The present invention relates to (cultivated) plants which produce unreduced microspores and/or pollen grains, to methods for producing said plants, and to the use thereof.

Crosses between plants of different ploidy often fail because seed development does not proceed normally and non - viable seeds are produced. It is assumed that abnormalities in growth and structure of the endosperm are the cause of triploid seed failure (Westoby & Haig, 1991 ), consistent with the proposed role of the endosperm in reproductive isolation and angiosperm speciation.

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed failure, a phenomenon that has been termed "triploid block" (Marks, 1966). Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions (Westoby & Haig, 1991 ).

In plants, sexual polyploidization by the formation of unreduced (2n) gametes is an important feature in both nature and plant breeding programs. For example, unreduced gametes have revolutionized strategies to improve important polysomic polyploid crops such as potato and alfalfa (Ortiz et al., 1997; Carputo et al., 2003; Capo et al., 2002; Motzo et al., 1994), as unreduced pollen can be used to overcome incompatibility barriers in interspecific or interploidy crosses. However, mutants producing unreduced pollen are only available in few crop species or varieties. Thus far, production of polyploid plants is mainly achieved by colchizine treatment, a laborious, time-consuming and inefficient technique, with less than 5% tetraploid plants recovered in treated populations (Lower and Johnson, 1969).

There is therefore an unmet need to provide alternative methods for producing polysomic polyploid crop plants.

The technical problem underlying the present invention is the provision of an alternative and more efficient method for producing unreduced gametes in plants in order to facilitate sexual polyploidization by maintaining parental heterozygosity.

The technical problem is solved by the provision of the embodiments characterized in the claims. In particular, it was surprisingly found within the scope of the present invention that a mutation in the Jason (JAS) gene leading to expression of a non-functional JASON protein or a protein with substantially reduced biological activity, results in the production of unreduced microspores and pollen at high frequencies. It was shown that in plants containing the mutated JASON (JAS) gene no chromosome separation occurs during meiosis I, leading to unreduced male gametes that have similar genetic composition as the parents.

The present invention thus relates in a first embodiment to a plant which produces unreduced microspores and/or pollen grains, optionally with preserved heterozygosity, comprising in its genome a JAS gene which is inactive or substantially inactive and/or expresses a protein product which is non-functional or substantially non-functional.

In a one embodiment, said JAS gene contains a mutation, which causes the JyAS gene to become inactive or substantially inactive and/or to express a protein product which is non-functional or substantially non-functional.

In a further embodiment, a plant according to any of the preceding embodiments is provided herein, which plant is homozygous for the JAS mutation.

In still another embodiment, the present invention further relates to a plant according to any of the preceding embodiments, which produces diploid and triploid, but no tetraploid progeny.

In a specific embodiment, the present invention provides a plant comprising the mutated JAS-1, JAS-2 or JAS-3 allele, wherein

(a) the mutated JAS-1 allele can be identified in the plant genome in a PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2: (b) the mutated JAS-2 allele can be identified in the plant genome in a

PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; and (c) the mutated JAS-3 allele which can be identified in the plant genome in a PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6.

In another specific embodiment of the invention, the PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 produces an amplicon of about 471 bp.

In still another specific embodiment of the invention, the PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4 produces an amplicon of about 1162 bp.

In one embodiment, the present invention relates to an orthologous gene, which is inactive or substantially inactive and/or expresses a protein product which is non-functional or substantially non-functional, for example, caused by a mutation in said orthologous gene.

In one embodiment, the present invention also provides seed of a plant according to any of the preceding embodiments, which is a homozygous plant and/or which seed grows a plant comprising in its genome a JAS gene which is inactive and/or expresses a protein product which is non-functional or substantially non-functional.

In a specific embodiment, the plant according to the invention and as described herein is a hybrid plant.

In a specific embodiment, the plant according to the invention and as described herein is a cultivated plant.

Furthermore, in one embodiment, the present invention relates to plant parts of a plant according to any of the preceding embodiments, particularly to plant parts comprising an unreduced microspore and/or pollen grain comprising in its genome a JAS gene, particularly a mutated JAS gene, which is inactive or substantially inactive and/or expresses a protein product which is nonfunctional or substantially non-functional.

In a specific embodiment, the invention relates to the unreduced microspore and/or pollen grain as such comprising in its genome a JyAS gene, particularly a mutated JAS gene, which is inactive or substantially inactive and/or expresses a protein product which is non-functional or substantially non-functional.

In one embodiment, a microspore and/or pollen grain is provided herein, which comprises the mutated JAS-1, JAS-2 or JAS-3 allele, which can be identified in the plant genome in a PCR reaction using a pair of primers comprising (a) a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; (b) a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; or (c) a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6.

The present invention further provides a method for producing a plant comprising unreduced microspores and/or pollen grains, particularly a plant with preserved heterozygosity, comprising in its genome a JAS gene which is inactive and/or expresses a protein product which is non-functional or substantially non-functional comprising (a) inactivating the JAS gene or close homologs, orthologs or paralogs of the JAS gene in the target plant such that the gene becomes inactive and/or expresses a protein product which is nonfunctional or substantially non-functional; particularly by mutagenizing the JAS gene in the target plant or reducing the transcript levels in the target plant; (b) identifying a plant comprising an inactive JAS allele, particularly in a PCR reaction; and (c) selecting and isolating said plant, particularly a plant comprising a mutated JAS allele.

In another embodiment, mutagenesis may be accomplished by TILLING and reduction in the transcript levels by gene silencing, particularly virus induced gene silencing or by RNA interference.

The present invention also relates to a plant or a method according to any of the preceding embodiments, wherein the plant is selected from a group consisting of monocots and dicots, including maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, sorghum, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.

In one embodiment, the present invention relates to a method for introducing at least one JAS allele, which is inactive and/or causes the expression of a protein product, which is non-functional or substantially non-functional, into a plant lacking said allele by (a) obtaining a first plant comprising in its genome at least one JAS allele, which is inactive and/or causes the expression of a protein product which is non-functional or substantially non-functional; (b) crossing said first plant with a second plant, wherein said second plant lacks said allele; and (c) identifying a plant resulting from the cross, which comprises said JAS allele and produces unreduced microspores and/or pollen grains with preserved heterozygosity; and (d) optionally, isolating said plant and (e) optionally, back-crossing said plant with the first or second plant.

The present invention further relates to a plant obtainable by any of the methods described herein.

In another embodiment, the present invention relates to the use of a microspore and/or a pollen grain according to the invention and as described herein for

(a) overcoming incompatibility barriers in interspecific or interploidy crosses; (b) the production of seedless fruit, particularly seedless fruit selected from the group consisting of watermelons, cucumber, grapes, bananas, citrus fruits, such as oranges, lemons and limes; (c) generation of stable tetraploid plants with preserved parental heterozygosity. (d) pollinating a plant comprising a mutation suppressing recombination, preferably a mutation in spoil.

In another embodiment, the present invention relates to an oligonucleotide primer which has at least 80%, particularly at least 85%, particularly at least 90%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity with a primer selected from the group of primers of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In a further embodiment, the present invention relates to an oligonucleotide primer selected from the group of primers of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In another embodiment, the present invention relates to an oligonucleotide primer, which has a nucleotide sequence which hybridizes with the primer selected from the group of primers of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In a further embodiment a polynucleotide is contemplated within the present invention comprising a nucleotide sequence which has at least 80%, particularly at least 85%, particularly at least 90%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity, but especially 100% sequence identity, with a polynucleotide obtainable in a PCR reaction by amplification of a DNA fragment from the genome of a plant according to the invention and as described herein, with a pair of PCR oligonucleotide primers comprising a forward primer of SEQ ID NO: 1 (ae481 ) and a reverse primer of SEQ ID NO: 2 (ae482), which nucleotide has a length of about 471 bp, or a pair of PCR oligonucleotide primers comprising a forward primer of SEQ ID NO: 3 (ae487) and a reverse primer of SEQ ID NO: 4 (ae488), which nucleotide has a length of about 1162 bp.

The present invention also relates to a polynucleotide, which has a nucleotide sequence which hybridizes with any of the above polynucleotides.

The present invention further relates to a kit for the detection of a plant comprising a mutated JAS allele, wherein said kit comprises at least one PCR oligonucleotide primer or a pair of PCR oligonucleotide primers of any of the preceding embodiments. The present invention further relates to the use of an oligonucleotide primer or of a kit according to the invention and as described herein, for identifying in a plant the presence of a mutated JAS allele or for monitoring the introgression of said mutated JAS allele into a plant lacking said allele.

Definitions

The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant breeding and cultivation if not otherwise indicated herein below.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes one or more plants, and reference to "a cell" includes mixtures of cells, tissues, and the like.

A "cultivated plant" is understood within the scope of the invention to refer to a plant that is no longer in the natural state but has been developed by human care and for human use and/or consumption.

The "JAS gene" is understood within the scope of the invention to refer to the At1g06660 gene and to homologs, orthologs and paralogs thereof.

A "homolog" is understood within the scope of the invention to refer to a gene related to a second gene by descent from a common ancestral DNA sequence. The term, homolog, may apply to the relationship between genes separated by the event of speciation (see ortholog) or to the relationship between genes separated by the event of genetic duplication (see paralog). An "ortholog" is understood within the scope of the invention to refer to a gene in a different species that has evolved from a common ancestral gene by speciation and has retained the same function in the course of evolution.

An "paralog" is understood within the scope of the invention to refer to a gene related by duplication within a genome. Paralogs may evolve new functions in the course of evolution, even if these are related to the original one.

By a "gene which is inactive or substantially inactive" a gene is understood within the scope of the invention, which is no longer capable of performing its envisaged function within the plant genome that is causing or controlling the expression of a functional protein product.

By a "a protein product which is non-functional or substantially non-functional" a protein is understood within the scope of the invention, which is no longer capable of performing its envisaged biological function within the plant.

By "polyploidy" is understood herein, the occurrence of related forms possessing chromosome numbers which are multiples of a basic number (n), the haploid number. Forms having 3n chromosomes are triploids; Λn, tetraploids; 5n, pentaploids, and so on.

An "allele" is understood within the scope of the invention to refer to alternative or variant forms of various genetic units identical or associated with different forms of a gene or of any kind of identifiable genetic element, which are alternative in inheritance because they are situated at the same locus in homologous chromosomes. Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation. In a diploid cell or organism, the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.

As used herein, the term "marker allele" refers to an alternative or variant form of a genetic unit as defined herein above, when used as a marker to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits.

As used herein, the phrase "diploid individual" refers to an individual that has two sets of chromosomes, typically one from each of its two parents. However, it is understood that in some embodiments a diploid individual can receive its "maternal" and "paternal" sets of chromosomes from the same single organism, such as when a plant is selfed to produce a subsequent generation of plants.

"Homozygous" or "Homozygosity" is understood within the scope of the invention and refers to a plant which is homozygous for a particular gene when identical alleles of the gene are present on both homologs.

"Heterozygous" or "Heterozygosity" is understood within the scope of the invention and refers to a plant which is heterozygous for a particular gene when two different alleles occupy the gene's position on the homologous chromosomes

As used herein, the term "progeny" refers to the descendant(s) of a particular cross. Typically, progeny result from breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) can be selfed (i.e., the same plant acts as the donor of both male and female gametes). The descendant(s) can be, for example, of the F-i, the F ₂ , or any subsequent generation.

As used herein, the terms "hybrid", "hybrid plant," and "hybrid progeny" refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).

"Backcrossing" is understood within the scope of the invention to refer to a process in which a hybrid progeny is repeatedly crossed back to one of the parents. Different recurrent parents may be used in subsequent backcrosses.

As used herein, the phrase "nucleic acid" refers to any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA, cDNA or RNA polymer), modified oligonucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. In some embodiments, a nucleic acid can be single-stranded, double- stranded, multi-stranded, or combinations thereof. Unless otherwise indicated, a particular nucleic acid sequence of the presently disclosed subject matter optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.

"PCR (Polymerase chain reaction)" is understood within the scope of the invention to refer to a method of producing relatively large amounts of specific regions of DNA or subset(s) of the genome, thereby making possible various analyses that are based on those regions.

"PCR primer" is understood within the scope of the invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of specific regions of DNA.

"Probe" as used herein refers to a group of atoms or molecules which is capable of recognising and binding to a specific target molecule or cellular structure and thus allowing detection of the target molecule or structure.

Particularly, "probe" refers to a labeled DNA or RNA sequence which can be used to detect the presence of and to quantitate a complementary sequence by molecular hybridization.

The term "hybridize" as used herein refers to conventional hybridization conditions, preferably to hybridization conditions at which 5xSSPE, 1 % SDS, ixDenhardts solution is used as a solution and/or hybridization temperatures are between 35 ^C C and 70 ⁰ C, preferably 65°C. After hybridization, washing is preferably carried out first with 2xSSC, 1 % SDS and subsequently with 0.2xSSC at temperatures between 35°C and 75°C, particularly between 45°C and 65°C, but especially at 59°C (regarding the definition of SSPE, SSC and Denhardts solution see Sambrook et al. loc. cit.). High stringency hybridization conditions as for instance described in Sambrook et al, supra, are particularly preferred. Particularly preferred stringent hybridization conditions are for instance present if hybridization and washing occur at 65°C as indicated above. Non-stringent hybridization conditions for instance with hybridization and washing carried out at 45°C are less preferred and at 35°C even less.

"Sequence homology" or "sequence identity" is used herein interchangeably. The terms "identical" or percent "identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wl 53711 ). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981 ), 482-489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. The deviations appearing in the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program "fasta20u66" (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson (1990), Methods in Enzymology 183, 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the "default" parameter settings may be used.

Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase: "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but to no other sequences.

The thermal melting point is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCI at 72°C. for about 15 minutes. An example of stringent wash conditions is a 0.2 times SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 times SSC at 45°C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6 times SSC at 4O ⁰ C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 times (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g. when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

A "plant" is any plant at any stage of development, particularly a seed plant.

A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

"Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

"Plant material" or "plant material obtainable from a plant" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

"Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

The present invention is based on the surprising observation that a mutation in the JASON (JAS) gene leading to expression of a non-functional JASON protein or a protein with substantially reduced biological activity, results in the production of unreduced microspores and pollen at high frequencies. It was further surprisingly shown that the JAS mutation does not cause any observable defects during meiosis II, but that chromosomes fail to separate properly during meiosis I, causing the formation of unreduced dyads. The consecutive mitotic divisions are not affected by the mutation and all pollen grains formed by plants incorporating the JyAS mutation contained two sperm cells and one vegetative cell.

The unreduced "JAS pollen" was shown to be fertile and to cause the formation of enlarged seeds at intermediate frequencies of between about 20% and 40%, particularly at a frequency of about 36%.

Examination of the progeny of diploid homozygous plants containing in their genome a JAS gene which is inactive or substantially inactive and/or expresses a protein product which is non-functional or substantially non-functional, revealed that only diploid and triploid, but no tetraploid progeny is produced by said plants, which is an indication that the JAS mutation only affects male meiosis.

A mutation in the JyAS gene such as to obtain a mutant JAS gene which is inactive or substantially inactive and/or expresses a protein product which is non-functional or substantially non-functional, may be accomplished by mutagenesis.

Any mutagenesis technique can be used to obtain cultivars according to the invention including, but not being limited to, chemical treatment, irradiation, or DNA insertion mutagenesis using, for example T-DNA or transposon inserts from the host plant or from a heterologous origin, using techniques well known to the skilled artisan in this field.

Chemical treatment for production of mutants of a plant cultivar can be carried out by known techniques with mutagens such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), methyl-N-nitrosourea (MNU), bleomycin, and the like. Mutations can also be effected by known techniques through irradiation with UV-irradiation, X-rays and fast neutrons (See, for example, Poehlman, 1987 or Malmbery, 1993).

In particular, chemical mutagenesis may be accomplished by using the TILLING technology.

TILLING (Targeting Induced Local Lesions in Genomes) is a method in molecular biology that allows directed identification of mutations in a specific gene. The method combines a standard technique, mutagenesis with a chemical mutagen such as Ethyl methanesulfonate (EMS), with a sensitive DNA screening-technique that identifies single base mutations (also called point mutations) in a target gene (McCallum (2000), Nat Biotechnology 18(4), 455-7). The method was made more applicable to high throughput techniques by using the restriction enzyme CeI-I combined with a gel based system to identify mutations (Colbert (2001 ), Plant Physiology 126(2), 480-4). Other methods of mutation detection, such as resequencing DNA, have been combined for TILLING and can also be used within the scope of the present invention. TILLING was introduced in 2000, using the model plant Arabidopsis. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, corn, wheat, rice, soybean, tomato and lettuce.

In the present invention, the mutations in the JAS gene may be randomly created by using the TILLING technology. The precise location of the mutation can then be confirmed by sequencing of the genome region containing the mutated gene, which is a process known to the person skilled in the art.

Alternatively, inactivation of desired genes as herein described can be, inter alia, accomplished by random mutagenesis in the plant by insertion of a mobile DNA sequence such as a transposable element or T-DNA into the plant genome.

T-DNA mutagenesis may be carried out by known methods via Agrobacterium (Hoekema et al., 1983; US Patent No. 5,149,645). Transposon insertion mutagenesis may be done by well-known methods (Fedoroff et al., 1984; US Patents No. 4,732,856 and No. 5,013,658). The transposable element may be an autonomous transposon, a non-autonomous transposon, or an autonomously non-autonomous transposon system. Large populations of plants, preferably at least thousands of plants, are screened for mutants. Identification of mutants can be done visually, or alternatively, by using known techniques used in plant biology.

For example, identifying a plant which carries a transposon inserted in the target JAS gene may be carried out by screening pools of transposon-carrying plants by PCR, using one primer having a nucleotide sequence corresponding to the target gene and a second primer corresponding to the transposon.

The activity of the JAS gene may also be regulated by transcriptional or post- transcriptional gene silencing techniques including for example, without however being limited thereto, virus induced gene silencing, an anti-sense approach or RNA interference. These techniques are well known to those skilled in the art and described, for example, in Burch-Smith (2006), Plant Physiology 142(1 ), 21-7; Wesley (2001 ), Plant Journal 27(6):581-90.

Virus-induced gene silencing (VIGS) is a known method which can be used to study the function of a gene by downregulating its expression and analyzing the resulting phenotype. The success of transient knockdown of JAS expression by Virus-induced gene silencing (VIGS) or stable knockdown by RNAi can be scored by determining the remaining JAS RNA levels by PCR.

In a specific embodiment of the invention, a JAS mutant plant is provided comprising a mutated JAS allele stably incorporated in its genome such that a non-functional or a substantially non-functional gene product is produced in the mutant plant. Such a mutant JAS allel may be induced in a plant by chemical mutagenesis using, for example ethylmethanesulfonate.

In a specific embodiment, the JAS mutant causes an increase in the transcript levels of the PHERES1 (PHE1) gene which is established by increased expression of the paternal PHE1 allele.

Jas mutant lines may, therefore, be identified in a genetic screen for increased expression levels of the PHE1 gene, which may be accomplished by providing to the suspected mutant plant line a PHE: reporter gene fusion construct such as, for example, a PHEv. GUS fusion gene construct and determining reporter gene expression in said plant line.

In an alternative approach for identifying a JAS mutant line, a PCR-based technique may be used. For example, a JAS mutant line comprising the mutated JAS-1, JAS-2 or JAS-3 allele, may be identified in a PCR reaction, wherein (a) the mutated JAS-1 allele can be identified in the plant genome in a PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2:

(b) the mutated JAS-2 allele can be identified in the plant genome in a PCR reaction using a pair of primers comprising a forward primer of

SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; and

(c) the mutated JAS-3 allele can be identified in the plant genome in a PCR reaction using a pair of primers comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6. Basically, the method of PCR amplification involves use of a primer or a pair of primers comprising two short oligonucleotide primer sequences flanking the DNA segment to be amplified or adapter sequences ligated to said DNA segment. Repeated cycles of heating and denaturation of the DNA are followed by annealing of the primers to their complementary sequences at low temperatures, and extension of the annealed primers with DNA polymerase. The primers hybridize to opposite strands of the DNA target sequences. Hybridization refers to annealing of complementary DNA strands, where complementary refers to the sequence of the nucleotides such that the nucleotides of one strand can bind with the nucleotides on the opposite strand to form double stranded structures. The primers are oriented so that DNA synthesis by the polymerase proceeds bidirectionally across the nucleotide sequence between the primers. This procedure effectively doubles the amount of that DNA segment in one cycle. Because the PCR products are complementary to, and capable of binding to, the primers, each successive cycle doubles the amount of DNA synthesized in the previous cycle. The result of this procedure is exponential accumulation of a specific target fragment that is approximately 2<n>, where n is the number of cycles.

Through PCR amplification millions of copies of the DNA segment flanked by the primers are made. Differences in the number of repeated sequences or insertions or deletions in the region flanking said repeats, which are located between the flanking primers in different alleles are reflected in length variations of the amplified DNA fragments. These variations can be detected, for example, by electrophoretically separating the amplified DNA fragments on gels or by using capillary sequencer. By analyzing the gel or profile, it can be determined whether the plant contains the desired allele in a homozygous or heterozygous state or whether the desired or undesired allele is absent from the plant genome.

In the alternative, the presence or absence of the desired allele may be determined by real-time PCR using double-stranded DNA dyes or the fluorescent reporter probe method.

Marker analysis can be done early in plant development using DNA samples extracted from leaf tissue of very young plants or from seed. This allows to identify plants with a desirable genetic make-up early in the breeding cycle and to discard plants that do not contain the desired, invention-relevant alleles prior to pollination thus reducing the size of the breeding population and reducing the requirements of phenotyping.

Further, by using molecular markers, a distinction can be made between homozygous plants that carry two copies of the desired, invention-relevant JAS allele and heterozygous plants that carry only one copy and plants that do not contain any copy of the favourable allele(s).

Thus, alternative markers can therefore be developed by methods known to the skilled person and used to identify and select plants with an allele or a set of alleles of a qualitative trait locus or loci according to the present invention and as disclosed herein before.

Accordingly, the markers specifically disclosed in the present invention may also be used in the identification and/or development of new or additional markers associated with the JAS gene, which in turn can then be used in marker assisted breeding and/or the search of recombinants bearing an inactive or substantially inactive JAS gene, particularly a mutated JAS gene such as, for example, mutated JAS-1, JAS-2 and/or JAS-3 alleles.

The present invention is further described by reference to the following non- limiting examples and figures.

The figures mentioned below show:

Figure 1 shows homozygous JAS plants producing significantly bigger seeds, (a) Silique from wild-type and JAS plants after self-fertilization. Triploid JAS seeds are enlarged (white asterisks) and sometimes abort (dark brown seeds). Lower panels show ripe seeds. Scale bars, 0.5 mm upper panels, 1 mm lower panels, (b) Seed weight of wild-type and JAS plants. Average seed weight is given in mg per 50 seeds. Significance was determined by two-tailed Student's t-test, ^* P < 0.01. Numbers above bars indicate numbers of scored seeds per genotype. Error bars, s.e.m.

Figure 2 shows progeny of homozygous JAS plants which are triploid. Representative flow cytometry histogram plots of nuclei from wild-type, tetraploid (4n) and JAS mutant seedlings.

Figure 3 shows homozygous JAS plants forming dyads and enlarged diploid pollen, (a) Quantification of meiotic products in Ler wild-type (n-69) and JAS - 1 (n=307) and JAS - 3 (n=167).Tet; tetrads, tri; triads, dy; dyads, (b) Tetrad formation in wild-type plants. Scale bar, 25 μm. (c) Dyad formation in JAS plants. Scale bar, 25 μm. (d) Pollen from JyAS plants was mixed with wildtype pollen marked by the sperm cell marker MGH3::H2B - GFP. GFP negative enlarged pollen grain is derived from JAS plants. Scale bar, 10μm. (e) DAPI staining of pollen shown in panel (d). Enlarged JAS pollen contains two sperm nuclei (sn) and one vegetative nucleus (vn). Scale bar, 10μm. (f) DNA content of sperm cells in mature pollen from wild type and JAS plants. Bars show mean relative DNA contents (DAPI fluorescence values) for pollen from wild type (WT) or JAS plants, with JAS pollen classified on the basis of size; normal sized (JAS) or enlarged (JAS enlarged). The mean fluorescence from the enlarged pollen was approximately twice that of the wild type and normal sized JyAS pollen, indicating that it has twice the DNA content, wt; wild type.

Figure4 shows that diploid jas gametes are heterozygous at centromeres. Diploid and triploid offspring derived from the pollination of the No-O accession with pollen from plants containing both the jas-1 (Ler) and jas-3 (CoI) alleles and displaying the jas phenotype were genotyped with trimorphic markers. For each marker plants containing only the Ler alleles are in red, plants containing only the CoI alleles are in blue and plants containing both the Ler and CoI alleles are in yellow. The No-O alleles are not shown as they are present in every plant. The position of markers (grey lines) and the centromere (blue oval) are indicated on the chromosome (red bars). In triploid plants markers are segregating away from the centromere and are heterozygous close the centromeres.

Figure 5 shows structure of JAS gene and location of mutations, (a) Protein sequence of JAS with site of JAS - 1 mutation indicated in red. (b) Exon-intron structure of JAS locus, with exons marked by black boxes, introns by black lines. Positions of JAS alleles are indicated.

Figure 6 shows PHE1 ::GUS and the endogenous paternal PHE1 allele are overexpressed in seeds of JAS plants. (a,c,e,g) PHE1 ::GUS expression in wild- type seeds, (b,d,f,h) PHE1 ::GUS expression in JAS seeds. Seeds were analyzed at 4 (a,b), 5 (c,d), 6 (e,f) and 10 DAP (g,h). Scale bars, 100 μm. (i) Quantitative RT-PCR analysis of PHE1 expression in wild-type and JAS seeds. Error bars, s.e.m. (k) PHE1 imprinting is not affected in JAS seeds. Allele specific PHE1 transcript levels were determined after crosses of C24 with wild type (Ler) or JAS plants. DAP; days after pollination.

wt; wild type. EXAMPLES

EXAMPLE 1 : General Methodology

1.1 Plant material and growth conditions

Plants were grown in a growth room at 70% humidity and daily cycles of 16 h light at 21 ⁰ C and 8h darkness at 18°C.

1.2 GUS expression analysis and phenotypic characterization of seeds and pollen

Siliques were harvested for GUS staining at the indicated time points (Fig. 6 a- h). Staining of seeds to detect GUS activity was done as described in Kόhler (2003), Genes and Development 17(12):1540-53. Mature pollen nuclei were visualized after staining with 4',6-diamidino-2 phenylindole (DAPI) as described in Park (1998), Development 125(19):3789-99. Buds were harvested for microscopic analysis of tetrad formation and fixed overnight in 3:1 ethanol:acetic acid for about 24 h. Buds were then separated and anthers dissected to release pollen into clearing solution (67% chloralhydrate in 8% glycerol) or into DAPI staining solution (100 mM sodium phosphate [pH 7.0], 1 mM EDTA, 0.1 % Triton X-100, and 0.4 mg/ml DAPI, high grade; Sigma). Microscopy imaging was performed using a Leica DM 2500 microscope (Leica) with either bright-field or epifluorescence optics. Images were captured using a Leica DFC300 FX digital camera (Leica), exported using Leica Application Suite Version 2.4.0. R1 (Leica Microsystems), and processed using Photoshop 7.0 (Adobe). For chromosome spreads, inflorescences were harvested and fixed in 3:1 (ethanol:acetic acid) at -20 ⁰ C overnight. Flower buds (0.3 - 0.8 mm) were fixed, equilibrated in citric buffer (1OmM sodium citrate, pH 4.8) and incubated with 1 % cytohelicase, 1% pectolyase and 1% cellulase in citric buffer for 3-4 hours at 37°C. Squashes made in 45% acetic acid were air-dried and mounted in antifade containing 4',6-diamidino-2-phenylindole (DAPI). Slides were analyzed with a Zeiss Axioscope fluorescence microscope (Zeiss, Germany) equipped with a cooled CCD camera (Visitron, Germany). Images were acquired using MetaView software (Universal Imaging Corporation, USA).

1^3 Flow cytometry

The ploidy levels of leaf cell nuclei were determined by flow cytometry using a PA ploidy analyzer (Partec). Leaves were chopped with a razor blade in CyStain extraction buffer (Partec), filtered through a 30-μm CellTrics filter (Partec) into a sample tube, and stained with CyStain Staining buffer (Partec). Data were collected for approximately 10,000 nuclei per run and were presented on a linear scale.

1.4 Genetic screen and mapping

The PHE1: GUS line, mutagenized by ethyl methanesulfonate (EMS), was screened for mutants by selecting M2 plants that showed GUS activity during late stages of seed development. For genetic mapping of the JAS mutation, an F2 mapping population was established by crossing JAS with the CoI-O accession. Analyzing 280 JAS F2 plants using PCR-based polymorphisms, the mutation was located on chromosome 1 in an area of 570 kb between polymorphisms SM104_106,6 and PA11.2 on BACs T21 E18 and F24B9, respectively. Open reading frames within this region were PCR-amplified and analyzed using the SURVEYOR® Mutation Detection Kit (Transgenomic). A polymorphism was detected in At1g06660 (JAS) and confirmed by sequencing.

EXAMPLE 2: Results

2.1 Induction and Detection of the JAS - 1 allele

The JAS - 1 allele was induced in the Landsberg erecta (Ler) accession by ethylmethanesulfonate mutagenesis and shown to cause an increase in the transcript levels of the PHERS1 (PHE1) gene which were established by increased expression of the paternal PHE1 allele.

Accordingly, JASon (JAS - 1) mutant was identified in a genetic screen for increased expression levels of a β - GLUCURONIDASE (GUS) reporter fused to the FERTILIZATION INDEPENDENT SEED (FIS) PcG target gene

PHERES1 (PHEI) (Kόhler et al., 2003). The JAS mutant had strongly increased GUS staining in the endosperm starting at 5 to 6 days after pollination (DAP), when embryos had reached late heart stage (Fig. 6a - h). Increased expression levels of the reporter gene were reflected by increased expression of the endogenous PHE1 gene (Fig. 6i).

2.2 Characterization of the JyAS - 1 allele

The JAS - 1 allele was shown to harbor a premature stop codon in the fifth exon at amino acid position 294 (C-to-T nucleotide substitution at position +1960 of the genomic sequence) of the At1g06660 gene, encoding an as yet unknown plant specific protein without described functional domains (Fig. 5). Additional alleles in the Columbia (CoI) accession were found in T-DNA insertion libraries: JAS - 2 (SALK_083575) and JAS - 3 (SAIL_813_H03) harbor insertions in intron 1 and exon 5, respectively. These two independent T-DNA insertion lines confirmed that the identified mutation is indeed the cause of the JiAS phenotype (Fig. 3).

2.3 Characterization of the JAS mutant

Development of JAS seeds is delayed compared to wild-type seeds (Fig. 6 g, h), but seed size is significantly increased (Fig. 1 ), resembling seeds derived from 2n x 4n crosses. Ploidy levels of the progeny of diploid homozygous JAS plants were measured and diploid and triploid (45%) but no tetraploid seedlings were found among the JAS progeny (10 triploids among 22 plants; Fig. 2). Karyotyping confirmed the presence of triploid seedlings. Enlarged seed formation was only observed when the JAS mutation was paternally transmitted (Table 1 ), suggesting that JAS pollen is diploid.

Table 1. Seed phenotype of reciprocal crosses between JAS and wild-type plants. Green or dry siliques resulting from self pollination or out-crosses to wild-type Ler plants were opened and the seeds classified as normal, enlarged, aborted, or unfertilized ovules, n (seeds), number of seeds scored.

Female Ler JAS JAS Ler

(selfed) (selfed)

X X

Male Ler JAS

Unfertilize 9% 11% 4% 10% d

Normal 91% 51% 95% 42%

Enlarged 0% 36% 0% 45%

Aborted 0% 2% 1 % 3%

n (seeds) 406 758 918 737

Consistent with this is the observation of 62 % enlarged pollen (n=144; Fig.

3c,d) and abnormalities after meiosis of microspore mother cells, with JAS plants forming dyads and triads at high frequency (64% dyads, 19% triads, n =307; Fig. 3a - c). Analysis of chromosome behaviour during meiosis in JAS mutants revealed no obvious defects during meiosis I, however, after metaphase Il chromatid separation was impaired, leading to the formation of unreduced dyads (Fig. 4). The subsequent mitotic divisions are not affected by the JAS mutation; all pollen grains formed by JAS mutant plants contained two sperm cells and one vegetative cell (Fig. 3e).

DNA content of sperm cells from wild type and enlarged JAS pollen was determined and indeed, the mean fluorescence from the enlarged pollen was approximately twice that of the wild-type and normal sized JAS pollen, indicating that it has twice the DNA content (Fig. 3f).

EXAMPLE 3: Potential applications for the generation of unreduced pollen

Generation of unreduced pollen gametes has several potential applications.

First, introducing the JAS mutation into crop species will greatly facilitate the formation of polyploid species, as the progeny of JAS plants will form unreduced gametes at high frequency. The JAS mutation could be introduced into different crop species by TILLING (Slade and Knauf, 2005) or virus induced gene silencing (Carrillo-Trip et al., 2006).

Secondly, the JAS mutation could also be applied to facilitate the production of seedless fruits. Seedless watermelons are the most prominent example where crosses between plants of different ploidy are currently used to produce seedless fruits. Thus, a cross is made between a tetraploid maternal parent and a diploid pollinator, resulting in a triploid plant that is self-infertile because of a gametic-chromosome imbalance. This triploid plant must be pollinated by a diploid plant in order to produce a seedless watermelon (Varoquoux et al., 2000). Seedless fruits have a growing popularity; however, producing seedless fruits is expensive and highly time consumptive. One obstacle is the production of tetraploid plants, which so far, is mainly done using colchizine. As stated above, this procedure is time consumptive and has low efficiency. This problem can be overcome by generating 2n gametes using the JAS mutation introduced into different crop species, either by TILLING or virus induced gene silencing.

Thirdly, the JAS mutation could be applied to generate stable tetraploid plants with preserved parental heterozygosity at high frequencies. Triploid jas mutant plants generate triploid pollen that can be used to pollinate wild-type plants, leading to the formation of tetraploid progeny. Thus far, tetraploids are predominantly generated by colchizine treatment of anther and microspore cultures, which is labor-intensive and of low-efficiency.

Fourthly, by combining the JAS mutation with mutations suppressing recombination, diploid pollen could be produced that is genetically identical to the parental plant. This could be achieved using the spoi l mutant that is defective in recombination (Grelon et al., 2001 ).

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