% in Hyperno to 64% in WID902. When response index and growth under control were considered together, WID902 was the most tolerant, while Hyperno the least. In both bread wheats and durum wheats, the highest performers under control were also highest performers under salinity suggesting that seedling vigour played a key role. This resulted in a significant correlation between growth under control and salinity (Figure 16). This is a positive outcome as far as breeders and farmers are concerned. It is not useful to have a salt tolerant bread wheat variety that does well under salinity but is an average performer under non-saline conditions. A well-known Australian Crop Physiologist Richard Richards, over three decades ago, argued that salinity was patchy in the field, and most of the yield came from non-saline parts of the paddock, hence breeders should select for yield under non-limiting conditions (Richards RA, 1983, Euphytica 32: 431-438). Likewise, most recently, Mujeeb-Kazi and colleagues make the important point that breeding wheat solely for salinity tolerance at the cost of yield loss in non-saline soils is unsuitable for farmers:“Breeders need to develop cultivars with high yield potential under both stress and nonstress conditions”, in other words vigorous cultivars (Mujeeb-Kazi A et al., 2019, “Chapter Four - Breeding strategies for structuring salinity tolerance in wheat”, Advances in Agronomy , 155: 121-187; https://doi: org/10.1016/bs.agron.2019.01.005). TABLE 12

Days to awn-visible stage, shoot dry weight (g pi ^-1 ) under control and salinity, salinity tolerance (ShootDW_salt/shootDW_control, %), sodium (Na), calcium (Ca), potassium (K), magnesium (Mg) and chloride (Cl) concentrations (mg kg ¹ DW) in penultimate leaves (salinity stressed plants) at harvest (awns visible) in 100 bread wheats, 12 durum wheats and barley cultivar Clipper (n=3)

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mg read wheat stage* control salt ST% transformed transformed Ca cone. K cone. cone. co 25 48 3.6368 2.0240 56 4.9 132 4946 32413 1356 14 GT Katana 40 2.3953 1.1073 46 5.4 218 7763 31733 1683 18 GT Scythe 52 3.3824 2.4099 71 4.7 110 4763 31839 1687 16 nnuello 43 2.3096 1.0315 45 4.7 106 7753 34281 1845 23 nza 44 3.0320 1.4624 48 4.4 80 6285 38005 1733 23 roona 42 2.5814 1.6585 64 4.3 75 5270 36208 1808 18 xe 38 2.3045 1.0832 47 5.4 211 5416 35896 1365 16 ZDH1 43 2.4750 1.4871 60 5.5 245 5364 36060 1720 17 aart-46 48 4.6012 2.6501 58 7.0 1058 6679 27088 2041 20 arham 46 3.1548 1.7154 54 5.8 323 8347 34547 2021 25 eckom 45 2.6600 1.4115 53 5.1 168 5978 35901 1852 21 erkut 51 4.4918 3.0532 68 5.5 235 5171 24868 2130 12 olac 51 3.4693 1.5487 45 5.6 266 5990 34866 1653 19 rookton 51 3.5682 2.3817 67 5.1 157 4834 33588 1639 10 alingiri 56 4.5087 2.5427 56 5.4 231 5414 34440 1495 15 arnamah 48 2.9094 2.1555 74 4.2 65 7223 34863 2264 14 ascades 46 2.8659 1.6254 57 4.4 84 5092 37315 1533 17 ondor 48 3.5253 1.8812 53 4.0 54 8131 33635 2073 21 orack 46 2.4119 1.2873 53 4.3 76 6334 42333 1802 18

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mgread wheat stage* control salt ST% transformed transformed Ca cone. K conc. cone. coorrell 47 3.2295 1.7004 53 4.8 116 6895 33199 1866 19osmick 52 3.8138 2.4291 64 6.0 402 4703 33018 1549 15utlass 50 3.1810 1.9835 62 4.7 105 7789 34535 2173 18 rysdale 43 2.5587 1.3458 53 5.7 286 4578 32333 1503 11 S Darwin 53 3.3401 1.6907 51 4.9 136 6945 38613 1781 13ga-Eagle-Rock 42 2.1907 1.4581 67 4.2 64 6900 34255 1731 19gret 61 4.9473 3.8408 78 6.1 463 4454 23254 2377 14mu Rock 40 2.4050 1.3336 55 4.3 74 7266 35456 1551 16spada 44 2.4468 1.3165 54 3.9 50 6426 39532 1705 18stoc 50 3.1840 1.7939 56 4.5 88 7963 33821 2075 17xcalibur 50 3.2349 2.2121 68 4.8 121 5566 35767 1710 13ang 52 3.5236 1.9274 55 4.0 53 8002 33668 2175 20ederation 53 4.9912 3.1378 63 6.5 684 4617 25564 1707 15ortune 56 4.3169 2.8175 65 5.8 318 6444 31850 1715 14rame 55 4.3640 2.6817 61 5.5 239 8809 25619 2169 18 abo 42 2.6686 1.1802 44 4.6 95 5530 31998 1484 16 ladius 44 2.6311 1.1806 45 4.5 94 6735 37961 1746 18 renade CL Plus 43 2.5074 1.7524 70 3.9 48 6172 36963 1862 15 alberd 49 4.4618 2.6952 60 5.0 149 3628 32845 1364 15 arper 54 4.5599 2.8476 62 4.0 52 6269 28171 1825 15 artog 44 2.7628 1.7603 64 6.0 423 6834 33030 1824 17 atchet CL Plus 38 1.9026 0.9700 51 5.0 141 5471 35594 1391 14nia 66R 50 4.1442 2.6422 64 4.8 119 7464 31741 2005 16anz 47 3.1996 1.6266 51 4.3 77 8146 35984 2089 27

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mgread wheat _ stage* control salt ST% transformed transformed Ca cone. K conc. cone. coustica CL Plus 44 2.5412 1.5369 60 4.3 70 9392 38610 2054 21harchia-65 42 3.3787 2.1496 64 5.2 183 10353 26663 2223 21ite 42 2.2268 1.3851 62 4.9 131 5981 35122 1586 17loka 46 4.2266 2.2064 52 7.1 1156 8322 29167 1785 18ord CL Plus 46 2.8633 1.3656 48 5.4 220 6613 33675 1778 18richauff 44 2.7311 1.3947 51 3.9 49 6238 34712 1564 17RL-19 41 2.3763 1.3690 58 4.0 53 7469 31259 1853 16ukri 53 4.8773 2.0992 43 5.4 212 5218 34162 1361 15erma Rojo 42 2.6965 1.4241 53 5.4 225 8719 28028 1990 16ongreach Catalina 40 2.0786 0.9614 46 5.4 229 10534 32683 2105 25ongreach Cobra 54 3.7320 2.2634 61 4.3 71 3969 34708 1681 10ongreach Dart 40 1.8309 0.8241 45 4.5 86 7843 35753 1701 18ongreach Impala 43 2.5066 0.9549 38 5.1 156 7118 31806 1727 17ongreach Orion 49 3.2812 2.0742 63 5.9 355 5690 34919 1998 18ongreach Phantom 48 2.7111 1.6902 62 5.2 183 6408 34632 1923 16ongreach Scout 52 3.5977 2.2623 63 5.1 159 4027 38982 1635 13ongreach Trojan 50 3.2176 1.7072 53 4.8 121 5128 39639 1789 17 ace 53 3.5854 1.9322 54 4.6 102 4309 40323 1661 15 achete 52 3.2779 2.2385 68 4.8 126 3094 40152 1318 14 agenta 53 3.4041 2.3766 70 5.3 201 7038 35017 2036 16 into 48 3.2722 1.8907 58 4.0 54 7268 39494 2078 22 olineux 49 3.5289 1.9703 56 5.8 326 7508 27757 2404 18 W#28 54 4.5514 2.7051 59 5.6 264 6169 29690 1797 15 W#293 65 6.8306 6.0916 89 8.2 3539 3433 26243 1558 9

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mgread wheat stage* control salt ST% transformed transformed Ca cone. K conc. cone. co W#451 58 4.6794 3.2764 70 8.3 3832 3359 28792 1547 lO W#491 48 3.0369 1.9534 64 5.3 209 6624 38178 2023 19ainari 60 49 4.0739 2.5161 62 5.5 247 4857 33713 1374 14lympic 65 7.8937 5.6414 71 7.0 1110 5208 23203 1782 15eake 41 2.3825 0.9912 42 4.0 54 5954 39970 1599 18itic-62 55 3.8783 2.9628 76 5.6 261 6938 26910 2141 18AC875 44 2.9232 1.7189 59 4.9 129 6056 39904 1613 20akha 61 38 2.0651 1.0531 51 5.3 210 8631 32062 1768 18akha 8 40 2.4944 1.2732 51 5.4 211 6142 32614 1867 15amnyt 16 45 3.3978 1.8957 56 6.2 509 9631 25797 2136 20cepter 48 3.2153 1.6465 51 3.8 45 6844 36536 2113 15hasta 43 2.6355 1.4383 55 4.3 74 5184 29631 1738 15hield 55 4.0976 2.7041 66 5.4 213 7179 34300 1863 9okoll 47 3.4809 2.0798 60 4.5 91 5153 32666 1548 13onora-64 40 1.8926 1.0937 58 4.2 66 6534 35349 1854 18tylet 52 3.5887 2.2791 64 3.4 30 6290 35302 1701 19uneca 51 3.3609 2.0324 60 3.4 31 6695 36590 1835 11unlin 49 3.6911 1.7293 47 3.5 35 5523 36094 1679 17ammarin-Rock 44 3.0511 1.4645 48 3.9 47 6830 39423 2019 14rident 51 3.2528 1.7797 55 4.7 106 5070 34012 1635 19M506 46 2.7659 1.6160 58 5.3 192 6579 40910 1975 21 arigal 52 4.1266 2.1638 52 5.2 189 4514 31012 1557 17 arimek 50 3.7516 2.2964 61 5.6 271 4562 35865 1442 15 ariquam 51 4.4085 2.6797 61 4.4 82 7165 32659 1949 15

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mgread wheat stage* control salt ST% transformed transformed Ca cone. K cone. cone. co entworth 45 2.5847 1.3553 52 4.9 134 5277 36351 1702 21 estonia 40 2.3287 1.1061 47 5.0 146 5670 32481 1487 14 estonia-Naxl 40 2.1554 1.1159 52 3.8 47 5895 30807 1816 15 estonia-Nax2 39 2.1011 1.1039 53 4.2 67 8468 30624 2136 15 orrakatta 44 2.5805 1.5797 61 4.7 108 9483 31992 2349 19 yalkatchem 44 2.1449 1.2358 58 4.6 104 7107 32548 1929 15ecora Rojo 39 1.8880 0.9171 49 5.4 213 7208 32026 2032 18itpi 51 3.4484 2.2429 65 4.8 120 4586 25590 1529 16oung 40 2.0192 0.9276 46 5.7 309 9403 31587 1954 24 urum wheat

aparoi 49 2.3521 1.2422 53 8.3 4205 6869 32071 1334 25BA-Aurora 49 3.3104 2.0860 63 8.4 4455 4896 31075 1072 16yperno 44 2.3436 1.0929 47 8.5 4889 6386 32757 1349 26alka 51 3.1222 1.6385 52 8.3 4142 7096 30927 1234 20ahel 44 2.1147 1.1784 56 9.2 9485 4223 27410 1083 18aintly 51 2.8706 1.6611 58 8.6 5477 7494 26684 1420 21amaroi 44 2.0839 1.1702 56 8.8 6865 6001 29099 1293 24amaroi_Nax2 44 1.8708 1.1601 62 6.2 513 5458 37780 1144 23jilkuri (WID 801) 54 2.8156 1.7151 61 9.1 9113 5852 23885 1081 19 ID 802 47 2.8292 1.6251 57 8.9 7062 4172 27815 996 20 ID 902 51 3.2706 2.0927 64 4.8 118 4876 34017 1090 15awa (WID 803) 44 1.7404 1.0478 60 8.2 3742 6014 32636 1353 24

Days to

awns Na cone. Na cone.

visible Shoot DW Shoot DW Log- Back- Mgread wheat _ stage* _ control salt ST% transformed transformed Ca cone. K conc. cone. coarley

lipper 36 1.4312 0.6142 43 8.4 4425 5353 32146 1079 16 *HSD 1.1109 0.9217 1.4 4027 10841 808 7 few awnless wheats may have been grown longer than wheats with awns. This would not have been an issue if they were all grown to heading as planned inowever, a decision was made to harvest plants earlier than heading to prevent nutrient deficiencies due to better than expected growth especially in the control treander increased light intensity which was trialled for the first time in this experiment. **HSD refers to Honestly significant difference.

TABLE 13

Response index and shoot DW (g pi ^-1 ) under control in bread wheats, durum wheats and barley

Shoot Response

Bread wheat

DW index

A25 3.6368 -0.1997

AGT Katana 2.3953 -0.1517

AGT Scythe 3.3824 0.3840

Annuello 2.3096 -0.1610

Anza 3.0320 -0.2913

Aroona 2.5814 0.2549

Axe 2.3045 -0.1052

AZDH1 2.4750 0.1662

Baart-46 4.6012 -0.3229

Barham 3.1548 -0.1337

Beckom 2.6600 -0.0532

Berkut 4.4918 0.1652

Bolac 3.4693 -0.5448

Brookton 3.5682 0.2114

Calingiri 4.5087 -0.3584

Carnamah 2.9094 0.4970

Cascades 2.8659 0.0008

Condor 3.5253 -0.2559

Corack 2.4119 0.0155

Correll 3.2295 -0.2068

Cosmick 3.8138 0.0680

Cutlass 3.1810 0.1140

Drysdale 2.5587 -0.0402

DS Darwin 3.3401 -0.3024

Ega-Eagle-Rock 2.1907 0.3581

Egret 4.9473 0.5988

Emu Rock 2.4050 0.0671

Espada 2.4468 0.0175

Estoc 3.1840 -0.0779

Excalibur 3.2349 0.3007

Fang 3.5236 -0.2083

Federation 4.9912 -0.1383

Fortune 4.3169 0.0654

Frame 4.3640 -0.1070

Gabo 2.6686 -0.2912

Gladius 2.6311 -0.2616

Grenade CL Plus 2.5074 0.4062 Halberd 4.4618 -0.1695 Shoot Response

Bread wheat

DW index

Harper 4.5599 -0.0934

Hartog 2.7628 0.2158

Hatchet CL Plus 1.9026 0.0939 Inia 66R 4.1442 0.0243 Janz 3.1996 -0.2573

Justica CL Plus 2.5412 0.1646

Kharchia-65 3.3787 0.1265

Kite 2.2268 0.2570

Kloka 4.2266 -0.4756

Kord CL Plus 2.8633 -0.2570 Krichauff 2.7311 -0.1253 KRL-19 2.3763 0.1248 Kukri 4.8773 -1.0884

Lerma Rojo 2.6965 -0.0690

Longreach

Catalina 2.0786 -0.0515

Longreach Cobra 3.7320 -0.0342

Longreach Dart 1.8309 0.0037

Longreach Impala 2.5066 -0.3905

Longreach Orion 3.2812 0.1268

Longreach

Phantom 2.7111 0.1859

Longreach Scout 3.5977 0.0690

Longreach Trojan 3.2176 -0.1908

Mace 3.5854 -0.2515

Machete 3.2779 0.2937

Magenta 3.4041 0.3337

Minto 3.2722 -0.0496

Molineux 3.5289 -0.1695

MW#28 4.5514 -0.2293

MW#293 6.8306 1.3863

MW#451 4.6794 0.2426

MW#491 3.0369 0.1959

Nainari 60 4.0739 -0.0472

Olympic 7.8937 0.1100

Peake 2.3825 -0.2578

Pitic-62 3.8783 0.5516

RAC875 2.9232 0.0497

Sakha 61 2.0651 0.0507

Sakha 8 2.4944 -0.0628

Samnyt 16 3.3978 -0.1423

Scepter 3.2153 -0.2496 Shoot Response

Bread wheat

DW index

Shasta 2.6S55 -0.0073

Shield 4.0976 0.1224

Sokoll S.4809 -0.0227

Sonora-64 1.8926 0.2253

Stylet 3.5887 0.0928

Suneca 3.3609 0.0231

Sunlin 3.6911 -0.5365

Tammarin-Rock 3.0511 -0.3040

Trident 3.2528 -0.1456

VM506 2.7659 0.0691

Warigal 4.1266 -0.4405

Warimek 3.7516 -0.0164

Wariquam 4.4085 -0.1435

Wentworth 2.5847 -0.0509

Westonia 2.3287 0.1011

Westonia-Naxl 2.1554 0.0433

Westonia-Nax2 2.1011 0.0736

Worra katta 2.5805 0.1768

Wyalkatchem 2.1449 0.1714

Yecora Rojo 1.8880 0.0523

Yitpi 3.4484 0.1656

Young 2.0192 -0.0391

Durum wheat

Caparoi 2.3521 0.0168

DBA-Aurora 3.3104 0.1160

Hyperno 2.3436 -0.1259

Kalka 3.1222 -0.1854

Sahel 2.1147 0.1375

Saintly 2.8706 0.0329

Tamaroi 2.0839 0.1532

Tamaroi_Nax2 1.8708 0.3087

Tjilkuri (WID 801) 2.8156 0.1295

WID 802 2.8292 0.0289

WID 902 3.2706 0.1536

Yawa (WID 803) 1.7404 0.2977

Barley

Clipper 1.4312 0.1044 [0385] On the one hand, harvesting all entries enabled us to make proper comparisons amongst varieties, but this led to entries being grown for different timeframes (2-3 weeks difference). This inevitably resulted in significant growth/maturity interaction (r ² =0.78 and 0.72 under control and salinity for bread wheat; r ² =0.62 and 0.58 under control and salinity for durum wheat). This correlation was less obvious when salinity tolerance (%) was regressed against maturity scores (r ² =0.37 and 0.07 for bread wheat and durum wheat, respectively). This influence of maturity is also the case in field trials. The ideal scenario is to test varieties within 7-10 days maturity range, which is not easy to do when dealing with a diversity panel like the one in the present study. Simply put, there are just not enough varieties within a short maturity range to analyse these data independent of maturity.

[0386] Sodium, potassium, calcium, magnesium and chloride are the main elements that affect plant growth under salinity. Of these, sodium has received the most attention. Sodium exclusion, leading to salinity tolerance has become a central dogma for this research field. As we have seen earlier with a fewer varieties, there was no correlation between low sodium concentration and either salinity tolerance (relative shoot dry weigh) or shoot dry weight under salinity (Figure 17). In fact, in contrast to the hypothesis, MW#293, the highest performer under salinity, and had the highest sodium concentration (3,500 mg Na kg ^-1 versus <500 mg Na kg ^-1 for the majority of varieties).

[0387] Potassium, calcium and magnesium concentrations were within the range for adequate growth, and did not correlate with salinity tolerance and shoot growth under salinity. Durum wheats had higher sodium concentrations than bread wheats with the exception of Tamaroi with Nax2 gene and WID902 with Nax1 and Nax2 genes. These two durum wheats had sodium concentrations similar to those seen in bread wheats (Table 12). However, the effects of Nax1 and Nax2 genes on growth were small (Table 12). The only other notable effect was that durum wheats had lower magnesium concentrations than bread wheats (Table 12).

[0388] One of the objectives of this study was to demonstrate the value of selecting for low sodium concentration to improve salinity tolerance. As the wheat diversity set was previously genotyped using 90 K SNP genotyping platform (see Experiment 2 above), this gave us the opportunity to search for genetic markers associated with sodium concentration and shoot growth using genome-wide association studies (GWAS).

[0389] For the 100 bread wheats, the BLUEs of Na ⁺ extracted from the fitted model were used in genome-wide association mapping, based on 41 ,035 SNR markers with minor allele frequency (MAF)> 0.05. For each of the SNRs, a mixed-linear model (MLM) was fitted where the fixed component of the model contained a numerical version of the SNR as well as a covariate to adjust for the confounding effects of population structure. The MLM also contained a random effect for the lines with an assumed variance structure equivalent to the kinship matrix centred using the IBS method and then compressed to optimum groups. This then allowed the PSD (population parameters previously determined) compressed MLM method to be used to speed up computation time (Zhang et ai, 2010, supra). From each of the fitted models, SNR effects were assessed using a significant p-va!ue threshold set at P=8.91e-5 equivalent to a level of 0.05 after Bonferroni correction. All genome wide association mapping and assessment was computationally conducted using TASSEL software (Bradbury et ai, 2007, supra).

[0390] From the GWAS study, genetic markers for leaf sodium and chloride concentrations, and shoot growth were identified (Tables 14, 15 and 16). There were three associated markers for sodium concentration on chromosomes 2A and 7 A and one associated marker of unknown location. Four markers were associated with chloride concentration, with two markers on 5A near a vernalisation gene, and two markers of unknown location. There were 25 and 48 genetic markers associated with shoot growth under control and salinity, respectively, most of which were located in close proximity to vernalisation gene on 5A. The effect of vernalisation on shoot growth was further validated with this analysis. Genetic markers associated with major sodium exclusion genes Nax1 and Nax2 could not be detected in the present study, possibly due to relatively small population size (100 bread wheats).

[0391] Some of these markers associated with leaf sodium concentration were also identified under sodicity in Example 2 above, and may play a role in tissue tolerance and osmotic adjustment.

[0392] In summary, shoot growth under control and salinity were significantly correlated indicating that seedling vigour played a key role. Low sodium concentration did not indicate salinity tolerance. Sodium excluding Westonia bread wheat with sodium exclusion genes Nax1 and Nax2 did not perform any better than Westonia. On the contrary, the novel wheat germplasm MW#293 had the highest sodium concentration, the highest biomass under salinity, and the second highest biomass under control. Therefore, improving sodium exclusion ability further is unlikely to improve salinity tolerance. In contrast, increasing sodium concentration to levels not inhibiting plant growth can contribute to tissue tolerance/osmotic adjustment, and hence salinity tolerance.

TABLE 14

Genetic markers associated with sodium concentration identified by Genome Wide

Association Studies

90K marker name Chromosome Position P-value tplb0058o04 1071 2A 762646301 4.51 E-05

BS00035033 51 7A 85293920 7.59E-05

RFL Contigl 1 15 264 Unknown 0 7.85E-05

TABLE 15

Genetic markers associated with chloride concentration identified by Genome Wide

Association Studies

90K marker name Chromosome Position P-value

BS00022071 51 Unknown 0 3.00E-05 wsnp_Ex_c22727_3193429

6 Unknown 0 3.45E-05

RAC875 rep c109716 67 5A 588774401 4.34E-05

4.49E-05

Excalibur c23354 187 5A 588647307

TABLE 16

Genetic markers associated with shoot dry weight under salinity identified by

Genome Wide Association Studies

90K marker name Chromosome Position P-value wsnp Ex C31799 40545478 _ 5A 589968707 4.85E-07

RAC875 C13931 205 _ 5A 590295774 8.33E-07

RAC875 rep c1 13313 607 _ 5A 590131280 8.91 E-07

IACX9410 _ Unknown 0 1 .47E-06

Excalibur c23354 187 _ 5A 588647307 1 .70E-06

RAC875 C30566 230 _ 5A 590351 1 14 1 .89E-06

RAC875 rep d 16420 103 _ 5A 590303159 2.02E-06

Excalibur_c7729_144 _ 5A 589959022 2.63E-06 wsnp_Ex_c22727_31934296 _ Unknown 0 3.46E-06

BS00022071 51 _ Unknown 0 3.69E-06

Kukri_c941 1 676 _ Unknown 0 4.13E-06

BS00031 1 17 51 _ 5A 586996071 4.14E-06

Kukri C10033 724 5A 590694085 4.27E-06 90K marker name Chromosome Position P-value wsnp_Ex_rep_c66689_650109 5A

88 589762638 4.39E-06 wsnp Ex c7729 13177883 5A 589962592 4.75E-06 wsnp Ex c5998 10513766 5A 588646248 5.20E-06

Excalibur c15263 570 4D 507281561 5.45E-06

IACX448 5A 586994636 5.47E-06

RAC875 C9984 1003 5A 589913426 9.64E-06 wsnp AJ612027A Ta 2 1 5A 586821599 1 .01 E-05

BS00009782 51 Unknown 0 1 .37E-05

BobWhite rep c58252 1 12 7A 54442030 2.59E-05

RAC875 rep c109716 67 5A 588774401 2.72E-05

Kukri c9080 257 6A 31370799 3.00E-05

Tdurum contig61934 60 Unknown 0 3.40E-05

BS00064143 51 7A 28023355 3.44E-05

BobWhite c2963 1532 Unknown 0 3.46E-05

GENE-401 1 673 Unknown 0 4.47E-05

IAAV619 5A 578891389 4.52E-05

Kukri c6669 145 5A 586499714 4.53E-05

TA004624-0679 6A 351 1 1086 4.66E-05 tplb0038h19 1394 5A 589940807 4.66E-05

Tdurum contig86202 145 Unknown 0 5.01 E-05

Tdurum contig42015 2187 Unknown 0 5.22E-05

RAC875 C51375 299 4B 655434085 5.42E-05

Ra C14405 782 Unknown 0 6.34E-05

Excalibur c1708 1975 6A 29559295 6.82E-05 wsnp_Ex_rep_c67468_660692

82 Unknown 0 7.04E-05

RAC875 rep c85909 299 6D 167686025 7.53E-05

Tdurum contig86202 175 Unknown 0 7.66E-05

Tdurum contig62141 93 Unknown 0 7.74E-05 wsnp_CAP8_rep_c9132_3977

980 2A 30204351 8.00E-05

Excalibur c58765 317 Unknown 0 8.04E-05

Kukri C35661 63 6A 33954717 8.07E-05

IACX5958 Unknown 0 8.21 E-05

BS00098062 51 5A 589882425 8.24E-05

Kukri c264 539 Unknown 0 8.26E-05

Kukri C35024 1 16 Unknown 0 8.86E-05