WO2010071431A1 - Method of breeding cysdv-resistant cucumber plants - Google Patents

Abstract

The present invention relates to a method of testing a nucleic acid sample for the presence of at least one nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3, comprising the step of determining the presence in said sample of a nucleic acid comprising a nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3.

Description

Title: Method of breeding CYSDV-resistant cucumber plants

FIELD OF THE INVENTION

The present invention is in the field of plant markers, in particular markers for use in plant breeding. The present invention provides methods of breeding CYSDV-resistant cucumber plants using marker- assisted selection. The invention further provides a nucleic acid detection method for detecting the presence of markers linked to resistance to CYSDV in the genome of a cucumber plant, a method for selecting a cucumber plant based on the presence or absence of said markers, and a method for producing a cucumber plant line using the said methods. The invention further provides novel markers for the detection of resistance to CYSDV in cucumber plants.

BACKGROUND OF THE INVENTION

Marker-assisted breeding provides a technological improvement to conventional breeding by allowing for the monitoring of the introgression of specific genetic material through backcrossing from a donor parent into the genetic background or genome of an elite variety. This allows for a significant acceleration of the breeding process. The technology uses genetic markers linked to a specific trait to be introgressed. Genetic markers are heritable and readily detectable polymorphic genetic properties linked to a genetic interval of interest whose precise boundary is unknown or a gene whose identity is unknown. The markers can be used to identify alleles at a locus on a particular chromosome to which they are linked. The allele will usually have a phenotypic effect that is more complex to detect than the marker.

Traditional plant breeding methods are based on mere cross- fertilization and selecting from the offspring a plant having desired trait combinations. During the crossing process, thousands of genes are mixed and many backcrossings to an elite line are required to remove the unwanted traits. This takes years. Marker-assisted breeding methods are more precise, more predictable and faster. By controlling the introgression or insertion of a well- defined genetic locus into a plant, an existing plant line can be provided with a specific new characteristic essentially without the transfer of undesirable traits. Types of molecular markers that have been successfully applied in plant breeding include RFLP (restriction fragment length polymorphism) (WO89/07647; WO90/04651), RAPD (random amplification of polymorphic DNA) ( WO95/19697; WO99/05903), AFLP (amplified fragment length polymorphism) (EPO 534 858; WO00/15852), VNTR (variable number tandem repeats) also known as minisatellite (WO98/42867), STR (short tandem repeat), also known as SSR (simple sequence repeats) or microsatellite (WO99/67421), SNP (single nucleotide polymorphism) (US2007/039065), SFP (single feature polymorphism) (Borevitz et al. 2003. Genome Res. 13: 513-523), EST (expressed sequence tag) (WO05/17158); RDA (representational difference analysis) (WO99/53100), GMS (genomic mis-match scanning) (US Patent 5,376,526), SCAR (Sequenced Characterized Amplified Region; WO98/56948), isozyme (Tanksley & Rick, 1980. Theor. Appl. Genet. 57: 161-170; Ammati et al 1985 Plant Disease 69(2): 112- 115), ASH (allele specific hybridization) (Coryell et al., (1999) Theor. Appl. Genet. 98:690-696), 3SR (self- sustained sequence-replication), and RAD (restriction site associated DNA) markers (Miller et al. Genome Res 17:240-248 (2007))

There are various consideration for preferring a particular type of marker. EFLP and AFLP markers are generally not the first choiso as they require tedious? restriction enzyme digestions of lar»e amounts of DNA and separation of many digests from different individuals in parallel using &^■! electrophoresis. Microsatellite (also known as simple sequence ropeat (SSRt! markers have the advantage of being highly polymorphic, even within a specie? and cultivar, inherited, multiallelic, and codominant However, the time, effort and great expense needed to screen and multiplex mierosatellites is a serious limitation fur the expanded use of microsatellites in plant genetics. RAPDs are considered faster and cheaper to develop, but thev often show less polymorphism. ASH markers are used as dominant markers where rhe presence of absence of only one allele is determined from hybridize Lion or lack of hybridization by only one probe. The alternative allele may be interred from the Jack of hybridization. The ultimate marker for any trait is the causal DNA polymorphism(s) or allelic variant directly responsible for the desired phenotype. The reason is that in the allele the genotype and associated phenotype are directly connected. However, knowledge about the genetic basis for a particular trait is not essential to a breeding program, as long as the predictive value of the marker allows for the selection of offspring plants that carry the trait with sufficient certainty. For this, the locus of the trait and the locus of the marker must be sufficiently close on the chromosome so that they are for ins LJ nee inherited together in preferably in ore than 95% ot raei> >sos,

Because thu number oi^' genetic markers i^'or a plan! species with a narrow genetic- base such as cucumber (Dijkhuizen ft al, 1996) iβ limited, the d.34-ei >\vry t >f additi' fiiai MXTR-HC markers associated with a trait wiJl facilitate genotvping applications including marker-trait association studies, gene mapping, '.vn^ discovery, iriarker-assisred selection, and 3oavk>-r assisted breeding. Assume that a recurrent parent is crossed with a donor parent that contains one or more genes to be introgressed. The resulting Fl is mated to the recurrent parent, thus generating the first backcross (BCl); this generation is crossed with the recurrent parent, generating the second backcross (BC2) and so on. In each backcross generation, the plants to be crossed with the recurrent parent are selected in such a way that all of them possess the whole set of marker alleles present in the recurrent parent, and the marker alleles associated with the gene(s) to be introgressed. This imposes a restriction on the markers to be used: they have to be codominant, or if dominant, as in the case of RAPDs, the donor parent must carry the dominant alleles. (Reyes-Valdes, Crop Science 40:91- 98 (2000))

Yellowing viruses may cause significant economic damage in cucumber production, with the species CYSDV (closterovirus family) posing the biggest threat. The virus is transmitted by whitefly and although control may entail the application of insecticides, control is preferably achieved by using virus resistant cultivars. At least two quantitative trait loci or QTLs in the genome of certain resistant cucumber varieties are believed to be associated with valuable closterovirus resistance traits (vide WO 02/22836). An example is the QTLs originating from the cucumber landrace Khira PI250147. Proper introgression of the genetic material associated with such closterovirus resistance into offspring plants may be accomplished by selecting for plants having markers associated with these QTLs.

SUMMARY OF THE INVENTION

The present inventors have discovered novel markers for detecting resistance to CYSDV in cucumber plants originating from the cucumber landrace Khira PI250147. These markers are located in very close proximity to the resistance locus of CYSDV-QTL-I from cucumber accession PI250147.

This invention relates to compositions and methods of use of genetic markers for characterization of cucumber germplasm with respect to resistance to CYSDV. Plants and lines identified as having highly heritable CYSDV resistance traits are useful in developing commercial cultivars of cucumber crops having valuable agronomic and/or seed quality traits.

In a first aspect, the present invention provides a method of testing a nucleic acid sample for the presence of at least one (polymorphic) nucleic acid sequences selected from SEQ ID NO: 1, 2 and 3.

In a preferred embodiment, said method comprising detecting the presence of at least one of the polymorphisms of SEQ ID NO: 1 relative to SEQ ID NO: 2.

In another preferred embodiment, said nucleic acid sample is a sample from a plant, a plant germplasm or a plant line.

In yet another preferred embodiment, said plant is a cucurbit, most preferably a cucumber.

In another preferred embodiment, the method of testing of the present invention is part of a method for genotyping a plant or germplasm at the genetic marker locus of the marker having the nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3 associated with resistance to CYSDV, said method comprising a) amplifying the marker locus of the nucleic acid sequences or a portion thereof by admixing an amplification primer or amplification primer pair with the nucleic acid sample, wherein the primer or primer pair is complementary or partially complementary to at least a portion of the marker locus or the said nucleic acid sequences, and wherein the primer or primer pair is capable of initiating DNA polymerization by a DNA polymerase using the said nucleic acid as a template, and b) extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and said template nucleic acid to generate at least one amplicon and optionally detecting the said nucleic acid sequence in the resulting amplified marker amplicon. In another preferred embodiment of a method of testing of the invention the primer or primer pair is selected from the group consisting of SEQ ID NO: 4 and 5.

In another aspect, the present invention provides a method of selecting a plant or germplasm comprising performing the method of testing of the invention on a nucleic acid sample from a plant, a plant germplasm or a plant line, and selecting the plant or germplasm having at least one nucleic acid sequence selected from SEQ ID NO: 1 and 3, and selecting said plant or germplasm for further breeding purpose. In another aspect, the present invention provides a method of breeding plants, comprising selecting a plant or germplasm by performing a method of selecting a plant or germplasm according to the present invention, and crossing said selected plant or germplasm with a second plant or germplasm, preferably from an elite plant line, to produce progeny plants or germplasms. In a preferred embodiment of a method of breeding plants, said method further comprises the step of analysing DNA samples isolated from one or more said progeny plants or germplasms for the presence of at least one (polymorphic) nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3, wherein said analysis identifies a plant comprising at least one marker linked to CYSDV resistance.

In another preferred embodiment of a method of breeding plants, said progeny plants or germplasms are from a segregating population with respect to the presence of said at least one nucleic acid sequence and/or with respect to resistance to CYSDV. In another preferred embodiment of a method of breeding plants, said method further comprises one or more steps of backcrossing, selfing, outcrossing, and selection of plants.

In still another preferred embodiment of a method of breeding plants, said method further comprises the step of molecular marker analysis of DNA samples isolated from one or more plants resulting from use of the method, wherein said analysis identifies a plant comprising at least marker of SEQ ID NO: 1 or 3 linked to CYSDV resistance.

A plant of the invention may optionally comprise a second QTL associated with closterovirus resistance (QTL-2 as described in WO 02/22836, reference is made explicitly to the specification of this document for the details on the localization and characteristics of this QTL). This QTL was shown to be located on a separate chromosome. Hence, a method of breeding plants of the invention comprising the step of molecular marker analysis of DNA samples isolated from one or more plants resulting from use of the method may comprise an analysis that identifies a plant comprising at least marker of SEQ ID NO: 1 or 3 linked to CYSDV resistance, optionally in combination with markers linked to the quantitative trait locus identified in WO 02/22836 as QTL-2. Suitable markers for the detection of QTL-2 include those described in WO02/22836.

In yet another preferred embodiment of a method of breeding plants, said identified plant displays an increased resistance to CYSDV when compared to the second cucumber plant or germplasm.

In yet another aspect, the present invention provides an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule having the sequence of any one of SEQ ID NO: 1, 2 and 3; b) a nucleic acid molecule having a length of between 10 and 50 nucleotides and having a sequence identity of at least 80% with any one of SEQ ID NO: 1, 2 and 3; c) a nucleic acid molecule capable of hybridizing under stringent hybridization conditions to a nucleic acid molecule having the sequence of any one of SEQ ID NO: 1, 2 and 3; d)a nucleic acid molecule complementary to the nucleic acid molecules of any of a) to c).

In still a further aspect, the present invention provides the use of a isolated nucleic acid molecule of the invention as a hybridization probe or amplification primer.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows distinct DNA fragments amplified from resistant and susceptible inbred breeding lines as described in Example 2. Fragments were visualized by electrophoresis and ethidium bromide staining. (R) indicates resistant phenotype, (S) indicates susceptibility.

Figure 2 indicates the position of the CYSDV resistance locus designated CYSDV-QTL-I herein. Indicated are the various marker loci for the markers identified herein and some additional markers described earlier in WO 02/22836 and WO2007/053015. The different notations for the QTL ("CYSDV-QTL-I" and "QTL-I") are not intended to indicate any difference between the two QTL, and both references are made interchangeably herein. For the purpose of the drawing, however, the region indicated "QTL-I" indicates the region flanked by the closely linked markers identified in the present invention.

Figure 3 shows the scoring of the markers as described herein and indicating that the newly identified markers predict the phenotype of inbred lines correctly.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the term "cucurbit" refers to a plant of the family Cucurbitaceae, a plant family commonly known as melons, gourds or cucurbits and includes crops like cucumbers, squashes (including pumpkins), luffas, melons and watermelons. In particular, cucurbit refers to cucumber.

As used herein the term "cucumber" refers to a plant, or a part thereof, of the species Cucumis satiυus including, but not limited to, plants commonly referred to as Cucumber, American gherkin, Cassabanana, Cuke, Gherkin, Hothouse cucumber, Lemon cucumber, Mandera cucumber, Pickling cucumber, Serpent cucumber, Slicing cucumber, Snake cucumber, and West Indian gherkin.

As used herein the term "plant line" refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.

As used herein the term "plant part" indicates a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like.

As used herein the term "Cucurbit yellow stunting disorder virus" and its abbreviation "CYSDV" refers to a particular species of the family of Closteriviridae commonly referred to as closterovirus. The virus was first detection in the United Arab Emirates in 1982; and has since spread throughout the Mediterranean region and North America in the Rio Grande Valley of southern Texas and northern Mexico. CYSDV is transmitted in a semi-persistent, non-circulative manner by whiteflies. The virus has a bipartite genome consisting of two separately encapsulated single strand plus sense RNA segments. The term "nucleic acid" as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids). A polynucleotide can be full- length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to a specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skilled in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. If double-stranded, a DNA molecule generally has both a coding or sense and a non-coding or antisense strand. As used herein the term "linkage group" refers to all of the genes or genetic traits that are located on the same chromosome. Within the linkage group, those loci that are close enough together will exhibit linkage in genetic crosses. Since the probability of crossover increases with the physical distance between genes on a chromosome, genes whose locations are far removed from each other within a linkage group may not exhibit any detectable linkage in direct genetic tests. The term "linkage group" is mostly used to refer to genetic loci that exhibit linked behaviour in genetic systems where chromosomal assignments have not yet been made. Thus, in the present context, the term "linkage group" is synonymous to (the physical entity of) chromosome. As used herein the term "gene", refers to a nucleic acid sequence, in particular a DNA segment, composed of a transcribed region and optionally one or more regulatory sequences that make transcription possible and which contains a template for a nucleic acid polymerase (in eukaryotes, RNA polymerase II). Genes, when expressed, are transcribed into mRNAs that are then translated into protein. Also a "gene" is defined herein as a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristics or trait in an organism. The term "trait" refers to characteristic or phenotype. As used herein, the term "allele(s)" means any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. The length of an allele can be as small as 1 nucleotide base but is typically larger. Allelic sequence can be amino acid sequence or nucleic acid sequence. Since the present invention relates to genomic regions that may comprise one or more genes or regulatory sequences, it is in some instances more accurate to refer to "haplotype" (i.e. an allele of a chromosomal segment) in stead of "allele", however, in those instances, the term "allele" should be understood to comprise the term "haplotype". As used herein the term "haplotype" refers to the genotype for multiple loci or genetic markers in a haploid gamete. Generally, the haplotype is a set of alleles or a group of closely linked genes that is inherited as a unit, and hence, their loci or markers linked thereto reside in a relatively small and defined region of a chromosome. Also, the term refers to a series of common marker scores between individuals having a certain phenotype. A preferred haplotype comprises the 10 cM region or the 5 cM region or the 2 cM region surrounding an informative marker having a significant association with resistance.

A "locus" is defined herein as the position that a given gene or a regulatory sequence occupies on a chromosome of a given species.

As used herein, the terms "introgression", " introgressed" and "introgressing" refer to both a natural and artificial process whereby genomic regions of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species. The process may optionally be completed by backcrossing to the recurrent parent. As used herein the terms "resistant" and "resistance" encompass both partial and full resistance to infection. A susceptible plant may either be non- resistant or have low levels of resistance to infection. The term is used to include such separately identifiable forms of resistance as "full resistance", "immunity", "intermediate resistance", "partial resistance", "hypersensitivity" and "tolerance".

As used herein the term "full resistance" is referred to as complete failure of the disease to develop after infection, and may either be the result of failure of the disease to enter the cell (no initial infection) or may be the result of failure of the agent to multiply in the cell and infect subsequent cells (no subliminal infection, no spread).

As used herein the term "susceptible" refers to a plant having no resistance to the disease resulting in the plant being affected by the disease, resulting in disease symptoms. The term "susceptible" is therefore equivalent to "non-resistant". As used herein the term "flanked" refers to situated in between (and including) genomic markers.

"Genotype" means the specification of an allelic composition at one or more loci within an individual organism. In the case of diploid organisms, there are two alleles at each locus; a diploid genotype is said to be homozygous when the alleles are the same, and heterozygous when the alleles are different.

"Phenotype" means the detectable characteristics of a cell or organism that are a manifestation of gene expression.

"Linkage" refers to relative frequency at which types of gametes are produced in a cross. For example, if locus A has alleles "A" or "a" and locus B has alleles "B" or "b," a cross between parent I with AABB and parent II with aabb will produce four possible gametes where the haploid genotypes are segregated into AB, Ab, aB and ab. The null expectation is that there will be independent and equal segregation into each of the four possible genotypes, i.e., with no linkage, 1/4 of the gametes will be of each genotype. Segregation of gametes into a genotypes differing from 1/4 are attributed to linkage. Two loci are said to be "genetically linked" when they show this deviation from the expected equal frequency of 1/4.

"Linkage disequilibrium" is defined in the context of the relative frequency of gamete types in a population of many individuals in a single generation. If the frequency of allele A is p, a is p', B is q and b is q', then the expected frequency (with no linkage disequilibrium) of genotype AB is pq, Ab is pq', aB is p'q and ab is p'q'. Any deviation from the expected frequency is called linkage disequilibrium.

As used herein, the term "linked" refers to a marker locus and a second locus being sufficiently close on a chromosome that they will be inherited together in more than 50% of meioses, i.e., not randomly. This definition includes the situation where the marker locus and second locus form part of the same gene. Furthermore, this definition includes the situation where the marker locus comprises a polymorphism that is responsible for the trait of interest (in other words the marker locus is directly "linked" to the phenotype). When two loci are inherited together in more than 50% of meioses, the percent of recombination observed between the loci per generation (centimorgans (cM)), will be less than 50. In particular embodiments of the invention, genetically linked loci may be 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1 or less cM apart on a chromosome. Preferably, the markers are less than 5 cM apart and most preferably about 0 cM apart.

As used herein the term "cM" refers to centimorgan and indicates the distance between two loci or markers on a chromosome or a genetic linkage map thereof. Distances between loci are usually measured by the frequency of crossing-overs between loci on the same chromosome. The farther apart two loci are, the more likely that a crossing-over will occur between them. Conversely, if two loci are close together, a crossing-over is less likely to occur (between them). As a rule, one centimorgan (Kosambi map function (cM)) is approximately equal to 1% recombination between loci (markers).

As used herein the term "marker", or its equivalent "genomic marker", refers to a heritable and readily detectable polymorphic DNA sequence linked to a gene or QTL of interest. A "polymorphism" is a variation among individuals in sequence, particularly in DNA sequence. Useful polymorphisms include a single nucleotide polymorphisms (SNPs) and insertions or deletions in DNA sequence (INDELs). A marker can be a gene and can be a section of DNA with no known function which is used as an indicator in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Markers are indicated as being 'cis'-linked when they are physically associated with the allele linked with the trait of interest both in the donor and in the recipient plant receiving the introgression. Dominant traits can readily be assessed by using cis markers as the presence of the allele is positively associated with the phenotype. Hence, cis markers are the most important markers for marker assisted breeding for dominant traits. Markers are indicated as being genetically linked 'in trans' when they are physically associated with the opposite allele, that is, the allele that does not confer the trait of interest in the donor plant. The skilled person is aware that trans-markers that are absent in plants having the introgression, may also be useful in an assay for detecting successful introgression in offspring plants, although testing the absence of a marker to detect the presence of a specific introgression is indirect. Trans markers that are however of relevance in marker assisting breeding for recessive traits, as the presence of the allele of interest in homozygous (phenotype expressed) form is best indicated when the marker is no longer present in the population. Markers may be dominant or co- dominant.

A "Marker assay" means a method for detecting a polymorphism at a particular locus using a particular method, e.g., phenotype (such as seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allelic specific hybridization (ASH), RAPID, etc. Preferred marker assays include single base extension as disclosed in US 6,013,431 and allelic discrimination where endonuclease activity releases a reporter dye from a hybridization probe as disclosed in US 5,538,848, the disclosures of both of which are incorporated herein by reference.

A "Quantitative Trait Locus (QTL)" means a locus that controls to some degree numerically representable traits that are usually continuously distributed.

The term "hybrid" in the context of nucleic acids refers to a double- stranded nucleic acid molecule, or duplex, formed by hydrogen bonding between complementary nucleotide bases. The terms "hybridise", "hybridization" or "anneal" refer to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary bases. "Hybridizing" means the capacity of two nucleic acid molecules or fragments thereof to form anti- parallel, double-stranded nucleotide structure. The nucleic acid molecules of this invention are capable of hybridizing to other nucleic acid molecules under certain circumstances. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if the molecules exhibit "complete complementarity," i.e., each nucleotide in one sequence is complementary to its base pairing partner nucleotide in another sequence. Two molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions. Similarly, the molecules are said to be "complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high-stringency" conditions. Nucleic acid molecules that hybridize to other nucleic acid molecules, e.g., at least under low stringency conditions are said to be "hybridizable cognates" of the other nucleic acid molecules.

Conventional stringency conditions are described by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and by Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D. C. (1985), each of which is incorporated herein by reference. Deviations from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. Thus, in order for a nucleic acid molecule to serve as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed. Appropriate stringency conditions that promote DNA hybridization, for example, 6.Ox sodium chloride/sodium citrate (SSC) at about 45° C, followed by a wash of 2.Ox SSC at 50° C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated herein by reference. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.Ox SSC at 50° C. to a high stringency of about 0.2x SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C, to high stringency conditions at about 65° C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.

"Sequence identity" refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity" is the identity fraction times 100. Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc. Burlington, Mass.). Polynucleotides of the present invention that are variants of the polynucleotides provided herein will generally demonstrate significant identity with the polynucleotides provided herein. Of particular interest are polynucleotide homologs having at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, and more preferably even greater, such as 98% or 99% sequence identity with polynucleotide sequences described herein.

The term "probe" refers to a single- stranded oligonucleotide sequence that will form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A/T en G/C content) of the primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification. It will be understood that "primer", as used herein, may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified. Hence, a "primer" includes a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing. The oligonucleotide primers may be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences, and direct chemical synthesis. Chemical synthesis methods may include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in e.g. US 4,458,066. The primers may be labeled, if desired, by incorporating means detectable by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical means. Template-dependent extension of the oligonucleotide primer(s) is catalyzed by a polymerizing agent in the presence of adequate amounts of the four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP and dTTP, i.e. dNTPs) or analogues, in a reaction medium which is comprised of the appropriate salts, metal cations, and pH buffering system. Suitable polymerizing agents are enzymes known to catalyze primer- and template-dependent DNA synthesis. Known DNA polymerases include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, and Taq DNA polymerase. The reaction conditions for catalyzing DNA synthesis with these DNA polymerases are known in the art. The products of the synthesis are duplex molecules consisting of the template strands and the primer extension strands, which include the target sequence. These products, in turn, serve as template for another round of replication. In the second round of replication, the primer extension strand of the first cycle is annealed with its complementary primer; synthesis yields a "short" product which is bound on both the 5'- and the 3'-ends by primer sequences or their complements. Repeated cycles of denaturation, primer annealing, and extension result in the exponential accumulation of the target region defined by the primers. Sufficient cycles are run to achieve the desired amount of polynucleotide containing the target region of nucleic acid. The desired amount may vary, and is determined by the function which the product polynucleotide is to serve. The PCR method is well described in handbooks and known to the skilled person. After amplification by PCR, the target polynucleotides may be detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions will be used. If some mismatching is expected, for example if variant strains are expected with the result that the probe will not be completely complementary, the stringency of hybridization may be lessened. However, conditions are chosen which rule out nonspecific/adventitious binding. Conditions which affect hybridization, and which select against nonspecific binding are known in the art, and are described in, for example, Sambrook and Russell, 2001. Generally, lower salt concentration and higher temperature increase the stringency of hybridization conditions.

The phrase "stringent hybridization conditions" refers to conditions under which a polynucleotide will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, stringent conditions are selected to be about 5-1O⁰C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O⁰C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O⁰C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are often: 50% formamide, 5xSSC, and 1% SDS, incubating at 42⁰C, or, 5xSSC, 1% SDS, incubating at 65⁰C, with wash in 0.2xSSC, and 0.1% SDS at 65⁰C. For PCR, a temperature of about 36⁰C is typical for low stringency amplification, although annealing temperatures may vary between about 32⁰C and 48⁰C depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references (e.g. in Ausubel, et al. 1999).

As used herein the term "propagating material" refers to any part of the plant, including single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, and calli, which part is capable of regenerating into a new plant. As used herein the term "selecting" refers to assessing the presence of a marker or trait in a plant or plant part and designating to said plant or plant part the status of being selected in so far as having the desired trait or marker, thereby providing a plant or plant part for future breeding purpose.

"Purified" and "isolated" refers to a nucleic acid molecule or polypeptide separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be greater than 60% free or 75% free or 90% free or 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The terms "isolated and purified" and "substantially purified" are not intended to encompass molecules present in their native state.

As used herein the term "marker assisted selection" refers to a process in modern plant breeding wherein offspring plants having favourable alleles are selected for future breeding purpose by detecting in those plants the presence of trait-associated markers, for instance markers defining a QTL or genomic region associated with a desirable trait.

As used herein the term "offspring plant" refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include selfings as well as the Fl or F2 or still further generations. An Fl is a first- generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfings of Fl's, F2's etc. An Fl may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of said Fl hybrids.

As used herein the term "transgressive segregants" refers to offspring (e.g., created within a breeding program) which possess significantly different traits/phenotypes than their parents. Such offspring is the results of the separation (from each other) of allele pairs during meiosis and the subsequent distribution into different germ cells.

As used herein the term "inbred" means a substantially homozygous individual or line.

As used herein the term "homozygous" means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes, as opposed to the term "heterozygous", which refers to a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.

As used herein the term "hybrid" in the context of plant breeding refers to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines.

Description of the preferred embodiments

In a first aspect, the present invention provides a method of testing a nucleic acid sample for the presence of at least one (polymorphic) nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3. Said method suitably comprises the step of detecting the presence of at least one nucleic acid sequences selected from SEQ ID NO: 1, 2 and 3 in a sample of (preferably isolated) nucleic acids by performing a nucleic acid analysis method, in particular a method of analysis that identify unique nucleotide (usually the polymorphic) sequences in a DNA molecule. A method of testing of the invention may involve any form of nucleic acid analysis known in the art. Suitably, the method comprises the step of amplifying the region of the DNA suspected of comprising the nucleic acid sequences of interest, which region or location may suitably be referred to as the marker locus for the marker having the said nucleic acid sequence and detecting the polymorphic nucleic acid sequence in the resulting amplified marker amplicon. Also, a portion of the marker locus may be amplified and the polymorphic nucleic acid sequence in the resulting amplified marker amplicon may thereafter be detected. Alternatively, the (polymorphic) sequences may be amplified directly from the nucleic acid sample by using amplification primers hybridizing specifically to the polymorphic marker sequence. The generation of an amplicon then indicates the presence of the nucleic acid sequences of interest.

Generally, the step of amplifying comprises admixing an amplification primer or amplification primer pair with the nucleic acid, suitably a nucleic acid isolated from the cucumber plant or germplasm, wherein the primer or primer pair is complementary or partially complementary to at least a portion of the marker locus or the said nucleic acid sequences, and wherein the primer or primer pair is capable of initiating DNA polymerization by a DNA polymerase using the said nucleic acid as a template. The step of amplifying then further comprises extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to generate at least one amplicon.

Nucleic acid amplification methods useful in the method of the testing according to the present invention are well known in the art. In principle, any nucleic acid amplification method may be employed in methods of the invention, such as the Polymerase Chain Reaction (PCR; Mullis 1987, U.S. Pat. No. 4,683,195, 4,683,202, and 4,800,159) or by using amplification reactions such as Ligase Chain Reaction (LCR; Barany 1991, Proc. Natl. Acad. Sci. USA 88:189- 193; EP Appl. No., 320,308), Self- Sustained Sequence Replication (3SR; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), Strand Displacement Amplification (SDA; U.S. Pat. Nos. 5,270,184, en 5,455,166), Transcriptional Amplification System (TAS; Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), Rolling Circle Amplification (RCA; U.S. Pat. No. 5,871,921), Nucleic Acid Sequence Based Amplification (NASBA), Cleavase Fragment Length Polymorphism (U.S. Pat. No. 5,719,028), Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acid (ICAN), Ramification- extension Amplification Method (RAM; U.S. Pat. Nos. 5,719,028 and 5,942,391) or any other suitable method for amplifying DNA.

In order to amplify DNA with a small number of mismatches to one or more of the amplification primers, an amplification reaction may be performed under conditions of reduced stringency (e.g. a PCR amplification using an annealing temperature of 38⁰C, or the presence of 3.5 mM MgCb). The person skilled in the art will be able to select conditions of suitable stringency.

The primers herein are selected to be "substantially" complementary (i.e. at least 65%, more preferably at least 80% perfectly complementary) to their target regions present on the different strands of each specific sequence to be amplified. It is possible to use primer sequences containing e.g. inositol residues or ambiguous bases or even primers that contain one or more mismatches when compared to the target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target DNA oligonucleotide sequences are considered suitable for use in a method of the invention. Sequence mismatches are also not critical when using low stringency hybridization conditions.

The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The amplicons may be directly or indirectly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, enzyme reagents or visualized with labelled oligonucleotides as reporter molecules. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide, Hoechst dyes. Suitable fluorescent labels include, but are not limited to fluoresceins, rhodamines (FAM, R6G, TAMRA, and ROX), Texas red, BODIPY, coumarins, cyanine dyes (thiazole orange [TO], oxazole yellow [YO], TOTO, YOYO; Cy3, Cy5), and Alexa dyes. Reporter molecules include, but are not limited to oligonucleotides labelled with fluorochromes such as FAM, ROX, Texas Red, TET, TAMRA, JOE, HEX, CAL Red, and VIC.

Alternatively, the DNA fragments may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels that may be associated with nucleotide bases for incorporation into the amplicon include e.g. fluorescein, cyanine dye or BrdUrd.

A method of testing a nucleic acid sample for the presence of at least one (polymorphic) nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3 may also be performed by using a probe-based detection system, optionally in combination with nucleic acid amplification. Suitable detection procedures for use in the present invention may for example comprise an enzyme immunoassay (EIA) format (Jacobs et al., 1997, J. Clin. Microbiol. 35, 791-795). In EIA procedures, either the forward or the reverse primer used in the amplification reaction comprises a capturing group, such as a bio tin group for immobilization of amplicons on e.g. a streptavidin coated microtiter plate, for subsequent EIA detection of amplicons.

Probes useful for the detection of the marker sequence as disclosed herein preferably bind only to at least a part of the marker sequence region as amplified by the DNA amplification procedure. Those of skill in the art can prepare suitable probes for detection based on the nucleotide sequence of the marker sequence without undue experimentation as set out herein. Also the complementary sequences of the marker sequence may be equally suitable as detection probes in a method of the invention, provided that such a complementary strand is amplified in the amplification reaction employed.

In yet other alternative embodiments, nucleic acid samples may be tested by southern blotting, wherein the nucleic acid sample is contacted with a probe that is complementary or partially complementary to at least a portion of the marker nucleic acid sequences, wherein the probe is capable of hybridizing to at least a portion of the marker nucleic acid sequences under stringent hybridization conditions when bonded to a filter material. Suitable procedures may thus comprise immobilization of the amplicons and probing the DNA sequences thereof. To facilitate the detection of binding, the specific amplicon detection probes may comprise a label moiety such as a fluorophore, a chromophore, an enzyme or a radio-label, so as to facilitate monitoring of binding of the probes to the reaction product of the amplification reaction. Such labels are well-known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), β-galactosidase, horseradish peroxidase, streptavidin, biotin, digoxigenin, ³⁵S or ¹²⁵I, ³H, and those described above. Other examples will be apparent to those skilled in the art. Detection may also be performed by a so called reverse line blot (RLB) assay, such as for instance described by Van den Brule et al. (2002, J. Clin. Microbiol. 40, 779-787). For this purpose RLB probes are preferably synthesized with a 5'amino group for subsequent immobilization on e.g. carboxyl-coated nylon membranes.

Alternatively, the method may comprise in situ hybridization tests, wherein the target nucleic acid is contacted in situ with a probe that is complementary or partially complementary to at least a portion of the said nucleic acid sequences, and capable of hybridizing thereto under stringent hybridization conditions. Such detection probes may be suitably labeled with a detectable label.

The use of nucleic acid probes for the detection of DNA fragments is well known in the art. Mostly these procedure comprise the hybridization of the target DNA with the probe followed by post-hybridization washings. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Tm = 81.5 °C + 16.6 (log M) + 0.41 (% GC)- 0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus, the hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with > 90% identity are sought, the Tm can be decreased 10 °C. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1,2,3, or 4 °C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6,7,8,9, or 10 °C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11,12,13,14,15, or 20 °C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 ⁰C (aqueous solution) or 32 ⁰C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used.

Nucleic acid sequences of SEQ ID NO:1, 2 and 3 represent markers linked to a QTL associated with resistance to CYSDV in cucurbits on linkage group 4. These markers are closely linked to the resistance locus, and, hence, exhibit very high predictive value for the presence of CYSDV resistance in cucurbit plants and germplasms.

A method of testing according to the present invention can thus suitably be employed as a method of genotyping plants, germplasms, and plant lines of cucurbits, such as cucumber plants, populations of cucumber plants and/or cucumber lines, for the presence of the subject genetic markers associated with CYSDV. In essence, the invention provides a method for molecular marker analysis of DNA samples isolated from one or more plants.

The molecular markers referred to herein indicate the presence of at least one allele having a polymorphic sequence linked to CYSDV resistance. SEQ ID NO:1 is an INDEL marker associated with resistance representing an 18 nucleotide insertion in the genomic DNA sequence relative to the sequence in the corresponding locus of the susceptible plant or germplasm. SEQ ID NOs:l and 2 corresponds to the marker locus E16/M55-F-112/130 for CYSDV-QTL-I as indicated herein. This locus is bi-allelic. The resistant locus (indicated by SEQ ID NO: 1) comprises the polynucleotide insertion 5'- CTGTGTTTATAATCCCAT-S', relative to the susceptible locus (indicated by SEQ ID NO: 2). Thus a method of the invention as described herein may comprise detecting the presence of at least one of the polymorphisms of SEQ ID NO: 1 relative to SEQ ID NO: 2.

A method of testing according to the present invention is advantageously employed for the purpose of reducing the size of the introgression comprising the CYSDV resistance alleles on linkage group IV derived from cucumber accession Khira PI250147. Reduction of the introgression with the use of closely linked markers facilitates identification of recombinants around this locus. An example illustrating the application of this is the recombination between CYSDV and PM resistance loci, described in earlier application WO2007/053015. Precise introgression of small genomic regions is essential to insert one trait without losing the other. Another example is removal of linkage drag associated with CYSDV, which may include such negative traits as necrosis and/or large blossom end. Linkage group IV is indicated by the RFLP markers (map position in cM in brackets) CsC477H3 (16.3), CsC032a/El (30.2), CsP357/H3 (36.9), CsC588/H3 (39.7), CsP046/El (43.1), CsC694/E5 (44.6), CsP347/H3 (44.6), CsC365/El (53.3), CsC386/El (53.3) and CsC230/El (54.8). The nucleic acid sample used in methods of the invention may thus suitably be a nucleic acid sample from a plant or a plant germplasm (e.g. a seed). Methods for isolating nucleic acids from plants or germplasms need not be described in detail here as they are well known in the art (see e.g. Kang et al. 1998. Plant Molecular Biology Reporter 16: 1-9; WO/2004/056984).

A plant used in aspects of the present invention is preferably a cucurbit, still more preferably a cucumber. It is contemplated that the marker may find utility not only in the instance of the PI250147 Khira resistance introgressed into Cucumis sativus, but that the methods find wider applicability once the Khira-landrace-based resistance to CYSDV crosses the species- bounderies, i.e. is introgressed into other cucurbits. CYSDV-QTL-I as detectable by the methods of the present invention is believed to provide at least partial resistance to CYSDV in cucumber plants. When full resistance in plants is required, in addition to the presence of CYSDV- QTL-I, the presence of CYSDV-QTL-2 as identified in WO02/22836 may be determined as part of an additional step in a method of the invention. Explicit reference is made to the description of WO02/22836 for the markers linked to QTL- 2 and the sequences useful as target and primer sequences in detection methods of the present invention.

The goal of plant breeding is to combine various desirable traits in a single variety or hybrid. For commercial crops, these desirable traits may include resistance to diseases and insects; tolerance to heat and drought; reduced time to crop maturity; uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant height; and improved agronomic qualities such as greater yield, flowering, plant growth and/or plant structure.

Usually, breeding for desirable traits culminates in the establishment of the breeding line or elite line, usually a parent for the generation of commercial hybrid seed. A preferred aspect of the invention therefore relates to elite lines as will be detailed later herein.

Current breeding programs involve extensive genotyping efforts. In fact, a method of the invention when performed on plants is advantageously performed as part of a method for genotyping a plant or germplasm at the genetic marker locus of the marker having a nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3, since these marker loci have been found to be coupled with resistance to CYSDV, in particular based on the genomic introgressions derived from cucumber accession Khira PI250147 associated with such resistance. A method of genotyping according to the invention comprises a first steps of amplifying the marker locus of the nucleic acid sequences or a portion thereof by admixing an amplification primer or amplification primer pair with the nucleic acid sample, wherein the primer or primer pair is complementary or partially complementary to at least a portion of the marker locus or the said nucleic acid sequence, and wherein the primer or primer pair is capable of initiating DNA polymerization by a DNA polymerase using the said nucleic acid as a template.

A method of genotyping according to the invention comprises a second steps of extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and said template nucleic acid to generate at least one amplicon and optionally detecting the said nucleic acid sequence in the resulting amplified marker amplicon.

Detection of the nucleic acid sequence in the marker amplicon is particularly beneficial when using non-specific primers for the amplification of (a part of) the marker. When using specific primers annealing only to the polymorphic sequences of SEQ ID NO 1, 2 or 3 or the polymorphic nucleotide in SEG ID NO:1 relative to SEQ ID NO:2 the generation of the marker amplicon indicates the presence of the nucleic acid sequence of SEQ ID NO: 1, 2 and/or 3. Examples of primers that amplify only the specific marker sequences are the primers of SEQ ID NO: 4 and 5. Use of these primers results in an amplicon having a size of 109 base pairs for the resistant allele and 91 base pairs for the susceptible allele as described in more detail above.

In another aspect, the present invention provides a method of selecting a plant or germplasm. The selection method involves performing the method of testing a nucleic acid sample for the presence of at least one (polymorphic) nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3 as described herein, followed by selecting the plant or germplasm having at least one nucleic acid sequence selected from SEQ ID NO: 1 and 3 for further breeding purpose. SEQ ID NO: 1 and 3 indicate plants that comprise the resistance allele. The plants thus selected have the desired genotype at at least one selected genetic marker locus associated with resistance to CYSDV. Such plants may be used as parents for creation of a breeding population. For instance, a method of selecting plants may provide cucumber plants or cucumber lines which, based upon their genotype, are predicted to produce transgressive segregants for resistance to CYSDV.

In another aspect, the present invention provides a method of breeding plants. Breeding methods as contemplated in the present invention are essentially marker- assisted methods. The method comprises the selection of a plant or germplasm using the above described selection method, and crossing the thus selected plant or germplasm with a second plant or germplasm, preferably from an elite plant line, to produce progeny plants or germplasms. This method of breeding plants is advantageous for producing cucumber plants comprising the said nucleic acid sequence and/or exhibiting resistance to CYSDV. In particular, such method make use of at least one genetic marker to select a cucumber plant from a cucumber breeding population by marker- assisted selection, and crossing the selected cucumber plant with a second cucumber plant wherein the progeny cucumber plants of the cross produce transgressive segregants for resistance to CYSDV of exhibit resistance to CYSDV.

The progeny plants may again be tested for the presence of the marker characterized by SEQ ID NO: 1, 2 and 3, or for any other marker associated with a desirable trait. In particular, the test identifies a plant comprising at least one marker linked to CYSDV resistance.

In yet other alternative embodiments, the progeny plants may be tested for exhibiting desirable traits, including resistance to CYSDV, using a bioassay.

Although the methods of the invention are by no means limited thereto, they are advantageously performed on progeny seed or progeny plants from a segregating population with respect to the CYSDA markers as defined herein and/or with respect to resistance to CYSDV. Once a suitable progeny plant or germplasm in a method of breeding of the invention has been identified and selected, it may be used for further breeding purpose. Further breeding purpose as defined herein may comprise one or more steps of backcrossing, selfing, outcrossing, and selection of plants. Preferably, backcrossing and selection is commenced for about 4 to about 9, suitably about 7 generations or cycles, in order to provide for an essentially homozygous breeding parent.

The plants or germplasm resulting from the method described above may again be analysed for the presence of the marker of SEQ ID NO: 1 or 3 linked to CYSDV resistance. Preferably the plant thus identified displays an increased resistance to CYSDV when compared to the second cucumber plant or germplasm, which was used as an original parent in the first cross.

The present invention also relates to novel and inventive isolated nucleic acid molecules. Such molecules comprise for instance a nucleic acid molecule having the sequence of any one of SEQ ID NO: 1, 2 and 3; nucleic acid molecules of between 10 and 50 nucleotides in length and having a sequence identity of at least 80%, preferably at least 85, 88, 91, 93, 95, 96, 97, 98, or 99 % with SEQ ID NO: 1, 2 or 3; nucleic acid molecules capable of hybridizing under stringent hybridization conditions to a nucleic acid molecule having the sequence of SEQ ID NO: 1, 2 or 3; and nucleic acid molecules complementary to any of the previous nucleic acid molecules.

The here described nucleic acid molecules may suitably be used as a hybridization probe or amplification primer in method of the present invention. In another aspect, the invention provides oligonucleotide probes or primers for the detection of markers linked to CYSDV-resistance as described herein. The detection probes and primers herein are selected to be "substantially" complementary to one of the strands of the double stranded DNA amplicons generated by an amplification reaction of the invention. It is allowable for detection probes and primers of the present invention to contain one or more mismatches to their target sequence. In general, sequences that exhibit at least 65%, more preferably at least 80% homology with the target DNA oligonucleotide sequences are considered suitable for use in a method of the present invention. The invention will now be described by the following non-limiting Examples. EXAMPLES

Example 1. Discovery of markers closely linked to QTLl for resistance to

CYSDV in cucumber.

Markers

The position of QTLl for CYSDV resistance in cucumber was previously studied by AFLP linkage mapping as described in WO02/22836. Fine- mapping of QTLl for CYSDV resistance in cucumber was performed by bulked segregant analysis (BSA) on multiple F2 pools with opposed phenotypes

(resistant and susceptible). This resulted in additional AFLP markers. Markers identified by BSA were mapped on F2 individuals and QTL isogenic recombinants (QIRs).

Phenotypic values for CYSDV resistance level were interpreted, resulting in the identification of three markers QTL 1 being more closely linked than those described in WO02/22836: E23/M60-F-187-P1, E16/M55-F-130-P1, and E16/M55-F-112-P2. The marker loc i posiπoned within QTL-I and the CYSDV resistance appeared I u be bo closely linked on the chromosome t hat they inherited together in mine than 95% of meioses, preferably more than 9 )% (l.o cM ώtran.-e b-psveen marker E23/M60-F-187-P1 and E16/M55-F-112/130-P1). It is contemplated herein άui, one of the markers E23/M60-F-187-P1 and E16/M55- F-130-P1 is even more elo^celv linked to the resistance Ioeu= that ir i= inherited toget her in more than 99% of meio&es TLa^, recombination froquoncj observed between the loci of marker and resistance per generation (in centimoruan? (cM)), Vv ill he less than δ In particular embodiments of the invention, genetically linked loci may be 2, or 1 or fewer cM apart on a chromosome.

Marker sequence

The base pair sequence of the most closely lined AFLP markers was determined. Marker E16/M55-F-130-P1 appeared to be allelic to E16/M55-F-112- P2, because both fragments were identical except for an insertion/deletion (INDEL) of 18 base pairs sequence difference. The sequences for AFLP markers E16/M55-F-130-P1 and E16/M55-F-112-P2 were determined to be as follows:

Resistant allele: E16/M55-F-130 (5' to 3'): GAATTCCCAA TGAACCCAAT TCAATTCCCT TTTCTTCTAC AGGAATCCCA TCTGTGTTTA TAATCCCATC TGTGTTTATA ATCCCATTTT GAATAAGGTC GTTAA

Susceptible allele: E16/M55-F112 (5' to 3'):

GAATTCCCAA TGAACCCAAT TCAATTCCCT TTTCTTCTAC AGGAATCCCA TCTGTGTTTA TAATCCCATT TTGAATAAGG TCGTTAA

Polymorphisms were not identified for marker E23/M60-F-187-P1. The sequence for AFLP marker E23/M60-F- 187-Plwas determined to be as follows:

Resistant alleles: E23/M60-187 (5' to 3'):

GAATTCTAAA ATTTAGAATA TAATTCGATA TTTCTCTAAA AAAGACAAAA TAAAAAGAAT GAGTAGAAAA CTATAGAAAG AGGCAGAATC GTGTGAATGA AAGATAAAAG AGAATAGTAA CGAAGAATTT TCCAGACGTG TTCAAATGGT TGATATGAGT TAA

Predictive value

Genetic linkage between traits and markers determines the predictive value markers for indirect selection. The predictive value of all AFLP markers was determined by screening a series of 38 inbred lines originating from distinct breeding programs (Figure 3). Subsequently, the genotypes were compared with corresponding phenotypes. The genotypes for marker E23/M60-F-187-P1 and the bi-allelic marker E16/M55-F-112/130 always corresponded with the phenotype of the tested inbred lines. This is consistent with the conclusion that marker E23/M60-F-187-P1 and the bi-allelic marker E16/M55-F-112/130 are closely linked to CYSDV QTLl. Furthermore, marker E23/M60-F-187-P1 and the bi- allelic marker E16/M55-F-112/130 have a predictive value of 100%. Thus, markers E23/M60-F-187-P1 and E16/M55-F-112/130 are particularly useful for indirect selection in marker- assisted breeding programmes.

Example 2. SCAR marker development

Marker E16/M55-F-130/112 was converted into a SCAR (sequence characterized amplified region) marker, which allowed its detection by a polymerase chain reaction assay. Primers were designed and thermal cycling conditions developed.

PCR Primers The following PCR primers were designed for the AFLP marker E16/M55-F- 130/112:

Forward primer:

5'- AGCGGATAAC AATTTCACAC AGGACACACT GGTACGAACC CAATTCAATT CCCTTTTC-3'

Reverse primer: δ'-AACGACCTTATTCAAAATGGGAT-S'

PCR Thermal cycling

The PCR reaction mixture consisted of 1.5 μl of 5.0 μM forward primer, 1.5 μl of 5.0 μM reverse primer, 0.20 μl of 20.0 mM (of each) dNTP mix, 2.0 μl 10x Taq PCR buffer, 1.2 μl of 25.0 mM MgCl₂, 0.1 μl of 5.0 Units/μl Taq DNA Polymerase (e.g. New England Biolabs, MA, USA) 10 pg-1 μg of template DNA and sterile deionized water to make up a volume of 20 μl. The PCR reaction consisted of 94⁰C denaturing (3.0 minutes), followed by 35 cycles of 94⁰C denaturing (0.5 minute), 54⁰C annealing (1.0 minute), and 72⁰C extension (1.0 minute); with a 72⁰C final extension (5 minutes) followed by storage at 4⁰C.

Results

The SCAR marker was tested on 25 inbred lines (Figure 1). The results were consistent with AFLP genotyping. AFLP score (A) corroborated with presence of the resistant allele, demonstrated by an amplified DNA fragment of 109 base pairs. AFLP score (B) correlated with the presence of the susceptible allele of 91 base pairs with a deletion of 18 base pairs. Thus, genotyping with the SCAR and AFLP E16/M55-F-112/130 markers always provided the same result. Therefore, the SCAR marker can be applied for marker- assisted breeding in a similar fashion as AFLP E16/M55-F- 112/130. SEQUENCE LISTING

<110> De Ruiter Seeds R&D B. V.

<120> Method of breeding CYSDV-resistant cucumber plants

<130> P86308EP00

<160> 5

<170> Patentln version 3.3

<210> 1

<211> 105 <212> DNA

<213> AFLP marker E16/M55-F-130

<400> 1

GAATTCCCAA TGAACCCAAT TCAATTCCCT TTTCTTCTAC AGGAATCCCA TCTGTGTTTA 60 TAATCCCATC TGTGTTTATA ATCCCATTTT GAATAAGGTC GTTAA

<210> 2

<211> 87 <212> DNA

<213> AFLP marker E16/M55-F-112

<400> 2 GAATTCCCAA TGAACCCAAT TCAATTCCCT TTTCTTCTAC AGGAATCCCA TCTGTGTTTA 60 TAATCCCATT TTGAATAAGG TCGTTAA

<210> 3

<211> 105 <212> DNA

<213> AFLP marker E23/M60-F-187

<400> 3 GAATTCTAAA ATTTAGAATA TAATTCGATA TTTCTCTAAA AAAGACAAAA TAAAAAGAAT 60 GAGTAGAAAA CTATAGAAAG AGGCAGAATC GTGTGAATGA AAGATAAAAG AGAATAGTAA 120 CGAAGAATTT TCCAGACGTG TTCAAATGGT TGATATGAGT TAA

<210> 4 <211> 58 <212 > DNA

<213> Forward primer for SCAR marker designed for biallelic AFLP marker

E16/M55-F-130/112

<400>

AGCGGATAAC AATTTCACAC AGGACACACT GGTACGAACC CAATTCAATT CCCTTTTC <210> 5 <211> 23 <212> DNA

<213> Reverse primer for SCAR marker designed for biallelic AFLP marker E16/M55-F-130/112

<400> 5

AACGACCTTA TTCAAAATGG GAT <210> 6

<211> 18

<212> DNA

<213> Polymorphic polynucleotide biallelic AFLP marker E16/M55-F-130/ 112

<400> 6

CTGTGTTTAT AATCCCAT

Claims

1. A method of testing a nucleic acid sample for the presence of at least one nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3, comprising the step of determining the presence in said sample of a nucleic acid comprising a nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3.

2. The method of claim 1, said method comprising detecting at least one of the polymorphisms of SEQ ID NO: 1 relative to SEQ ID NO: 2.

3. The method of claim 1 or 2, wherein said nucleic acid sample is a sample from a plant, a plant germplasm or a plant line.

4. The method of claim 3, wherein said plant is a cucurbit.

5. The method of claim 4, wherein said cucurbit is a cucumber.

6. The method of any one of claims 1 to 5, wherein said method is part of a method for genotyping a plant or germplasm at at least one genetic marker locus associated with resistance to CYSDV harboring a marker having the nucleic acid sequence selected from SEQ ID NO: 1, 2 and 3.

7. The method of claim 6, wherein said method comprises: a) amplifying the marker locus of the nucleic acid sequences or a portion thereof by admixing an amplification primer or amplification primer pair with the nucleic acid sample, wherein the primer or primer pair is complementary or partially complementary to at least a portion of the marker locus or the said marker nucleic acid sequences, and wherein the primer or primer pair is capable of initiating DNA polymerization by a DNA polymerase using the said sample nucleic acid as a template nucleic acid, and b) extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and the said template nucleic acid to generate at least one amplicon and optionally detecting the said nucleic acid sequence in the resulting amplified marker amplicon.

8. Method according to claim 6, wherein said method comprises the steps of: a) optionally amplifying the marker locus of the nucleic acid sequences or a portion thereof by admixing an amplification primer or amplification primer pair with the nucleic acid sample, wherein the primer or primer pair is complementary or partially complementary to at least a portion of the marker locus or the said marker nucleic acid sequences, and wherein the primer or primer pair is capable of initiating DNA polymerization by a DNA polymerase using the said sample nucleic acid as a template nucleic acid, and b) determining the presence of the said marker nucleic acid sequence in the genome of said plant or germplasm or in the optionally amplified marker locus by determining a melting curve for the genomic nucleic acid or said optionally amplified marker locus and establishing the presence of the said marker nucleic acid sequence in the marker locus in case the melting curve indicates the presence of the polymorphism.

9. The method of any one of claims 6-8, wherein the primer or primer pair is selected from the group consisting of SEQ ID NO: 4 and 5.

10. A method of selecting a plant or germplasm comprising performing the method of any one of claims 3-9 and selecting the plant or germplasm having at least one nucleic acid sequence selected from SEQ ID NO: 1 and 3 for further breeding purpose.

11. A method of breeding plants, comprising selecting a plant or germplasm by the method of claim 10, and crossing said selected plant or germplasm with a second plant or germplasm, preferably from an elite plant line, to produce progeny plants or germplasms.

12. The method of claim 11, further comprising the step of analysing DNA samples isolated from one or more of said progeny plants or germplasms for the presence of at least one nucleic acid sequences selected from SEQ ID NO: 1, 2 and 3, wherein said analysis identifies a plant comprising at least one marker linked to CYSDV resistance.

13. The method of claim 12, wherein said progeny plants or germplasms are from a segregating population with respect to the presence of said at least one nucleic acid sequence and/or with respect to resistance to CYSDV.

14. The method of any one of claims 11-13, further comprising one or more steps of backcrossing, selfing, outcrossing, and selection of plants.

15. The method of any one of claims 11-14, further comprising the step of molecular marker analysis of DNA samples isolated from one or more plants resulting from the use of the method, wherein said analysis identifies a plant comprising at least marker of SEQ ID NO: 1 or 3 linked to CYSDV resistance, optionally in combination with markers linked to the quantitative trait locus identified in WO02/22836 as QTL-2.

16. The method of claim 15, wherein said identified plant displays an increased resistance to CYSDV when compared to the second cucumber plant or germplasm.

17. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule having the sequence of any one of SEQ ID NO:

1, 2 and 3; b) a nucleic acid molecule having a length of between 10 and 50 nucleotides and having a sequence identity of at least 80% with any one of SEQ

ID NO: 1, 2 and 3; c) a nucleic acid molecule capable of hybridizing under stringent hybridization conditions to a nucleic acid molecule having the sequence of any one of SEQ ID NO: 1, 2 and 3; d) a nucleic acid molecule complementary to the nucleic acid molecules of any of a) to c).

18. Use of a nucleic acid molecule of claim 17 as a hybridization probe or amplification primer.