AGENTS AND METHODS FOR ALTERING WAX PRODUCTION IN PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application number 62/370,306, filed August 3, 2016.

FIELD

[0002] The present invention relates to the modulation of wax production in plants. BACKGROUND

[0003] A major factor limiting yields of wheat and other major crops is water availability. Precipitation is frequently insufficient to support maximum yields in the Canadian prairies and many other geographic areas. Therefore, an important goal of plant breeding is to improve the efficiency with which water is used by wheat and other crops. In land plants, a hydrophobic coating called a cuticle covers the surfaces of the plant. The cuticle plays important roles in plant development, growth and defense against water loss, heat, UV radiation and pathogenic attacks. The cuticle consists of a framework of cutin, intracuticular wax and epicuticular wax overlaid on top. Significant accumulation of specific epicuticular waxes confers a 'glaucous' or blue-white coloration to leaves. The epicuticular wax layer on the surface of leaves, stems and floral organs reduces non-productive water loss from plants. In dry climates, high epicuticular wax production in wheat and other major crops is thought to contribute to improved performance [Mondal et al. Euphytica 201, 123-130 (2015), and references cited therein].

[0004] In grassy monocotyledonous species, such as barley and wheat, hentriacontane-14,16-dione (β- diketone) and its hydroxy derivatives are the major and unique components of cuticular wax in the upper parts of adult plants [Tulloch et al., Can. J. Botany 58, 2602-2615 (1980)]. In older wheat plants, the wax layer consists almost entirely of C31 β-diketone waxes that are produced from fatty acids [Adamski et al. 2013, Plant J. 74 989].

[0005] In wheat cultivars, there is substantial variability in wax deposition. In common (bread) wheat (T. aestivum L., genomes AABBDD), two genetic loci associated with wax production (W1 and W2) and two wax production inhibitors {Iw1 and Iw2) have been identified and mapped: Iw1 on chromosome arm 2BS [Adamski et al., supra], Iw2 on 2DS [Tsunewaki et al., Genes Genet. Syst. 74, 33-41 (1999)], W1 on 2BS [Lu et al Theor. Appl. Genet. 128, 1595-603 (2015)] and W2 on 2DS [Tsunewaki et al., supra]. In durum wheat (T. turgidum subsp. durum, genomes AABB), the locus of a third wax inhibitor, Iw3, has been found in the distal region of chromosome arm 1 BS [Wang et al., Theor. Appl. Genet. 127, 831 -841]. The identity and sequences of genes at the W1 locus have recently been reported [Hen-Avivi et al. 2016, Plant Cell, June 2016 vol. 28 no. 6: 1440-1460].

[0006] The genetic and biochemical basis for diketone wax deposition in wheat and the relationship between wax production and inhibition remain poorly understood. Because of the lack of knowledge surrounding diketone wax deposition, plant breeders have not been able to systematically control and optimize wax content in wheat cultivars and other crops. Therefore, the amount of desired surface wax is selected among breeding lines by visually assessing glaucousness. This provides breeders a rough estimate of the amount of wax present and enables them to select for or against wax content when they choose lines for germplasm development or commercialization. Visual selection of wax content among different lines is descriptive and approximate and does not allow fine distinctions. Precise quantitation of wax content using analytical methods such as gas chromatography and/ or mass spectrometry is too expensive and time consuming to be applied to large populations that are screened by plant breeders. Furthermore, the trait is controlled by more than one gene (at the l l and Iw loci) so heritability is difficult to predict from the glaucous appearance of the parents. Since visual measurement of wax is unreliable and the genetic basis of wax deposition is unclear, the results of genetic crosses cannot be reliably predicted.

[0007] Accordingly, there is a need for novel approaches for modulating wax production in plants such as cereals, in particular wheat, and for the identification of the genetic basis for the production, inhibition, and modulation of epicuticular wax in wheat.

[0008] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

SUMMARY

[0009] The present inventors have identified a microRNA precursor and microRNAs, Iw1 and variants thereof as described herein, that can be employed to regulate wax production in cereal plants. Accordingly, the present application provides vectors, nucleic acid molecules, and methods for modifying wax production in cereal plants. The application further provides genetically modified plants, plant cells, and kits comprising the vectors and nucleic acid molecules.

[0010] A first embodiment is a vector for use in transforming plant cells, said vector comprising a promoter and a nucleic acid molecule, said nucleic acid molecule comprising a sequence which has at least 80% sequence identity to the full length sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof, wherein the promoter is operably linked to the nucleic acid molecule. A further embodiment is a plant comprising the vector.

[0011] Another embodiment is a genetically modified plant cell, said cell comprising an exogenous nucleic acid molecule, wherein said exogenous nucleic acid molecule comprises a sequence which has at least 80% sequence identity to the full length sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof. A further embodiment is a plant comprising the genetically modified plant cell. In an embodiment, the plant is a wheat plant.

[0012] Another embodiment is a vector comprising a nucleic acid molecule, said nucleic acid molecule comprising a sequence that has at least 80% sequence identity to at least 15 contiguous nucleotides of the sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof. In a further embodiment, the nucleic acid molecule comprises a sequence that has at least 80% sequence identity to the full length sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof. In another embodiment the nucleic acid molecule comprises a sequence that has at least 90% sequence identity to at least 15 contiguous nucleotides of the sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof. In a still further embodiment, the nucleic acid molecule comprises a sequence that has at least 90% sequence identity to the full length sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof. In yet another embodiment, the nucleic acid molecule comprises a sequence as set forth in any one of SEQ ID NOs: 3 to 9. In another embodiment, the nucleic acid molecule is a modified nucleic acid molecule comprising up to 10 base pair substitutions relative to the sequence of any one of SEQ ID NOs: 3 to 9, wherein said modified nucleic acid molecule has reduced binding to SEQ ID NO: 1 relative to a corresponding nucleic acid molecule comprising the sequence of any one of SEQ ID NOs: 3 to 9. In yet another embodiment, the nucleic acid molecule comprises a sequence as set forth in any one of SEQ ID NOs: 3 to 9 having 0, 1 , or 2 nucleotide substitutions; 0, 1 , or 2 nucleotide insertions; and 0, 1 , or 2 nucleotide deletions, and wherein the total number of nucleotide substitutions, insertions, and deletions is between 1 and 4. In an embodiment, the nucleic acid molecule comprises 0 or 1 nucleotide substitutions; 0 or 1 nucleotide insertions; and 0 or 1 nucleotide deletions, while in a still further embodiment the nucleic acid molecule comprises a single nucleotide substitution, insertion, or deletion. In another embodiment, the nucleic acid molecule comprises a sequence that has at least 95% sequence identity to the full length sequence of any one of SEQ ID NOs: 3 to 9, or a complement thereof.

[0013] An embodiment is a genetically modified plant cell, said plant cell comprising an exogenous nucleic acid molecule, wherein said exogenous nucleic acid molecule is a nucleic acid molecule as defined above.

[0014] Another embodiment is a genetically modified plant cell comprising a vector as described above.

[0015] Yet another embodiment is a plant comprising a genetically modified plant cell as described above, or comprising a vector as described above. In an embodiment, the plant is a wheat plant.

[0016] Another embodiment is a method of modulating wax production in a plant comprising: transforming a plant cell with a vector as described above to produce a transformed plant cell; regenerating a plant from the transformed plant cell; and cultivating the plant under conditions suitable for plant growth and development.

[0017] Another embodiment is a method of modulating wax production in a plant cell, wherein the modulation is effected by altering the sequence and/or expression of at least one of SEQ ID NOs: 3 to 9 in the plant cell by T-DNA activation, gene editing technology, TILLING, site-directed mutagenesis, introducing of a nucleic acid sequence, introducing sequences encoding hairpin formations, directed evolution, or any combination of one or more thereof. In a further embodiment said gene editing technology uses a meganuclease, a Zinc finger nuclease (ZFN), a Transcription Activator-Like Effector- based Nucleases (TALEN), or the CRISPR-Cas system. In an embodiment, the modulation of wax production is a decrease in wax production. In another embodiment, the modulation of wax production is an increase in wax production. In an embodiment of the method, the plant is wheat.

[0018] Yet another embodiment is a method for creating a population of plants with enhanced or increased wax production, the method comprising: a) providing a first population of plants; b) determining the presence or absence of a nucleic acid molecule having at least 80% sequence identity to the full length sequence of any one of SEQ ID NOs: 3-9 in one or more plants within the first population of plants; c) selecting one or more plants lacking said nucleic acid molecule from the first population of plants; and d) producing a population of offspring from at least one of said selected plants.

[0019] Another embodiment is a method for creating a population of plants with decreased wax production, the method comprising: a) providing a first population of plants; b) detecting the presence or absence of a nucleic acid molecule having at least 80% sequence identity to the full length sequence of any one of SEQ ID NOs: 3-9 in one or more plants within the population of plants; c) selecting one or more plants containing said nucleic acid molecule from the first population of plants; and d) producing a population of offspring from at least one of said selected plants. [0020] An embodiment is a kit for modulating wax production in a plant, wherein the kit comprises: a) a nucleic acid molecule having at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 3-9; or a vector as described above; and b) instructions for modulating wax production in the plant.

[0021] A still further embodiment is a vector as described above, for use in transforming a plant cell, wherein the vector further comprises a promoter, and wherein the promoter is operably linked to the nucleic acid molecule.

[0022] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only, with reference to the accompanying drawings.

LISTING OF SEQUENCES

[0023] The following are a list of sequences appearing in the document:

[0024] SEQ ID NO:1 is the full length sequence of WAX1 {W1) cDNA, with the coding DNA sequence (CDS) underlined:

gccgaaccat aatcgagctc tgacgacttt tattagcttg tactcgtagt tgaagtgcat

caacatatcg tacctaggtc gccatgcctg caaacaagac ttacccctcc cataaaaatg

ccaacggtga ggtggacgac gaattctacc cattaatccg caagtacaag gacggccgga

tcgagcggtt catgagctca ttcgtgccgg cgtcggagga cccggcggcc agccgtggtg

tggcgacgag ggacgtcgtc gtcgaccagg gcaccggtgt gtccgtgcgc ctgttccttc

ctgcccaggc tgccgaggcc ggcgcgaggc tcccccttgt tgtgtacgtc catggtggtt

ccttctgcac ggagagtgcc ttctcccgga cgtaccaccg ttacgccact tccctcgccg

ccagcgcagg ggcgctcatc gtgtccgtgg agtaccgtct ggcgccggaa tatcccgtgc cgacgtccta cgatgacaca tgggccgcgc tgcggtgggt ggcgtccttg tccgaccctt ggctcgccaa atacgcagac cctagccgca cgttcctcgc cggcgacagc gctggcggca

acatcgtgta ccacacggcc gtgcgcgcca cacatgatga cagcatcatg gacatccagg

ggttggtcat ggtgcatcca ttcttctggg ggcccgagcg tctcccggcg gagaaggtct

tggacggcga cgccatgttc ccaccagtgt gggtggataa gctgtggccg ttcgtgacgg

cgggcggggc tggcaacgat gatcctcgga tcaatcctcc ggacgaggag atcgcgttgc

taactggcag gcgggtgctt gtggccgttg cagagaagga caccctgcgc gaccgggggc

gccagtttgt gtgcagcatg cgcaggtgtg ggtgggttga tggcagcctc accgtggtgg

agtcggaggg tgaggaccat ggcttccact tgtacgcccc cctacgtgcg accagcaaga

agcttatgaa gagcatcgtg cagttcataa accatcgcgc caccttgccg tcaccggcca

tggtgatccc agaaggctcg gccgaaacta tgctaggcgt ccctagtagg ccatttaagg

acatatttgg ctacgggatg cgcatgaaac gttggagtgg cacgagtttt gggctcaaag

ttggtcgtgc aaaagcatcg acgacgagct atgggttacc tttgaagcaa gctcgcacct

tcggagaccc tgtttcagca ccaacttcgg taagattcgt gatgaggaac tgtttctaga

accagttccc ggggtgtgtt aagttaattt aaatttacct caataaatgt attcttttca

ttattgcttg cacgttgagt ttgtatacgt caaggttatg atcacttctg gagtcgtgta

agtgcttttg ctattgcact ggcgcattgt atctgccaga aacgtgcaag ttcacgttgc

ggctaagtcg ttgcttggca tgttgtagtc tcgtcgccac gatccaccgc gtgcaggatg

gcgcacgctt tgtgtggatc gatcacggtg agctttgtgt gtctggttgt caagtgttag

aataaataaa ggaaatgaaa gaaagataga aaagatgcaa ttacggcgtc ac

[0025] SEQ ID NO: 2 is the amino acid sequence of a polypeptide encoded by

MPANKTYPSHKNANGEVDDEFYPLIRKYKDGRIERFMSSFVPASEDPAASRGVATRDVWD QGTGVSVRLFLP

AQAAEAGARLPLWYVHGGSFCTESAFSRTYHRYATSLAASAGALIVSVEYRLAPEYP VPTSYDDTWAALRWV

ASLSDPWLAKYADPSRTFLAGDSAGGNIVYHTAVRATHDDSIMDIQGLVMVHPFFWG PERLPAEKVLDGDAMF

PPVWVDKLWPFVTAGGAGNDDPRINPPDEEIALLTGRRVLVAVAEKDTLRDRGRQFV CSMRRCGWVDGSLTV

VESEGEDHGFHLYAPLRATSKKLMKSIVQFINHRATLPSPAMVIPEGSAETMLGVPS RPFKDIFGYGMRMKRW

SGTSFGLKVGRAKASTTSYGLPLKQARTFGDPVSAPTSVRFVMRNCF

[0026] SEQ ID NO: 3 is the sequence of a small-RNA-producing long-noncoding (Inc), inverted-repeat hairpin RNA (shown as a cDNA), which is called Inhibitor of Wax1 (Iw1) and is the precursor of the microRNA termed miRWl

ATCAGAGGTCCGCCTTACAACAAAGCAGAGAAAGGTGTCCGCTATCCTCACGCTGCT TTG GAGGCCGTAGGCCGTGTCCAGGTGGT CGGGCGGCAGGGCATCA CAGAGG CCGCCACA CAAATTCCTTGTTGGGTACCCGACGGTGTGCCGGAAGATGCTCGTCGCCGAGAGAGCCAA GGATCTGGGCACCAGCTCGGT CCCGCGACTGCATGGTCAGTACGTAGTTCAGTGTCTCTA

CCTTCGTACTG GAC CA AC G G C AT GAG G G G AT A A A A A A AC GAAT AT GAT C C CAT G T C T G C AC AAT C AG G T AG G T C AC CAT AC C C T CAT TAG G C C AG T T C T AAAG GGAGAGTGCGTTCT GCCGGACTCGTACCCTCAT TAGGCCAGTTCCCTCACCACGAGTCCA GGGGGCTCATCGTGTCCATGGAAGCACCAGAATATCCCATGCATGGCGACGT CCTACGAC AACACATGGGCCCAAATGTGGGCTCTGTCCTTGTACTAT CCTTGT GAGACCAGTTGTGCC GGTTAGTGTGCAGCATGTGCAAGTCGGGTGTGT TGGCGACAACCT CGGGCTCGGAGGGTG AGGACCAT CACTTCCACCTGTATGCCCCCTTCATACATGCGACCAGCAAGAAGCCTAT GT AGAGCATCCTGCGGTTCATAAACCATT GCGTCATGTTGCCGCCGGACAAGGT GATATCAG AGGACTCGACCGAACCTATGCTCGGCATGCCAATATTTT CTTGAAAGGCATACTAGGCAC GCAGAGCACAGGTT CGGCCAAGCCCTCTAGGAT CACCAT GCCCTGTGACAGCAAGGTGGC GT GATAAT TTATGAACTGCACGATGCT TTTCAGAAGCTT CTTGCT GGTTGCATGTAGACG GGCATAGGTGGAAGCCATGCCCTTAACCCTCGGATTTAACCATGGTGAGCCT GTCGTCAA CACACCCGCACTTGCGCATGGTGCACATATACT GGCGCCCCCGGTATCATAAGGTGTCCT C T G C AAC T AC C AC C AC AC CTGCTTACCGTCA

[0027] SEQ ID NO: 4 is the sequence of the most abundant microRNA termed miRW1 and is derived from

SEQ ID NO: 3 (miRW1 hairpin IncRNA or Iw1)

TTTATGAACTGCACGATGCTT

[0028] SEQ ID NO: 5 is the sequence of a variant of miRW1 :

TTTATGAACTGCACGATGC

[0029] SEQ ID NO: 6 is the sequence of a variant of miRW1 :

TTATGAACTGCACGATGCTTT

[0030] SEQ ID NO: 7 is the sequence of a variant of miRW1 :

TTATGAACTGCACGATGCTT

[0018] SEQ ID NO: 8 is the sequence of a variant of miRW1 :

TTTATGAACTGCACGATGCTTT

[0019] SEQ ID NO: 9 is the sequence of a variant of miRW1 :

TTTATGAACTGCACGATGCT

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0020] The present invention relates to methods of modulating wax production by modulating the expression of at least one, two, or more nucleic acid sequence(s) that is/are involved in wax production in plants, preferably in cereals, and more preferably in wheat. Nucleic acids as used herein include DNA, RNA (e.g., miRNA, mRNA), or derivatives of either DNA or RNA (e.g., cDNA), including naturally occurring molecules and synthetic analogues. The nucleic acids of the invention encompass all forms of nucleic acids including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0021] As used herein, "expression" or "expressing" refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence. The term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors. Thus, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide).

[0022] By "modulating wax production" it is intended that wax production is increased, decreased or suppressed relative to the level in a plant that has not been altered by the methods described herein. By "modulate expression of a target nucleic acid sequence" it is intended that the expression of the target nucleic acid sequence is increased or decreased relative to the expression level in a plant that has not been altered by the methods described herein. Expression levels may be assessed by determining the level of a gene product- by any method known in the art including, but not limited to determining the levels of the RNA and protein encoded by a particular target nucleic acid sequence. For nucleic acid sequences that encode proteins, expression levels may be determined, for example, by quantifying the amount of the protein present in plant cells, or in a plant or any part thereof. Alternatively, if a target nucleic acid sequence encodes a protein that has a known measurable activity or has a specific-associated phenotype (e.g., glaucousness), then activity levels or phenotypic traits may be measured/quantified to indirectly assess expression levels.

[0023] Identification of the W1 gene

[0024] The inventors of the present application have identified the genes corresponding to the loci previously identified as W1 (wax producing gene) and Iw1 (wax repressing gene) in wheat. With this knowledge, the inventors have invented genetically modified cells and plants, methods of genetically modifying plant cells and plants to alter the level of epicuticular wax produced by the plants, as well as methods to select for desirable wax traits from natural populations. Genetic modification includes transgenic modification, induced mutation and gene editing. Such genetically modified plants are useful in that they are specifically adapted for their environmental conditions regarding water availability, heat and pathogens.

[0025] Using near-isogenic pairs of wheat lines, in which one of each pair demonstrated glaucousness and one of which did not, whose creation is described by Clarke et al, 1994, Crop Science 34: 327-330, the inventors compared expression of large (transcripts from RNAseq) and small RNAs (e.g., miRNA and siRNA) to identify differences that were consistently associated with loss of wax production and glaucousness. Using the methods further described herein, potential wax-related genes that were strongly downregulated in non- glaucous lines were identified and the location of the differentially expressed unigenes were then determined. Using this methodology, the inventors identified seven down-regulated target genes on chromosome 2BS of wheat. One of the targets - referred to herein as WAX1 (W1) - was most significantly down-regulated in the non-glaucous F1 progeny of glaucous X non-glaucous crosses and was therefore specific to glaucous lines. The inventors confirmed the role of this gene in producing epicuticular wax by both transiently and stably silencing the gene in a glaucous line, and thereby producing a non-glaucous phenotype with β-diketone waxes decreased or absent.

[0026] In accordance with one aspect of the invention, there are provided methods of modulation of wax production in a plant by impacting the expression of the nucleic acid sequence of W1. Modulating wax production refers to an increase or a decrease in wax production of the plant, preferably in cereals, and more preferably in wheat. It may result in an increase of wax production, as when the expression of W1 gene is enhanced. Inversely, it may result in a reduction or suppression of wax production, as when the expression of W1 gene is silenced or prevented by a mutation of the W1 gene, or when a nucleic acid which inhibits expression of W1 is expressed.

[0027] In an aspect, the present invention provides nucleic acids, e.g., antisense nucleic acid molecules that inhibit the expression of the W1 gene. Inhibition of the W1 gene would have the effect of reducing the production of diketone epicuticular wax. The term "antisense nucleic acid molecule" as used herein refers to any nucleic acid molecule, such as a short RNA molecule, capable of inhibiting the expression of W1, for example, inducing degradation of a RNA molecule, blocking its translation and/or stopping its replication, and includes for example microRNA (miRNA), decoys, aptamers, small nuclear (sn) RNAs, ribozyme (see, e.g., PCT publication No. WO 2006/002547), antisense oligonucleotides (ASONs), small or short interfering RNA (siRNAs) and short hairpin RNAs (shRNAs). In an embodiment, the above-mentioned antisense nucleic acid molecule is an atypical miRNA. miRNAs are small non-coding RNA molecules (typically containing about 20-22 nucleotides) that function via base-pairing with complementary sequences within mRNA molecules, usually through cleavage of the target mRNAs. In this case, the miRNA of the invention is termed "atypical" because it is produced by a long inverted repeat hairpin RNA, rather than a typical miRNA gene.

[0028] An antisense nucleic acid molecule comprises or consists of an oligonucleotide at least a portion of which is complementary to the target nucleic acid sequence (e.g., SEQ ID NO:1 ) to which it is capable of hybridizing under physiological conditions.

[0029] As further discussed below, the inventors of the present application have discovered certain specific miRNAs which are associated with a decrease in the expression of the l l i transcript and therefore inhibit the production of diketone wax in non-glaucous lines. Therefore, in an embodiment of the invention, the antisense nucleic acid molecule targets, or is complementary to, the nucleotide sequence of the binding sites of the most prevalent of these miRNA (SEQ ID NOs: 4-9), specifically residues 1030-1052 of SEQ ID NO: 1. In a further embodiment, the antisense nucleic acid molecule targets, or is complementary to, a nucleotide sequence corresponding to residues 1030-1052 of SEQ ID NO:1 , or to its complement. In an embodiment, the antisense nucleic acid molecule comprises 0, 1 , 2 or 3 mismatches relative to a sequence of SEQ ID NO:1 , or its complement. In an embodiment, the antisense nucleic acid molecule comprises 0 or 1 mismatch relative to a sequence of SEQ ID NO:1 , or its complement. One or more nucleotides (or linkages) within the sequences described herein can be modified, for example by chemical or other modification as described below.

[0030] In embodiments, the present invention relates to the use of edited or modified versions of the miRNAs described herein (e.g., SEQ ID NO: 4 to 9) to alter the binding of the miRNA to the target nucleic acid (W1 transcript of SEQ ID NO:1). For example, gene editing approaches may be used to modify the binding site of miRW1 in W1 in a non-glaucous wheat. In another embodiment, an edited version of Iw1 may be introduced into glaucous wheat, the edited version having a miRW1 sequence having reduced binding to W1 relative to native Iw1.

[0031] In another embodiment, the present invention relates to the use of a non-functional pseudo-copy of W1 (or a fragment thereof) that would act as a target mimic and compete for miRW1 binding.

[0032] The antisense nucleic acid molecules according to the present invention may comprise modified products produced by chemically modifying the constitution moieties to, for example, alter intracellular stability and half-life, such as phosphate backbone and/or ribose and/or base etc., of the molecule. The modification methods are known in the art, which can be thio-modification and/or sterol modification and/or PEG- modification and/or glyco-modification and/or LNA-modification etc., as described for example in Dykxhoorn DM et al., Annual Review of Biomedical Engineering, 2006, Volume 8: pages 377-402 and Behlke MA et al., Molecular Therapy, 2006, Volume 13: pages 644-670. In addition, one or more ribose groups may be modified to add a methyl moiety to the 2'-OH to form a 2'-methoxy moiety (referred to as 2'O-methyl-modified). Also, the 2'-OH moiety can be linked to the 3' or 4'-carbon of ribose by a methylene or ethylene linker, typically a methylene linker to the 4'-carbon, to form a "locked nucleic acid" (see WO 98/39352 and WO 99/14226). Examples of other modifications include instances where the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, where R is C1 -C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.

[0033] In certain embodiments, chemical modification also includes the use of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and other similarly modified forms of adenine, cytidine, guanine, thymine, and uridine, which are not as easily recognized by endogenous endonucleases. Examples of modified bases include uridine and/or cytidine modified at the 5-position, e.g., 5- (2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8- bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6- methyl adenosine. "Analogs" also include sequences in which one or more thymine (T) bases have been substituted for uracil (U) base and vice versa.

[0034] Further, chemical modification can encompass modified backbones such as morpholino and/or further non-natural internucleoside linkages such as siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate; formacetyl and thioformacetyl; alkene-containing; methyleneimino and methylenehydrazino; amide, and the like.

[0035] In an embodiment, the above-mentioned antisense nucleic acid molecule is from about 5 to about 100 nucleotides in length, in further embodiments the antisense nucleic acid molecule is from about 10 to about

100, from about 5 to about 50, from about 10 to about 50, from about 15 to about 50, from about 10 to about

30, from about 18 to about 29, from about 19 to about 27, from about 18 to about 25, from about 19 to about

25, or from about 19 to about 23 nucleotides in length, e.g., 19, 20 , 21 , 22 or 23 nucleotides in length. [0036] Identification of miRW1

[0037] The inventors of the present application have further identified a family of related 20-22 nucleotide (nt) sequences in non-glaucous lines of wheat, which consist of small RNA sequences. Most of these small RNA sequences could not be mapped to known wheat genome sequences. However, 6 of them, including the most abundant 21 nt sequence were uniquely mapped to residues 1030-1052 of the l l i sequence with up to 1 mismatch (SEQ ID NOs: 4-9). Since these small RNAs were complementary to specific sequences in l l i, the - most abundant sequence was designated as microRNA specific to l l i (miRW1) (SEQ ID NO: 4), while the remaining five sequences were termed miRW1 variants (SEQ ID NOs: 5-9).

[0038] Expression of miRW1 was virtually absent in glaucous isolines and in glaucous cultivars but present in non-glaucous isolines including the non-glaucous F1 progeny of crosses between glaucous and non-glaucous isolines. The most significant homolog of the miRW1 sequence in the wheat NCBI unigene set or in genome survey sequences was the l l i sequence with 1 base mismatch, raising the possibility that miRW1 was derived from l l i and targeted an undiscovered gene. However, structure prediction software did not indicate that the l l i transcript could fold to form a hairpin loop structure characteristic of miRNA-producing precursors, and the concept of l l i as a miRNA precursor is inconsistent with the inverse correlation between l l i and miRW1 expression (W1 is only expressed in glaucous plants). Therefore, it was deduced that miRW1 is produced from an unknown precursor gene and targets W1.

[0039] miRNAs are normally encoded by a nucleic acid such as an miRNA precursor, also referred to as a

"primary miRNA" or "pri-miRNA" (Current Opinion in Plant Biology 18:87 (2014)) that is transcribed and processed into the mature miRNA. "pri-miRNAs" or "primary miRNAs" are long, polyadenylated RNAs transcribed by RNA polymerase II that encode miRNAs. A "pre-miRNA" is a primary miRNA that has been processed to form a shorter sequence that has the capacity to form a stable hairpin and is further processed to release a miRNA. Similarly, long-noncoding, inverted-repeat containing hairpin RNAs have been discovered that result in the generation of miRNA or siRNA. These long-noncoding, inverted-repeat containing hairpin

RNAs can be endogenously present in the plant or generated artificially to manipulate mRNA levels. Therefore, a precursor miRNA, primary miRNA, or long-noncoding, inverted-repeat containing hairpin RNA is a nucleotide sequence that is capable of producing one or more miRNAs. Given this functional definition, a precursor miRNA, primary miRNA, long-noncoding, inverted-repeat containing hairpin RNA and/or a miRNA of the invention can be represented as a ribonucleic acid or, alternatively, in a deoxyribonucleic acid form that "corresponds substantially" to the precursor miRNA, primary miRNA, long-noncoding, inverted-repeat containing hairpin RNA, and/or miRNA. It is understood that a DNA in its double-stranded form will comprise a strand capable of being transcribed into the miRNA precursor or hairpin molecule described. Expression constructs, recombinant DNA constructs, and transgenic organisms incorporating the miRNA encoding DNA that results in the expression of the described long-noncoding, inverted-repeat containing hairpin RNA are encompassed by the present invention.

[0040] The inventors have determined the sequence of the precursor mRNA (SEQ ID NO: 3) that is further processed to give SEQ ID NO: 4 (and/or SEQ ID NOs: 5-9). This precursor appears to be Inhibitor of Wax1 (Iw1). In an embodiment, the miRW1 IncRNA precursor of the invention comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 3, or its complement.

[0041] In the studies described herein, the present inventors have shown that the expression of W1 is negatively regulated by miRW1 and its variants, all of which are encoded by Iw1. Accordingly, the increased expression of W1 described herein may also be achieved by reducing the expression of the miRWIs and/or the Iw1 described herein. For example, the expression of the Iw1 described herein may be reduced using an antisense nucleic acid molecule (siRNA, shRNA, miRNA, etc., as described herein) targeting the Iw1 transcript (SEQ ID NO:3), i.e. having a nucleotide sequence substantially complementary to a region of the miRNA IncRNA precursor transcript and being capable of inducing its degradation.

[0042] The miRW1 and Iw1 nucleic acids of the invention can be introduced into wheat cells to reduce the production of diketone wax by binding to W1. In an embodiment, the reduction of the expression of W1 gene in plant cells or plant organs/tissues is achieved by increasing the endogenous expression of the miRWIs or Iw1 described herein, or by inducing the exogenous or recombinant expression of the miRW1 or Iw1. For example, a construct comprising a promoter functional in a plant cell operably linked to a sequence encoding the Iw1 may be introduced in a plant cell. Introduction of the construct into the host cells is effected under conditions such that the Iw1 transcript is produced and mature miRW1 is then excised from Iw1 by an endogenous ribonuclease. In another embodiment, the introduced construct is a mature miRW1 expressed from a vector designed for artificial or synthetic miRNAs [Tiwari et al., Plant Molecular Biology 86: 1 -18 (2014)]. The resulting mature miRW1 induces degradation of an mRNA transcript in the cell or otherwise inhibits translation of the mRNA. In an embodiment, the Iw1 (SEQ ID NO: 3) comprises the coding sequence and the miRW1 is at least one of SEQ ID NOs: 4-9, for example SEQ ID NO:4.

[0043] The levels of inhibition can be altered by the amount of miRW1 available in the cell. Accordingly, miRW1 and Iw1 constructs can be designed to vary the level of W1 expression by various means known in the art. In one embodiment, overexpression can be achieved by placing the DNA sequence encoding the Iw1 under the control of a promoter, examples of which include but are not limited to endogenous promoters, heterologous promoters, inducible promoters and tissue specific promoters. Thus, depending on the promoter used, increased expression or overexpression can occur throughout a plant, in specific tissues of the plant, or in the presence or absence of different inducing or inducible agents, such as hormones, chemical agents or environmental signals. In another embodiment, the increased expression of miRW1 and Iw1 is achieved by the use of multiple copies of the same gene to provide for increased expression and/or the use of multiple vectors to increase the amount of miRW1 and Iw1 available in the cell.

[0044] Other methods of varying the amount of diketone wax include introducing mutations into the sequence of the miRW1 or Iw1. These modifications can include mismatches which reduce the binding of the miRW1 and thereby limit the extent to which it inhibits expression of W1. Mutations can be introduced into genes using methods known in the art such deletion (knockdown) or disruption of the gene by site-directed mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING) in order to introduce mutation(s) inhibiting activity, or by gene-editing technologies based on engineered nucleases such as Meganucleases, Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based Nucleases (TALEN), and the CRISPR-Cas system.

[0045] The invention further relates to methods of expressing the nucleic acids described here in plant cells, and to expression vectors and constructs comprising the nucleic acids described herein. The isolated nucleic acid molecules of the invention may be incorporated into a vector, such as a recombinant expression vector

(e.g., a eukaryotic expression vector, a viral expression vector). In an embodiment, the vector comprises an expression cassette which includes transcriptional regulatory sequences and/or a promoter operably-linked to the nucleic acid. A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.

"Transcriptional regulatory sequences" or "transcriptional regulatory elements" are generic terms that refer to

DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably-linked. As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting transcriptional regulatory element, a.k.a. cis-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked polynucleotide.

[0046] As used herein, the term "promoter" refers generally to a DNA molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter can be initially isolated from the 5' untranslated region (5' UTR) of a genomic copy of a gene. Alternately, promoters can be synthetically produced or manipulated DNA molecules. Promoters can also be chimeric, that is a promoter produced through the fusion of two or more heterologous DNA molecules. Plant promoters include promoter DNA obtained from plants, plant viruses, fungi and bacteria such as Agrobacterium and Bradyrhizobium bacteria.

[0047] The vectors or constructs of the invention include expression cassettes including a "transit peptide" or "targeting peptide" or "signal peptide" molecule located either 5' or 3' to or within the gene(s). These terms generally refer to peptide molecules that when linked to a protein of interest directs the protein to a particular tissue, cell, subcellular location, or cell organelle. Examples include, but are not limited to, chloroplast transit peptides (CTPs), chloroplast targeting peptides, mitochondrial targeting peptides, nuclear targeting signals, nuclear exporting signals, vacuolar targeting peptides, and vacuolar sorting peptides. For description of the use of chloroplast transit peptides see US Patent No. 5,188,642 and US Patent No. 5,728,925. For a description of the transit peptide region of an Arabidopsis EPSPS gene, see Klee, H.J. Et al (MGG (1987) 210:437-442).

[0048] Expression cassettes of the invention may contain a DNA near the 3' end of the cassette that acts as a signal to terminate transcription from a heterologous nucleic acid and that directs polyadenylation of the resultant mRNA. These are commonly referred to as "3'-untranslated regions" or "3'-noncoding sequences" or

"3'-UTRs". The "3' non-translated sequences" means DNA sequences located downstream of a structural nucleotide sequence and include sequences encoding polyadenylation and other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the mRNA precursor. The polyadenylation signal can be derived from a natural gene, from a variety of plant genes, or from T-DNA. An example of a polyadenylation sequence is the nopaline synthase 3' sequence (nos 3'; Fraley et al, Proc. Natl. Acad. Sci. USA 80: 4803-4807,

1983). The use of different 3' non-translated sequences is exemplified by Ingelbrecht et al., Plant Cell 1 :671-

680, 1989. Well-known 3' elements include those from Agrobacterium tumefaciens genes such as nos 3 ', tml

3', tmr 3', tms 3', ocs 3', tr7 3', for example disclosed in US Patent No. US 6,090,627; 3' elements from plant genes such as wheat (Triticum aestivum) heat shock protein 17 (Hspl7 3'), a wheat ubiquitin gene, or a wheat fructose-1 ,6-biphosphatase gene.

[0049] Expression cassettes of the invention may also contain one or more genes that encode selectable markers and confer resistance to a selective agent such as an antibiotic or an herbicide. A number of selectable marker genes are known in the art and can be used in the cassettes/constructs. For example, selectable marker genes conferring tolerance to antibiotics like kanamycin and paromomycin (nptll), hygromycin B (aph IV), spectinomycin (aadA), US Patent Publication No. 2009/10138985A1 and gentamycin (aac3 and aacC4) or tolerance to herbicides like glyphosate (for example, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), US Patent Nos. 5,627,061 ; US 5,633,435; US 6,040,497; US 5,094,945), sulfonyl herbicides (for example, acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide (US Patent Nos. US 6,225,105; US 5,767,366; US 4,761 ,373; US 5,633,437; US 6,613,963; US 5,013,659; US 5,141 ,870; US 5,378,824; US 5,605,011)), bialaphos or phosphinothricin or derivatives (e.g., phosphinothricin acetyltransferase (bar) tolerance to phosphinothricin or glufosinate (US Patent Nos. US 5,646,024; US 5,561 ,236; US 5,276,268; US 5,637,489; US 5,273,894); dicamba (dicamba monooxygenase, Patent Application Publication No. US2003/01 15626A1), or sethoxydim (modified acetyl-coenzyme A carboxylase for conferring tolerance to cyclohexanedione (sethoxydim)), and aryloxyphenoxypropionate (haloxyfop, US Patent No. US 6,414,222).

[0050] A recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd edition; Cold Spring Harbor Laboratory). A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences such as for reporter genes are well known to persons skilled in the art.

[0051] "Identity" refers to sequence similarity between two polypeptides or two nucleic acid molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid or polypeptide sequence is "identical" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved. Two nucleic acid or polypeptide sequences are considered "substantially identical" if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity and/or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% with any of the polypeptide or nucleic acid sequences of the invention. "Substantially complementary" nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule.

[0052] Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics

Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et ai, 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National

Center for Biotechnology Information. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl.

Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or

0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.

[0053] A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a cell, e.g., a host cell, which may include a living cell capable of expressing the nucleic acid inhibitor from the defined recombinant expression vector. Accordingly, the invention also provides genetically modified cells containing the nucleic acid or recombinant expression vector described herein. Such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0054] In an embodiment, the present invention provides a vector comprising the miRW1 nucleic acid inhibitor or Iw1 IncRNA precursor thereof described herein. In an embodiment, the vector is a viral vector. In an alternative embodiment, the vector may be a regular plant overexpression vector, such as Gateway-compatible destination vectors disclosed in Mann DG, et al. (Plant Biotechnol J. 2012 Feb;10(2):226-36). In another embodiment, the vector is a vector for Virus-Induced Gene Silencing (VIGS), for example a vector derived from Tobacco mosaic virus (TMV), Tobacco rattle virus (TRV), Apple latent spherical virus (ALSV), Bean pod mottle virus (BPMV), Brome mosaic virus (BMV), Pea early browning virus (PEBV), Rice tungro bacilliform virus, Tomato bushy stunt virus, Turnip yellow mosaic virus (TYMV) or barley stripe mosaic virus (BSMV), as exemplified in the Examples below.

[0055] The cells that have been transformed or gene-edited may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81 -84. These plants may then be pollinated with either the same transformed strain or a different strain, and the resulting progeny having desired expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present disclosure provides transformed seed (also referred to as "transgenic seed") having the nucleic acid disclosed herein, for example, an expression cassette disclosed herein, stably incorporated into their genome.

[0056] In addition to direct transformation of a plant material with a recombinant DNA, a transgenic plant can be prepared by crossing a first plant comprising a recombinant or modified DNA with a second plant lacking the recombinant or modified DNA. For example, recombinant DNA can be introduced into a first plant line that is amenable to transformation, which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing increased or reduced wax production as described herein may be crossed with a genetically modified plant line having another recombinant DNA, or modified gene that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant or modified DNA that confers both traits. Typically, in such breeding for combining traits the transgenic or modified plant donating the additional trait is the male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, for example, marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant DNA, by application using a selective agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as the original transgenic parental line but for the recombinant DNA of the other transgenic parental line.

[0057] The present invention may be used for transformation of crop plants (for example, cereals). In an embodiment, the plant is a diketone wax-containing cereal. In a further embodiment, the plant is wheat, or any species of the genus Triticum such as Triticum aestivum or Triticum durum or a closely related species such as Hordeum vulgare (barley).

[0058] In another aspect of the present invention, there is provided a plant cell having the nucleic acids, vectors, cassettes and/or constructs described herein. A further aspect of the present invention provides a method of making such a plant cell involving introduction of the nucleic acid, vector, cassette and/or construct described herein into a plant cell. [0059] As used herein, "plant" includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. As used herein, the term "plant parts" includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture. As used herein, the term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant (e.g., leaves, stems, roots, etc.).

[0060] The plant breeding methods used herein are well known to one skilled in the art. For a discussion of plant breeding techniques, see Poehlman (1987) Breeding Field Crops. AVI Publication Co., Westport Conn.

[0061] Backcrossing methods may be used to introduce the nucleic acids described herein into the plants. This technique has been used for decades to introduce traits into a plant. In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the nucleic acid to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred nucleic acid from the nonrecurrent parent. Any person skilled in the art readily appreciates the methods described herein are applicable to any other crops which have the potential to outcross. By way of example, but not limitation it can include barley or any plant with the capacity to outcross.

Identification of wax phenotype in plant populations

[0062] Using any of a variety of approaches, the cells encoding the introduced recombinant nucleic acid encoding polypeptides that are involved in wax production described herein can be selected (e.g., markers such as diketone production, or DNA markers such as KASP, SSR), can be used to select for cells and tissues containing the nucleic acid).

[0063] The nucleic acids described herein may be used as markers to identify plants that exhibit reduced or increased wax production. For example, the expression of a nucleic acid (e.g., nucleic acid of SEQ ID NO:1) may be used to identify plants having the ability to produce wax or producing increased/high levels of wax, whereas the presence of the miRNAs or miRNA precursor described herein may be used to identify plants not having the ability to produce wax or producing decreased/low levels of wax.

[0064] The presence and/or level of the nucleic acids described herein may be evaluated at the genomic DNA or transcript (RNA or cDNA) level according to the methods disclosed below, e.g., with or without the use of nucleic acid amplification methods. Examples of such methods include polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), in situ PCR, SAGE, quantitative PCR (q-PCR), digital PCR, in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, or other DNA/RNA hybridization platforms.

[0065] The present invention includes kits comprising the polypeptide, nucleic acids, miRNA precursor and/or miRNAs (and constructs comprising such nucleic acids) of the invention as described herein. Such kits can further comprise, for example, ancillary reagents (e.g., buffers, solutions), other physical components (e.g., primers, probes) such as those necessary to carry out the instant methods, and container means. The kit can further comprise instructions for using the kit, i.e. to identify markers of wax production.

[0066] The present invention also provides a kit or package comprising reagents useful for determining the presence and/or amount/level of the nucleic acid encoding the polypeptide, such as an oligonucleotide (e.g., primer and/or probe). Such kit may further comprise, for example, instructions for screening for the presence of the nucleic acid sequence(s) that are involved in wax production in a plant described herein, control samples (e.g., samples to which the test sample may be compared to perform the assay), containers, reagents useful for performing the methods (e.g., buffers, enzymes, immunodetection reagents, etc.). The kit may further include where necessary agents for reducing background interference in a test, agents for increasing signal, software and algorithms for combining and interpolating marker values to produce a prediction of clinical outcome of interest, apparatus for conducting a test, calibration curves and charts, standardization curves and charts, and the like.

[0067] Further detailed description is provided below by way of examples and is not intended to limit the scope of the invention.

EXAMPLES

[0068] Plant materials and Growth Conditions [0069] Four pairs of glaucous (G) and non-glaucous (N) near-isogenic lines (NIL) of wheat, AE3 vs. AE3N, AG1 vs. AG1 N, AG2 vs. AG2N, and D051 vs. D051 N, were obtained from the Swift-Current Research and Development Centre of Agriculture and Agri-Food Canada (Clarke et al, 1994, Crop Science 34: 327-330; Clarke et al. 1995 Can. J. Plant Sci. 73, 961-967). GxN crosses were made for 3 of the near-isogenic lines to produce F1 generation heterozygous plants for AE3, AG1 and AG2. The bread wheat cultivar Bobwhite and durum wheat cultivar Strongfield were used in the production of transgenic plants. Nicotiana benthamiana were used as host plants in the VIGS procedure documented below.

[0070] Growth of wheat and N. benthamiana plants was carried out in 4-in-square or 6-in-round pots containing Sunshine mix #8 (Sun Gro™ Horticulture) mixed with a slow-release 14-14-14 fertilizer. Plants were maintained in a growth cabinet or sunroom with a 16 h day/8 h night, light intensity of 600-800 mol m ² s ¹ , and day/night temperatures of 22°C/18°C.

[0071] Wax extraction for GC-MS Profiling of Wax Composition

[0072] Cuticular wax was extracted from flag leaf sheath at booting-heading stage by submerging tissues in a glass tube containing 10 ml of HPLC grade chloroform (Fisher Scientific®) and 10ng of tetracosane (Sigma- Aldrich®) as an internal standard and agitating manually for 1 min. The tissue was rinsed with an additional 5 ml of chloroform, and the two extracts were combined. The wax extract was dried under a nitrogen stream and resuspended in 250 μΙ of toluene. Wax components were separated and identified by GC-MS using an Agilent® 6890N GC equipped with a 15 m MXT™-1 capillary column (Restek®) and an Agilent® 5973N mass selective detector. Samples were injected in split mode with hydrogen as carrier and an initial column temperature of 125°C. To enable separation of multiple components ranging from long chain fatty acids (>Ci ₄ ) to wax esters (>Cs2), the oven temperature was increased at 5 °C/min to a maximum of 350 °C and held at that temperature for 5 minutes. Components were identified by comparison of retention time and mass spectra to standards.

[0073] Four pairs of near-isogenic lines (as mentioned above) exhibited differences between isolines in the presence or absence of bluish-white glaucousness. Glaucous isolines showed bluish-white coloration from the booting stage in the stems, leaves, floral tissue and seeds whereas non-glaucous isolines were green and glossy. The primary constituent of epicuticular wax from glaucous lines was hentriacontane-14, 16-dione (β- diketone), some of which was hydroxylated to form a 25-hydroxy- -diketone (Tulloch et al, 1980, Canadian J.

Botany 58: 2602-2615). In each case the non-glaucous isolines contained no detectable diketone waxes, whereas these waxes predominated in the glaucous isolines. The data from the GC-MS is summarized below in Table 1.

Table 1 : Wax content in glaucous and non-glaucous isolines. Representative quantities of the wax components are denoted as YES for present, No for not present, and Low for peaks close to the detection limit. Peaks 17 and 18 are the diketone waxes that are the subject of the application.

22 C44 wax ester YES YES

23 C46 wax ester YES YES

24 C48 wax ester YES YES

25 C50 wax ester YES YES

[0074] Crosses between each of the near-isogenic (NIL) pairs (glaucous X non-glaucous) resulted in F1 plants that show no bluish-white coloration and were therefore 100% non-glaucous, confirming the dominance of the non-glaucous trait, as observed previously (Clarke et al, 1994, Crop Sci. 34, 327-330; 1995 Crop Sci. 35, 1241).

[0075] Identifying small RNAs and transcripts associated with wax production

[0076] Stranded RNA sequencing libraries for the NIL lines, using 5-6 μg total RNA as starting material, were prepared using the Ribo-Zero™ rRNA Removal Kit for Plants and the ScriptSeq™ v2 RNA-Seq Library Preparation Kit with ScriptSeq™ Index PCR primers according to the manufacturer's instructions (Epicentre, an lllumina Company). For all subsequent RNA library preparations, the TruSeq™ RNA Library Preparation Kit v2 was used according to manufacturer's instructions (lllumina®). For small RNA, the TruSeq™ Small RNA Library Preparation kit was used, respectively, according to the manufacturer's instructions (lllumina®). RNA and library quality control was accomplished using the RNA 6000 Pico kit (Agilent Technologies®).

[0077] Paired-end sequencing with 100 bp reads was performed on a HiSeq2500 (lllumina, Inc., San Diego, CA). A variety of tools in CLC genomics workbench (Qiagen Bioinformatics) were used to process and analyze the RNA-seq data. Quality control was performed to remove the sequencing adaptors and low quality sequences. For NILs, reads were then mapped to the NCBI Triticum aestivum Unigene Set (Build # 63, http://www.ncbi. nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=4565), consisting of 178,464 sequences and IWGSC Chromosome Survey Sequences from the AABB genome with 66,307 annotated genes, as a references. For miRW1 overexpression lines, reads were mapped to the NCBI Triticum aestivum Unigene Set (Build # 63) and IWGSC Chromosome Survey Sequences from AABBDD genome with annotated genes. The expression levels of unigenes/ genes were calculated as total and unique read counts per unigene using the CLC genomics workbench RNA-seq analysis module. Unique read counts per unigene were used to calculate differentially expressed genes (DEGS) of significance, p < 0.05, between each isogenic pair using the R Bioconductor package edgeR (Robinson et al. 2010, Bioinformatics 26, 139-140). DEGS common to all isogenic pairs were selected using the R package dplyr (Wickham and Francois, 2015, https://CRAN.R-project.org/package=dplyr (2015).

[0078] Small RNA sequencing was performed on a Hiseq2500 (lllumina) with read lengths of 50 bp. For NIL pairs including F1 plants, reads were trimmed and filtered for quality and size (19-28 bp) using CLC genomics workbench. Significant miRNAs, p < 0.05 and a FC > 2, between each isogenic pair were selected using the R Bioconductor package DESeq2 (Love et al, 2014, Genome Biol. 15:550). Differentially expressed miRNA common to all isogenic pairs were selected using the R package dplyr [Wickham and Francois, 2015, https://CRAN.R-project.org/package=dplyr (2015)]. Mapping of small RNAs to various references were performed in CLC genomic workbench.

[0079] For qPCR expression measurements of potential target genes in isogenic lines including the F1 generation, 1.6 μg of RNA was used with the Superscript™ III First-Strand Synthesis SuperMix™ for qRT-PCR (ThermoFisher Scientific) according to the manufacturer's protocol except that the synthesis reaction occurred in 40 \ I at 50°C for 60 min. cDNA was diluted 12.5-fold in water and 8 μΐ of this dilution was used for qRT- PCR expression analysis. For qPCR, the Power SYBR Green Master Mix (ThermoFisher Scientific) was used in a total reaction volume of 20 μΐ and 0.2 μΜ primer concentration. Cycle threshold (CT) values were collected on an ABI StepOne Plus using the StepOne v2.3 Software and relative quantitation was calculated based on using the 2 ^"MCT method (Pfaffl, 2001 , Nucleic Acids Res. 29, e45.). The gene, Ta.46201 , Cell Division Control protein 48 homolog E-like, was selected as a reference based on Paolacci et al. (2009) BMC Mol. Biol. 10, 1 1 and Gimenez et al. (2011) Planta 233, 163-73. Measurement of W1 expression following VIGS treatments utilized the procedure described above except that the High-Capacity RNA-to-cDNA (ThermoFisher Scientific) was used to synthesize cDNA from 1 μg of RNA following the manufacturer's instructions.

[0080] Expression of large (transcripts from RNAseq) and small RNAs (miRNA and siRNA, 19-28 bp) were compared between isogenic pairs to identify differences that were consistently associated with loss of wax production and glaucousness. Potential wax-related genes that were strongly downregulated in non-glaucous lines were identified by mapping reads to the NCBI-unigene set (build 63) and the IWGSC wheat survey sequence 1.0 (AABB). The location of the differentially expressed unigenes was then determined by BLASTing the gene sequences against the NCBI-unigene set and the IWSSC AABB survey sequence to search for the corresponding scaffolds. The BLAST algorithm is described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis is available through the National Center for Biotechnology Information. Seven down -regulated target genes were identified on chromosome 2BS. One of the targets - designated as WAX1_ (W1) - was most significantly down regulated in the non- glaucous F1 progeny of glaucous X non-glaucous crosses and was therefore specific to glaucous lines (see Table 2). The sequence of W1 was located in a scaffold associated with chromosome 2BS.

Table 2. Expression changes of W1 and other target genes located on chromosome 2BS between glaucous, non-glaucous, and non-glaucous F1 progeny.

[0081] Identification of miRW1

[0082] Differential expression analysis of small RNAs revealed a family of related 20-22 nucleotide (nt) sequences in non-glaucous lines (see Table 3). Criteria for selection were based on significance level of p < 0.05 using DESeq2 [Love et al. 2014, Genome Biol. 15:550].

Table 3. Small RNA reads from AG1 , AG2, AE3, and D051 Isogenic line pairs which map to the /iv transcript with greater than 1 average read per 10 million. F1-NonWax represents average reads from F1 crosses of AG1 , AG2, and AE3 (± standard error). Mapping position, 100% homology, within the Iw transcript is indicated. Small RNA which map to the W gene, with up to 3 mismatches, are also shown. * denotes significant difference, p < 0.05, between wax versus non-wax. SEQ ID NOs: 4-9 are in bold italics.

W Map

Total Average Reads per 10 Million Iw Map

Length Position Counts Position

(mismatch)

Small RNA Sequence NonWax Fl-NonWax Wax

TTTA TGAA CTGCA CGA TGCTT* 21 12555 463±83 257±33 1±0 848 1031(1)

CATGCGACCAGCAAGAAGCCT* 21 941 46±12 3±1 0 636 1005(2)

AAGAAGCCTATGTAGAGCATC* 21 963 31±4 26±5 0 648 1017(2)

G CAGCATGTG CAAGTCG GGTG * 21 416 20±3 2±1 0 550 -

TCG ACCG AACCTATG CTCG G C* 21 410 18±3 4±1 0 726 1098(3)

AGAGCACAGGTTCGGCCAAGC* 21 368 16±3 4±1 0 783 -

GACAAGGTGATATCAGAGGAC* 21 339 14±3 4±2 0 705 -

ATCAGAGGACTCGACCGAACC* 21 392 12±2 12±4 0 716 -

ACAAG GTG ATATCAG AG G ACT* 21 237 10±3 4±1 0 706 -

TATCAGAGGACTCGACCGAACC* 22 267 10±2 5±2 0 715 -

TCAGAGGACTCGACCGAACCT* 21 287 9±2 8±1 0 717 -

TTCGGCCAAGCCCTCTAGGAT* 21 161 8±1 1 0 793 1086(3)

AG CATGTG CAAGTCGG GTGTG * 21 156 7±2 1±1 0 552 -

ACAAG GTG ATATCAG AG G AC 20 132 6±1 2±1 0 706 -

TTCGGCCAAGCCCTCTAGGATC* 22 142 6±1 1±1 0 793 1085(3)

CCTCTAGGATCACCATGCCCT 21 104 5±1 1 0 804 -

TTTA TGAA CTGCA CGA TGCTTT* 22 120 5±1 1 0 848 1030(1)

TTTA TGAA CTGCACGA TGCT* 20 138 5±1 3 0 848 1032(0)

ATGCGACCAGCAAGAAGCCT 20 73 3 2±1 0 637 1006(2)

TTTA TGAA CTGCA CGA TGC 19 76 3±1 1 0 848 1033(0)

AG AAG CCTATGTAG AG CATC 20 93 2 4 0 649 1018(2)

AAG CCTATGTAG AG CATCCT 20 45 2 1±1 0 651 1020(3)

G G ACAAG GTG ATATCAG AG G AC 22 35 2 0 0 704 -

GACAAGGTGATATCAGAGGACT 22 37 2 0 0 705 -

ACTCGACCGAACCTATGCTCG 21 54 2±1 0 0 724 -

TCGACCGAACCTATGCTCG 19 57 2±1 1 0 726 1098(3)

TCG ACCG AACCTATG CTCG G 20 40 2 0 0 726 1098(3)

TTG G CCG AACCTGTG CTCTG C 21 53 2±1 1 0 781 -

TTA TGAA CTGCA CGA TGCTTT 21 68 2 2 0 849 1030(1)

TTA TGAA CTGCA CGA TGCTT 20 202 2 13±1 0 849 1031(1)

AGCAAGAAGCCTATGTAGAGC 21 16 1 0 0 645 1014(2)

CAAGAAGCCTATGTAGAGCAT 21 20 1 0 0 647 1016(2)

AGGTGATATCAGAGGACTCGA 21 40 1 1 0 709 -

AGGTGATATCAGAGGACTCGACCGAACC 28 13 1 0 0 709 -

TATCAGAGGACTCGACCGA 19 19 1 0 0 715 -

TCAGAGGACTCGACCGAACC 20 57 1 3±1 0 717 -

ACGCAGAGCACAGGTTCGGCC 21 17 1 0 0 779 -

AGAGCACAGGTTCGGCCAAG 20 27 1 0 0 783 - CCTAGAGGGCTTGGCCGAACC 21 27 1 0 0 791

[0083] Most of these small RNAs sequences could not be mapped to the IWGSC wheat genome survey sequences. However, 6 of them, including the most abundant 21 nt sequence (9365 total reads) were uniquely mapped to l l i with 1 mismatch or less:

[0084] SEQ ID NO: 4 - TTTATGAACTGCACGATGCTT;

[0085] SEQ ID NO: 5 - TTTATGAACTGCACGATGC;

[0086] SEQ ID NO: 6 - TTATGAACTGCACGATGCTTT;

[0087] SEQ ID NO: 7 - TTATGAACTGCACGATGCTT;

[0088] SEQ ID NO: 8 - TTTATGAACTGCACGATGCTTT;

[0089] SEQ ID NO: 9 - TTTATGAACTGCACGATGCT

[0090] Identification of miRW1 Precursor

[0091] We hypothesized that the putative miRW1 precursor would have weak homology to l l i since miRNAs and their targets often have sequence similarities that extend beyond the sequence of the miRNA itself (Axtell and Bowman, Trends Plant Sci. 13, 343-349 (2008); Nozawa et al, 2012 Genome Biology Evolution 4, 230-9). Therefore, using only non-waxy isolines, in which the putative miRNA precursor would be expressed but not W1, all RNAseq reads were pooled and aligned against W with low stringency (requiring that contiguous sequence comprising 20% or more of any read align to W with at least 80% homology). The mapped sequences were extracted and collected for de novo assembly. Three contigs of 150+ nt were obtained. Several of the differentially expressed small RNA sequences could be perfectly mapped to one or two of these contigs, including the most abundant miRNA sequence, indicating that one or more of the contigs was part of the miRW1 precursor. These contigs were the starting point for genome walking to obtain a complete sequence of the presumed precursor, and RACE to obtain a full-length cDNA (SEQ ID NO: 3). Finally, a full length transcript of 1051 bp and a genome region of 3207 bp were obtained. The structure prediction program in CLC Genomics software (from the Qiagen company) showed that the presumed miRNA precursor transcript could fold into a hairpin loop structure typical of miRNA precursors, with the miRW1 sequence (SEQ ID NO: 4) in the stem of the stem-loop.

[0092] Total genomic DNA was isolated from the NIL wheat pairs using the GenElute® Plant Genomic DNA Miniprep Kit (Sigma®) following the manufacturer's instructions, and used subsequently for PCR, genome- walking, cloning and DNA-seq. Leaf sheaths in 100-150 mg amounts were ground to a fine powder on liquid N2 prior to extraction. RNAs were extracted and small RNAs enriched from all samples with the mirVana™ miRNA isolation kit in combination with Plant RNA Isolation Aid according to the manufacturer's protocols which included a phenol extraction step (ThermoFisher Scientific®). Samples for RNA extraction were typically the leaf sheaths between the flag leaf and the penultimate leaf at the stage of head emergence unless otherwise noted. Contaminating DNAs were removed using the TURBO DNA-free™ Kit (ThermoFisher Scientific®) prior to RNA-seq, qPCR, cDNA RACE and full-length cDNA cloning. The quantity of DNA, RNA and small RNA were measured spectrophotometrically on a Synergy H1 plate reader with Take3 adapter (Biotek®). RNA quality was assessed using the Agilent® 2100 BioAnalyzer with the RNA 6000 Nano kit (large RNA) according to the manufacturer's instructions (Agilent Technologies®).

[0093] Cloning of the genomic region previously described as containing Iw1 (miRW1 IncRNA) was accomplished using the Universal GenomeWalker™ 2.0 kit following the manufacturer's protocol (Clontech Laboratories®). AG1 N and D051 N genomic DNA were digested with four separate restriction enzymes (EcoRV, Dral, Pvull, and Stul) and subsequently ligated to the GenomeWalker adaptors. GenomeWalker DNA was then used as a template for primary PCR amplification. Gene specific primers (GSP) were designed from contigs assembled from RNA-seq reads which loosely mapped to W1. Nested PCR was performed using outer and inner adapter primers provided in the kit and nested primers specific to the known Iw sequence (SEQ ID NO: 3). A series of PCR products, representing the genome walking steps, were cloned into the pCR™-Blunt II TOPO vector (ThermoFisher Scientific®), sequenced sequentially, and assembled to eventually obtain a 3kbp fragment.

[0094] A rapid amplification of cDNA ends (RACE) approach was used to clone the lw1/miRW1 transcript using the GeneRacer™ kit following the manufacturer's protocol (ThermoFisher Scientific®). Briefly, two to 3 of total RNA from AG1 N and AG2N leaf sheaths was dephosphorylated and de-capped prior to ligation of the GeneRacer RNA oligo to full-length mRNA. The RNA was reverse transcribed using Superscript™ III and PCR products were cloned using the Zero-Blunt™ TOPO PCR Cloning Kit. Five- and 3'-ends were obtained using nested PCR with GeneRacer 5' and 3' primers, including nested versions, together with 5' and 3' targeted GSP that were designed from the Iw1 template obtained from genome walking. Following analysis of the RACE PCR fragments, the full-length Iw1 transcript was cloned into the Gateway-cloning vector pDONR221 using BP Clonase II (ThermoFisher Scientific®) for use in subsequent analyses (see Wheat transformation). PCR amplification for genome walking, RACE, and full-length cDNA cloning was accomplished using the high-fidelity enzyme PfuUltra™ II Fusion HS DNA polymerase (Agilent Technologies).

Table 4: Primers used for PCR, Genome walking, RACE, qPCR, cloning, plasmid construction and sequencing in the studies described herein

T7S2 AACCACCACCACCGTGGAACGGAGGGAGTACATCT Cloning for VIGS

T7S3 AACCACCACCACCGTATATATTGGGGAGGCCGGT Cloning for VIGS

T2S5 AACCACCACCACCGTGCTTGCTCTCCTTCCTCCT Cloning for VIGS

BT1 S1 AAGGAAGTTTAAGCCTGCAAACAAGACTTACCCCT Cloning for VIGS

BT1 S2 AACCACCACCACCGTCCGCCATGGACGTACACAACA Cloning for VIGS

BT1 S3 AAGGAAGTTTAACCACGTCCTACGATGACACAT Cloning for VIGS

BT1 S4 AACCACCACCACCGTGCTTGCCAGATAGCAATGCGAT Cloning for VIGS

BT1 S5 AAGGAAGTTTAAGGCCGTTGCCCTGAAGGACA Cloning for VIGS

BT1 S6 AACCACCACCACCGTCGTTCCAACGTTTCATCCGCAT Cloning for VIGS

BT1 S9 AACCACCACCACCGTTCACGAACGGCCACAGCTTGT Cloning for VIGS

BT1 S7 AAGGAAGTTTAAGGCAAGCTCCTTGAAACTTGGA Cloning for VIGS

BT1 S8 AACCACCACCACCGTCACACGTGGTGAATTGTGGCGA Cloning for VIGS

BT2S1 AAGGAAGTTTAAGGAGCACCATGGTCTCTATAGTGA Cloning for VIGS

BT2S2 AACCACCACCACCGTGCATTTGCTCCACTTGAGATGGA Cloning for VIGS

BT2S3 AAGGAAGTTTAAGTTCCATGGTGTCTGCCACCA Cloning for VIGS

BT2S4 AACCACCACCACCGTGCTTGCATGGAGCTCGCCATT Cloning for VIGS

BT2S5 AAGGAAGTTTAACGTGCCCCACGGTTGTTTTCA Cloning for VIGS

BT2S6 AACCACCACCACCGTCACACGGATGAACTGCTCAACT Cloning for VIGS

BT2S7 AAGGAAGTTTAAAGTAAGGTGCTCGCTCTTGGTGA Cloning for VIGS

BT2S8 AACCACCACCACCGTTGTTCACGTTGACCCACATTTTCA Cloning for VIGS

M1 S1 AAGGAAGTTTAAGCACCAGAATATCCCATGCA Cloning for VIGS

M1 S2 AACCACCACCACCGTACTTGCACATGCTGCACAC Cloning for VIGS

M1 S3 AAGGAAGTTTAAATTCCTTGTTGGGTACCCGA Cloning for VIGS

M1 S4 AACCACCACCACCGTTGCATGGGATATTCTGGTGC Cloning for VIGS

M1 S5 AAGGAAGTTTAAGCCGGACTCGTACCCTCATT Cloning for VIGS

M1 S6 AACCACCACCACCGTAATGAGGGTACGAGTCCGGC Cloning for VIGS

BT1_F1 GCCCAAACATAATATAGATCTGACAGCTTT PCR and Cloning

BT1_R1 GTGATGCCCCAATTGCATCTTTTCTGT PCR and Cloning

BT1_F2 ATGCCTGCAAACAAGACTTACCCCTC PCR and Cloning

BT1_R2 GAAACAGTTGTTCATCATGGATCTTACCGA PCR and Cloning

BT2_F1 CAGCAACACAGTCCATAATTGTTTAGTCTG PCR and Cloning

BT2-R1 GGTTACAGATCCAATCAAAATAACATTTGGGT PCR and Cloning

BT2-F2 ATGGCAGGCAGCTCACCCAAGGTTA PCR and Cloning

BT2-R2 TTTTTTGTTGAGAGCGCCGGCTGCA PCR and Cloning

GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCTGCAAAC

attB1_T1 F1 ORF cloning

AAGACTTACCCCT

GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAAACAGTT

attB2_T1 R1 ORF cloning

CCTCATCACGAAT

GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCAGGCAGC

attB1_T2F1 ORF cloning

TCACCGAAGGTTAG

GGGGACCACTTTGTACAAGAAAGCTGGGTCTATTTTTTCTTG

attB2_T2R1 ORF cloning

AGAGCGCCGGTTG

attB1_BT1 F1 GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCCTGCAAAC ORF cloning AAGACTTACCCCT

GGGGACCACTTTGTACAAGAAAGCTGGGTTTAGAAACAGTT

attB2_BT1 R1 ORF cloning

CTTCATCATGGAT

GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCAGGCAGC

attB1_BT2F1 ORF cloning

TCACCCAAGGTTA

GGGGACCACTTTGTACAAGAAAGCTGGGTCTATTTTTTGTTG

attB2_BT2R1 ORF cloning

AGAGCGCCGGCT

M1-F1 AGCAAGAAGCCTATGTAGAGCATCCT Genome walking

M2-R4 GATGCTCTACATAGGCTTCTTGCT Genome walking

M2-F1 ACCAGCAAGAAGCCTATGTAGAGCATCCT Genome walking

M2-F3 GCAAGAAGCCTATGTAGAGCATC Genome walking

M2-R2 GATGCTCTACATAGGCTTCTTGCTGGT Genome walking

M2-R1 AGGATGCTCTACATAGGCTTCTTGCTGGT Genome walking

M2-R3 GGATGCTCTACATAGGCTTCTT Genome walking

M3-F1 CATGCGACCAGCAAGAAGCCT Genome walking

M3-R1 AGGCTTCTTGCTGGTCGCATG Genome walking

NG-M1 F1 CCGTCTACATGCAACCAGCAAGAAGCT Genome walking

NG-M1 F2 AAGCATCGTGCAGTTCATAAATTATCACGCCA Genome walking

NG-M1 F3 CAGATCAATCCTTTGGACGAGGAGATCGT Genome walking

NG-M1 F4 CCTGGCGGAGAAGGACTTGGACAGAAA Genome walking

NG-M1 F5 CGAGGGGTTGGTCATTGTGCATCCATT Genome walking

NG-M1 F6 GACACTTAGCTCGCCAATACACAGACCT Genome walking

NG-M1 R1 CAGGTTCGGCCAAGCCCTCTAGGAT Genome walking

NG-M1 R2 GTGGCGTGATAATTTATGAACTGCACGATGCT Genome walking

NG-M1 R3 ATGGATGCACAATGACCAACCCCTCGA Genome walking

NG-M2F1 CTATCCTTGTGAGACCAGTTGTGCCGGT Genome walking

NG-M2F2 CAAGAAGCCTATGTAGAGCATCCTGCGGT Genome walking

NG-M2F3 AGGTGATATCAGAGGACTCGACCGAACC Genome walking

NG-M2R1 GGTTCGGTCGAGTCCTCTGATATCACCT Genome walking

NG-M2R2 CAGGATGCTCTACATAGGCTTCTTGCT Genome walking

NG-M1 R4 GGTGTGCACAGTGTGCTACATGATGTT Genome walking

NG-M1 R5 CCGGAGGTCTGTGTATTGGCGAGCTAA Genome walking

NG-M1 R6 GATGTTGCCACCAAGGAGGGGAACGT Genome walking

NG-M1 F7 CGGACCTCTGATTCTTCTCCGGTGGCT Genome walking

NG-M1 F8 CTTCTCCGGTGGCTGGTTTGGGTATTTA Genome walking

NG-M1 F9 CACCTTTCTCTGCTTTGTTGTAAGGCGGA Genome walking

M1 F10 CGGCAATGTGATCTAGATTATTGACTCTAGT Genome walking

M1 R7 ACTAGAGTCAATAATCTAGATCACATTGCCG Genome walking

M1 R8 TAAATACCCAAACCAGCCACCGGAGAAGA Genome walking

M1 F11 CGACAAAGAAGATGTCCTGAGTTGGTCA Genome walking

M1 F12 CGGAATAAACTATCACCCGGGTAAAGCT Genome walking

M1 R9 GCAACCACATTAGCTTTACCCGGGTGAT Genome walking

M1 R10 CCGATGTCATAATCCTTGACCAACTCAGGA Genome walking 35Spl Internal

CCTGTTGTTTGGTGTTACTTC primers for sequencing F2

PZP internal F GATGTGCTGCAAGGCGATTAAG primers for sequencing

ZmUbM

ATGCAGCAGCTATATGTGGAT primers for sequencing internal R

SC350 TCTCCAGAAGAAGATGCAGGA primers for sequencing

BS10 GGTGCTTGATGCTTTGGATAAGG primers for sequencing

BS32 TGGTCTTCCCTTGGGGGAC primers for sequencing

M2F2 CAAGAAGCCTATGTAGAGCATCCTGCGGT RACE3'

M1 R1 CAGGTTCGGCCAAGCCCTCTAGGAT RACE3'

M1 R2 GTGGCGTGATAATTTATGAACTGCACGATGCT RACE3'

M2R1 GGTTCGGTCGAGTCCTCTGATATCACCT RACE 5'

M2R2 CAGGATGCTCTACATAGGCTTCTTGCT RACE 5'

RACE5_6 CAAGGATAGTACAAGGACAGAGCCCA RACE 5'

RACE5_7 AGAACGCACTCTCCCTTTAGAACTGG RACE 5'

Gpri_F1 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATCAGAGGTCC Full-length Iw1 cloning

A

GGGGACCACTTTGTACAAGAAAGCTGGGTATGACGGTAAGC

Gpri_R1 Full-length Iw1 cloning

AGGTGTGGTGGTAGTT

pri_F1 AATCAGAGGTCCGCCTTACAACAAAGCA Full-length Iw1 cloning pri_R1 TGACGGTAAGCAGGTGTGGTGGTAGTT Full-length Iw1 cloning

T1_q1 AGTAGGCCATTTAAGGACATATTTG qPCR

T1_q2 CTCATCACGAATCTTACCGAAGTT qPCR

T2_q1 GAACAAGGTGTCCCAAGAAGAGTA qPCR

T2_q2 TGCCCGTTAGACCACATATTATCT qPCR

CDC48_q3 GCTTATCTACATCCCTCTTCCTGA qPCR

CDC48_q4 GACCTCATCCACCTCATCCTC qPCR

T3_q9 ATCATGTTCATCATCCTCCAAG qPCR

T3_q10 CGAAGAAGTCCATATTTCCTCC qPCR

T4_q3 ATAGCGACCATCATCAAGAGTTTC qPCR

T4_q4 AGTTTATAGATGTAGCGGGAGGA qPCR

T5_q5 ATGGAGAGGAGGAAGATGAATAC qPCR

T5_q6 CTGGGCGAGTTTGTAGAAGAT qPCR

T6_q1 AAGTTTCTCCTTCTCCCTGTCC qPCR

T6_q2 TTGTTCCGTATGTGAATTATACCCA qPCR

T7_q3 ACTCGTTAGTCGGTCAAATG qPCR

T7_q4 CACATATTACAGGGATCGGAAGA qPCR

[0095] The R statistical computing language, running within the RStudio integrated development environment, was used for data analysis (R Core Team, 2015; RStudio Team, 2015). For some datasets, preliminary data arrangement and summary occurred in Microsoft Excel. The following R packages contributed to the analysis of data: dplyr (Wickham and Francois, 2015 https://CRAN.R-project.org/package=dplyr); stringr (Wickham, 2015 https://CRAN.R-project.org/package=stringr (2015)); tidyr (Wickham, 2016 https://CRAN.R- project.org/package=tidyr).

[0096] Confirming that W1 is involved in wax production

[0097] In order to establish whether W1 is involved in diketone wax production, virus-induced gene silencing (VIGS) was used to transiently block W1 expression in wheat. Fragments of W1 were incorporated into barley stripe mosaic virus and applied to leaves of glaucous AG2 plants at the tillering stage, before visible glaucousness was apparent. Development of glaucousness was then monitored for 4-6 weeks. W1 fragments all produced a large reduction in visible glaucousness relative to waxy controls and in total diketone wax accumulation. Control infections with a phytoene desaturase (PDS) fragment produced a slight reduction in wax content which may be attributed both to the general effects of viral infection and to the reduction in pigment accumulation due to inhibition of PDS. The levels of W1 expression in VIGS-treated plants was measured by qPCR and showed that all four of the tested fragments substantially reduced expression of the gene. The involvement of W1 in wax production led us to believe that it is the W1 gene that is also located on chromosome 2BS. A similar conclusion about the role of W1 was made by Hen-Avivi et al. 2016, Plant Cell, vol. 28 no. 6: 1440-1460".

Table 5. Suppression of W1 expression through Virus-Induced Gene Silencing results in reductions in diketone wax accumulation.

Target2 Fragmentl 904 ± 455

Target2 Fragment2 1127 ± 873

[0098] The BSMV-VIGS system developed by Yuan et al. {PLoS ONE 6, e26468. doi: 10.1371/journal.pone.0026468 (2011) comprising three T-DNA binary plasmids, pCaBS-a, pCaBS-β, and pCa- YbLIC, was used with modification. The pCa-YbLIC vector was modified by introducing ccdB gene for efficient selection of recombinant clones (Buhrow et al. Plant Methods. 2016 12:12. doi: 10.1186/s13007-016-0112-z). The resulting vector pCa-YbLIC-ccdB was used to clone a series of target gene fragments by LIC cloning strategy. The target gene fragments were PCR amplified from wax line AG2 genomic DNA using PfuUltra® II Fusion HS DNA Polymerase (Agilent Technologies®). The PCR fragments were gel purified with QIAquick® Gel Extraction buffer and MinElute column (Qiagen®). The purified PCR fragments were cloned into the pCa- vbLIC -ccdB vector by using LIC strategy. The resulting clones were verified by DNA sequencing. The plasmid DNAs of pCaBS-a, pCaBS-β, and pCa-vbLIC with target gene fragments were transformed into Agrobacterium tumefaciens strain c58. A. tumefaciens strains harboring pCa-vbLIC vector with PDS (Yuan et al. 2011, PLoS ONE 6, e26468. doi: 10.1371 /journal.pone.0026468) and GFP fragment were used as controls. Agroinfiltration into 3- to 4-week-old N. benthamiana plants and viral inoculation of wheat and barley at tillering stages were carried out as described previously (Yuan et al. 2011, PLoS ONE 6, e26468. doi: 10.1371/journal.pone.0026468).

Target! Fragmentl (T1 F1) sequence residues 754..1051 of SEQ ID NO:1 (298bp): tggataagctgtggccgttcgtgacggcgggcggggctggcaacgatgatcctcggatca atcctccggacgaggagatcgcgttgctaactggcaggcg ggtgcttgtggccgttgcagagaaggacaccctgcgcgaccgggggcgccagtttgtgtg cagcatgcgcaggtgtgggtgggttgatggcagcctcaccg tggtggagtcggagggtgaggaccatggcttccacttgtacgcccccctacgtgcgacca gcaagaagcttatgaagagcatcgtgcagttcataaa

Target! Fragment2 (T1 F2) sequence residues 754..1158 of SEQ ID NO:1 (405bp) : tggataagctgtggccgttcgtgacggcgggcggggctggcaacgatgatcctcggatca atcctccggacgaggagatcgcgttgctaactggcaggc gggtgcttgtggccgttgcagagaaggacaccctgcgcgaccgggggcgccagtttgtgt gcagcatgcgcaggtgtgggtgggttgatggcagcctca ccgtggtggagtcggagggtgaggaccatggcttccacttgtacgcccccctacgtgcga ccagcaagaagcttatgaagagcatcgtgcagttcataa accatcgcgccaccttgccgtcaccggccatggtgatcccagaaggctcggccgaaacta tgctaggcgtccctagtaggccatttaaggacatatttgg ctacggga

Target! Fragment3 (T1 F3) sequence residues 904..1158 of SEQ ID NO:1 (255bp) agtttgtgtgcagcatgcgcaggtgtgggtgggttgatggcagcctcaccgtggtggagt cggagggtgaggaccatggcttccacttgtacgccccccta cgtgcgaccagcaagaagcttatgaagagcatcgtgcagttcataaaccatcgcgccacc ttgccgtcaccggccatggtgatcccagaaggctcggc cgaaactatgctaggcgtccctagtaggccatttaaggacatatttggctacggga

Targetl Fragment4 (T1 F4) sequence residues 904..1051 of SEQ ID NO:1 (148bp) agtttgtgtgcagcatgcgcaggtgtgggtgggttgatggcagcctcaccgtggtggagt cggagggtgaggaccatggcttccacttgtacgccccccta cgtgcgaccagcaagaagcttatgaagagcatcgtgcagttcataaa

[0099] Transformation of wheat with Iw1

[00100] The full length of Iw1 cDNA (SEQ ID NO: 3) in pDONR221 was transferred into the pANIC 5E vector (Mann et al. 2012) using LR Clonase II (ThermoFisher Scientific) following the manufacturer's protocol. The pANIC 5E vector contains the ZmUbM promoter (maize ubiquitin 1 promoter and intron) to drive the expression of wheat Iw1 gene and a selectable marker bar gene which was placed under the transcriptional control of the rice actin 1 gene (OsActl) promoter.

[00101] Macrocarriers (0.6 μ gold particles) were prepared and coated with plasmid DNA according to the protocol by Jordan (2000) Plant Cell Rep 19, 1069-1075). Bread wheat (cultivar Bobwhite) immature embryos of 2-3μηι in size were isolated and arranged in the center of a petri dish of Osmotic Medium with the scutellum up. The embryos were bombarded with plasmid DNA coated gold particles at 650 psi rupture disks using the Biolistic PDS-1000/He Particle Delivering System (Bio-Rad, USA) according to manufacturer's instructions. The media and the culture of explants after bombarded and the selection of transformants were prepared and performed as described by Jordan (2000) supra with modifications.

[00102] The miRW1 lonq-noncodinq RNA is Iw1 and the D-qenome homeoloq is Iw2

[00103] Transgenic overexpression of the miRW1 precursor in wheat cultivar Bobwhite resulted in reduced glaucousness in 20 of 29 transgenic plants and a corresponding reduction in W1 gene expression (Table 6) thus confirming the predicted effect of miRW1 as a repressor of wax production by inhibition of W1 expression through the production of miRW1 (SEQ ID NO: 4) and associated small RNAs (Table 6). W1 and a homologous sequence from chromosome 2DS were the only genes with significantly changed expression

(Table 5). RNA sequencing analyses of the over-expression lines suggest that the miRW1 precursor is the wax inhibitor Iw1. However, due to the incomplete status of wheat genome assembly, the putative Iw1 sequence could not be physically located in existing genetic and physical maps of chromosome 2BS and therefore could not be shown to map close to region near the gene (Tsunewaki and Ebana, 1999, Genes Genet. Syst. 74, 33-41).

Table 6: Genes (Unigenes from build 63) significantly changed in Iw1 overexpression transgenic lines. Sequences representing W1 or W2 were the only significantly differentially-regulated unigenes in the non- glaucous TO generation of transgenic wheat over-expressing /i i For each gene/ feature ID, results are expressed as the log of the fold-change in expression (logFC) and values are all negative indicating that there is strong downregulation in non-glaucous lines. The scaffold or gene shows the sequence position reference and the E-value is a probability measure.

Table 7: The 39 /i i i-derived small RNA from the isogenic lines (Table 3) showed greater abundance in Iw transcript over-expression transgenic lines. NonWax and Wax represent the average reads in the TO plant generation (± standard error) in the glaucous and non-glaucous lines, respectively. Mapping position, 100% homology, within the Iw transcript is indicated. Small RNA which map to the W1 gene, with up to 3 mismatches, are also shown. * denotes significant difference, p < 0.05, between wax versus non-wax. SEQ ID NOs: 4-9 are in bold italics.

Average Reads per 10 W Map

Total Million Iw Map

Small RNA Sequence Length Position

Counts Position

NonWax Wax (mismatch)

TTTA TGAA CTGCA CGA TGCTT 65793 21 9147±2804 22±10 848 1031(1)

AGAGCACAGGTTCGGCCAAGC 12659 21 1736±745 5±1 783

CATGCGACCAGCAAGAAGCCT 9631 21 1342±253 2±1 636 1005(2)

AAGAAGCCTATGTAGAGCATC 4526 21 623±217 2±1 648 1017(2)

TCGACCGAACCTATGCTCGGC 3474 21 483±127 2±1 726 1098(3)

ACAAG GTG ATATCAG AG G ACT 3028 21 415±116 1±1 706

TCAGAGGACTCGACCGAACCT 2532 21 349±128 0 717

ATCAGAGGACTCGACCGAACC 2128 21 290±137 0 716 GCAGCATGTGCAAGTCGGGTG 1922 21 266±110 1 550 -

GACAAGGTGATATCAGAGGAC 1842 21 254±71 1±1 705 -

CCTCTAGGATCACCATGCCCT 1822 21 245±81 2±1 804 -

TATCAGAGGACTCGACCGAACC 1344 22 186±60 0 715 -

ACAAG GTG ATATCAG AG G AC 1315 20 180±48 0 706 -

TCGACCGAACCTATGCTCG 1162 19 162±42 0 726 1098(3)

TTCGGCCAAGCCCTCTAGGATC 1127 22 155±63 0 793 1085(3)

ACTCGACCGAACCTATGCTCG 916 21 125±59 0 724 -

TTCGGCCAAGCCCTCTAGGAT 863 21 118±41 0 793 1086(3)

AG CATGTG CAAGTCGG GTGTG 613 21 84±35 0 552 -

TTTA TGAA CTGCA CGA TGCT 570 20 79±23 0 848 1032(0)

AGAGCACAGGTTCGGCCAAG 569 20 79±27 1±1 783 -

TTG G CCG AACCTGTG CTCTG C 522 21 68±45 0 781 -

CAAGAAGCCTATGTAGAGCAT 513 21 70±22 0 647 1016(2)

AG AAG CCTATGTAG AG CATC 456 20 62±23 0 649 1018(2)

ATGCGACCAGCAAGAAGCCT 451 20 62±17 0 637 1006(2)

TTTA TGAA CTGCA CGA TGC 406 19 57±13 0 848 1033(0)

TCG ACCG AACCTATG CTCG G 360 20 51±14 0 726 1098(3)

TTTA TGAA CTGCA CGA TGCTTT 350 22 49±13 0 848 1030(1)

TTA TGAA CTGCA CGA TGCTTT 297 21 41±12 0 849 1030(1)

GACAAGGTGATATCAGAGGACT 284 22 39±12 0 705 -

G G ACAAG GTG ATATCAG AG G AC 256 22 35±12 0 704 -

TCAGAGGACTCGACCGAACC 254 20 35±13 0 717 -

AGGTGATATCAGAGGACTCGA 224 21 31±12 0 709 -

TTA TGAA CTGCA CGA TGCTT 192 20 27±8 0 849 1031(1)

ACGCAGAGCACAGGTTCGGCC 136 21 18±9 0 779 -

AGCAAGAAGCCTATGTAGAGC 136 21 18±9 0 645 1014(2)

AAG CCTATGTAG AG CATCCT 116 20 16±8 0 651 1020(3)

CCTAGAGGGCTTGGCCGAACC 61 21 8±5 0 791 -

TATCAGAGGACTCGACCGA 55 19 8±3 0 715 -

AGGTGATATCAGAGGACTCGACCGAACC 42 28 6±1 0 709 -

[00104] Mapping of the Iw location in the D genome was accomplished using the Aegilops tauschii genome sequences from Jia et al, 2013 Nature 496, 91-55 and the W7984 synthetic wheat genome sequence (Chapman et al. 2015 Genome Biology 16, 26.), and sequence resources available from EnsembI Plants (http://plants.ensembl.org/index.html; Kersey et al. 2016, Nucleic Acids Res. 44, D574-80). Marker sequences in the Iw2 region from A. tauschiiwere obtained from Nishijima et al., 2014, BMC Plant Biology 14, 246.

[00105] Since the putative Iw1 was not present in the Chinese Spring Wheat reference (IWGSC Chromosome Survey Sequences from AABBDD genome, v. 1.0), the putative Iw1 was BLASTed against two genome references which display a non-glaucous trait due to the presence of the Iw2 inhibitor. A sequence homologous to the putative Iw1 was identified on chromosome 2DS of synthetic wheat W7984 and in the sequenced genome of Aegilops tauschii (ancestor of the D-genome of hexaploid wheat; Jia et al, 2013 Nature 496, 91-55). Iw2 was previously fine mapped to contigs harbouring Iw1 homeologs in A. tauschii (Nishijima et al, 2014 BMC Plant Biology 14, 246) and contains the identical miRW1 sequence. Comparative analysis of the collinear regions of Iw1 homologs in three genomes revealed that the miRW1 precursor sequence was located close to markers associated with Iw2. Therefore, based on the dominant-negative effect of miRW1 on glaucousness and the co-localization of the D-genome miRW1 precursor sequence with Iw2 markers, we propose that the two miRW1 precursor genes on chromosomes 2BS and 2DS are Iw1 and Iw2.

[00106] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.