Disease-resistant cucumber plants

FIELD: agriculture.

SUBSTANCE: specified plant is a plant of Cucumis sativus type and includes at least one area at one chromosome, which gives resistance to closterovirus, and at least one area, which gives resistance to powdery mildew. The area of the chromosome, which gives resistance to closterovirus, is linked to at least one marker selected from the group, made of markers E16/M50-244, E16/M50-188, and E11/M48-251. The area of the chromosome, which gives resistance to powdery mildew, is linked to at least one marker selected from the group made of the following components: a marker of single-nucleotide polymorphism 39TG in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29GA in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193CT in SEQ ID NO:3, mutation of an insert 5'-AATTT-3' in position 221 in SEQ ID NO:4, and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.

EFFECT: production of the plant resistant to closterovirus and to powdery mildew of cucumbers.

25 cl, 8 dwg, 4 tbl

 

The technical field to which the invention relates

The present invention relates to the field of plants resistant to diseases, and to the cultivation of plants, in more detail, to the breeding of cucumber plants resistant to powdery mildew and to closterovirus.

The prior art inventions

Commercial production of cucumbers (Cucumis sativusmay incur losses because of a number of diseases. Viruses cause jaundice one of these diseases and can cause significant economic damage of cucumber cultivation. Family closterovirus (Closteroviridae), which forms the highest taxonomic group of viruses jaundice, affecting cucumber, includes more than 30-shaped and filamentous plant viruses are transmitted by insects. The family consists of three genera, of which the genus Crinivirus, carried by the whitefly, includes species that are particularly sensitive cucumbers. This genus includes, among others, types of virus disease yellow stunting cucumber (CYSDV)virus, infectious jaundice salad (LIYV), virus pseudoalhagi beet (BPYV). The greatest threat to producers of cucumbers are CYSDV and BPYV. Viruses are usually found in the insect-vector and transferred to the plant by the nutritional activity of the insects.

Therefore, closterovirus cucumbers control group is a rotary application of insecticides. However, preferably, the control closterovirus in agriculture and horticulture is achieved by obtaining a resistant virus cultivars of the host plant. Currently, at least two quantitative trait locus or QTL in the genome of resistant variants cucumber recognized as associated with resistance to closterovirus (see WO 02/22836). The corresponding introgression of genetic material associated with resistance to closterovirus, in the lines of plants descendants can be monitored by detecting specific markers associated with QTL. Therefore, knowledge of QTL may be important for breeding cultivars resistant to closterovirus.

Another important disease that affects commercial production of cucumbers is powdery mildew of cucumbers (PM). RM cucumbers can be caused by fungiS.fuligineaandE.cichoracearum. The disease is widespread and can occur year-round. The symptoms begin with small patches of thin, white fungal threads on the surface of infected leaves, which can then grow and eventually cover the stems and leaves a white powdery mass of spores and hyphae. Severe infection leads to discoloration and loss of leaves and an accompanying decrease in the number and size of fruits. Although the number of fungicide which is effective against powdery mildew, found fungus resistance against chemicals.

Different strains of cucumber was shown some level of resistance to powdery mildew. Such strains include, for example, sample PI 197088 wild Indian cucumber and samples PI 200815, PI 200818, as well as the cultivars Natsufushinari and Asomidori (Morishita and others, 2003). Also known other genes associated with resistance RM (Fanourakis, 1984; Fujieda and Akiya, 1962; Kooistra, 1968, 1971; Shanmugasundarum and others, 1971, 1972), includingpm-1andpm-2in Natsufushinari,pm-3in PI 200815 and PI 200818 and "pm-h" multigene Wis. SMR 18 (gene "pm-h" is without limitation to the locus pm detectedpm-hlater in this document). Although there are several commercial varieties of cucumbers with partial resistance RM, the majority of commercial producers still rely on deciduous fungicides. This is partly due to the fact that the resistance RM difficult to bring into line, have shown other types of resistance. For example, breeders are faced with such a lack of sensitivity to closterovirus, and Vice versa, in line with sustainable WP alleles, although they were not sensitive. In fact, experiments breeding have not given lines cucumbers, have shown resistance to both the RM and closterovirus. Today, the effective recombinants with dual resistance is not obtained. It will surprise you what) because the Association of different traits in one genome can be achieved relatively simple breeding procedures.

The aim of the present invention to provide a plant of cucumber, which have shown resistance to viruses collection closterovirus that affect the cucumber, in particular BPYV and CYSDV, and the plant which, in addition, shows resistance to powdery mildew of cucumber caused by fungiS.fuliginea and/or E.cichoracearum.

The INVENTION

The authors of the present invention was discovered a number of cucumber plants that have shown resistance as to CYSDV and RM. In addition, they discovered that these plants cucumber with dual resistance include two quantitative trait locus (QTL)associated with resistance to powdery mildew (found in this document aspm-landpm-h), and one large QTL associated with resistance to closterovirus (denoted herein as QTL-1). Most importantly, the inventors have discovered that all QTL localized in the same genetic group cohesion. Thus, the authors of the present invention mainly found that in these plants the genes of resistance to closterovirus and powdery mildew are on the same chromosome. Such plants are extremely effective for plant breeding.

The AOC is e, the inventors have discovered a possible mechanism in the absence of which could not be obtained plants of cucumber with dual resistance (with the help of experiments on crossing). Not being tied to any theory, I believe that the introgression of the genetic element, essential for resistance to one disease, led to the loss of genetic elements essential for resistance to other disease. Thanks to this discovery, the inventors have developed a scheme of selection, by which it is possible to solve this problem, while receiving the required plants with double resistance.

In the first aspect of the present invention relates to a plant of the formCucumis sativusthe plant , which shows resistance to closterovirus cucumbers, and resistance to powdery mildew of cucumbers.

In a preferred embodiment, such a plant according to the invention genome(s) region(s)responsible(s) for resistance to closterovirus (QTL-1)and genomic(s) region(s)responsible(s) for resistance to powdery mildew of cucumbers (pm-hand/orpm-l), are located on the same chromosome. In an even more preferred embodiment, such plants are present in bothpm-handpm-l,as QTL-1 is located betweenpm-handpm-l.

In another embodiment, the plant according to the present invention is the development of QTL-1 reveals the presence of at least one flanking marker selected from the group consisting of markers E16/M50-244, E16/M50-188, and E11/M48-251, as described in more detail in document WO 02/22836, to which reference is given in this context.

In another embodiment, the plant according to the invention the presence of a QTL, denoted bypm-hreveal the presence of a nucleic acid sequence comprising at least one mutation resulting from single nucleotide polymorphism (SNP)associated with resistance to powdery mildew in the specified plant, where the specified at least one single nucleotide polymorphism (SNP) selected from SNP1 and SNP2, as shown in table 2 below.

In another embodiment, the plant according to the invention the presence of a QTL, denoted bypm-hreveal the presence of a nucleic acid sequence comprising at least one mutation resulting from single nucleotide polymorphism (SNP), referred to as SNP 3 in table 3 hereinafter, associated with resistance to powdery mildew in the specified plant or comprising at least one mutation, referred to as the 5-bp insertion 5'-AATTT-3' table 3 further associated with resistance to powdery mildew in the specified plant.

Thus, the invention preferably relates to a plant that is resistant to closterovirus cucumber powdery mildew of cucumber, where this plant PR is dstanley a plant of the form Cucumis sativus,moreover, this plant contains one chromosome at least one chromosomal region that confers resistance to closterovirus and at least one chromosomal region that confers resistance to powdery mildew, where the specified at least one region that confers resistance to closterovirus, associated with at least one marker selected from the group consisting of markers E16/M50-244, E16/M50-188 and E11/M48-251, and

where the specified at least one chromosomal region that confers resistance to powdery mildew, associated with at least one marker selected from the group consisting of:

marker single nucleotide polymorphism 39T→G in SEQ ID NO:1,

marker single nucleotide polymorphism 29G→A in SEQ ID NO:2,

marker single nucleotide polymorphism 193C→T in SEQ ID NO:3,

the insertion mutation 5'-AATTT-3' at position 221 in SEQ ID NO:4, and

markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.

The plant according to the invention can optionally contain a second QTL associated with resistance to closterovirus (QTL-2, described in WO 02/22836, the reference in the present description is given for more detailed information on the localization and characteristics of this QTL). It is shown that the QTL is located on a separate chromosome.

In another aspect the invention relates to the plant part on which the URC according to the invention, as explained above. Preferably, a specified part of a plant selected from the pollen of semjazachatki, leaves, roots, root tips, anthers, flowers, fruits, stems, shoots, grafting cuttings, root sprouts, seeds, protoplasts and calli, and most preferably, from a seed.

In another aspect the invention relates to the F1 seed obtained by crossing plants of the cucumber according to the invention with dual resistance (resistance to closterovirus plus resistance to RM [including RM-sheet (pm-land RM-hypocotyl (pm-h) resistant phenotype]), as described previously, with a second cucumber plant or other plants, preferably, from a number of cucumbers, which includes desirable from a commercial point of view characteristics. Preferably, the specified second cucumber plant includes at leastpm-hin the form of a recessive gene. The specified second cucumber plant represents in this case at least the heterozygote, preferably, homozygotes for the recessivepm-hthe symptom. In another preferred embodiment, the specified second cucumber plant is sensitive to closterovirus and, more preferably, is an inbred plant.

In another aspect the invention relates to hybrid plant obtained by growing the seed of F1 according to the invention, as the op is Sano previously.

In another aspect the invention relates to the part of the hybrid plant according to the invention, as described previously. Preferably, a specified part of a plant selected from the pollen of semjazachatki, leaves, roots, root tips, anthers, flowers, fruits, stems, shoots, grafting cuttings, root sprouts, seeds, protoplasts and calli and, preferably, the part of the plant is a fruit.

As indicated above, the introgression of the genetic element, essential for resistance to the RM or to closterovirus, by crossing plants, each of which is stable only for one disease, it is believed, leads to the loss of genetic element, essential for resistance to other disease. With this discovery, the inventors have developed a scheme of selection by which this problem can be solved with obtaining this required plants with dual resistance. The authors of the present invention revealed that for the efficient creation of cucumber plants with dual resistance of hybrid between the parent plant of cucumber line, resistant to closterovirus, where this resistance is due to QTL-1, and a plant of cucumber line, resistant to PM, where this resistance is due topm-h and pm-l,the process of crossing should include the stage of the resolution of the images is of the recombinants (i.e. allow for the implementation of acts of homologous recombination in a single chromosome), followed by a stage of selection of plants with QTL-1 andpm-lor QTL-1 andpm-hin one chromosome. Preferably, the specified stage of sampling involves the selection of plants with QTL-1 andpm-landpm-hin one chromosome. Even more preferably, QTL-1 injected in the genomic region between regions containingpm-handpm-l,so in effect QTL-1 closterovirus placed between two QTL resistance RM.

The presence of any QTL described herein, associated with resistance to the RM or to closterovirus may be detected by detecting one or more genetic markers associated with the corresponding QTL on chromosome plants, genetic markers unbalance communication with the corresponding QTL on chromosome plants or their combination.

A suitable method of detecting the presence and/or location of these QTL in these plants includes the use of AFLP markers to characterize these QTL. Preferably, the specified stage is the selection of plants with QTL-1 andpm-landpm-hin one chromosome, includes the detection of at least one AFLP marker that characterize quantitative trait loci QTL-1, associated with resistance to closterovirus on the same chromosome, which contains a quantitative locus is th trait (QTL) for pm-lor, alternatively, on the same chromosome, which contains a quantitative trait locus (QTL) forpm-hassociated with resistance to RM, preferably on the same chromosome, which contains quantitative trait locipm-handpm-l,and in this case, the specified marker to the QTL-1 is located in the chromosomal region between QTLpm-hand QTLpm-l.Thus, the selection process may include detecting one or more markers selected from the group of markers consists of markers of table 2, 3 and 4.

In another aspect, the present invention relates to a method of selecting plantsCucumis sativus, which has shown resistance to closterovirus cucumbers and mildew of cucumbers, comprising detecting in the specified plant presence on one chromosome QTL-1 and at least one QTLpm-landpm-h.

In one of the embodiments of this method the method involves the following stages:

a) providing a sample of genomic DNA from a plant of cucumber, this sample contains fragments of genomic DNA of sufficient length;

b) conducting the reaction cleanup for the selection of fragments that contain at least the first QTL and molecular marker associated with it from the group consisting of QTL-1,pm-landpm-h;

(C) performing the amplification reaction Nikolayevich acids on these selected fragments of the La detection of fragments, which include at least a second QTL and molecular marker associated with it from the group consisting of QTL-1,pm-landpm-hand

d) detecting the reaction product of stage (C) of the amplified DNA fragment having the predicted length or projected sequence of nucleic acids.

In a preferred embodiment, the specified stage b) includes applying at least one set of primers for the determination of molecular marker for the specified first QTL or applying at least one probe nucleic acid with a primary sequence that is essentially complementary to the sequence of the nucleic acid, determining the marker, and a probe for nucleic acid that specifically hybridizes in stringent conditions of hybridization with the sequence of the nucleic acid, determining the marker(s).

In another preferred embodiment, the specified stage (C) includes applying at least one set of primers for determining the specified second QTL or applying at least one set of primers that specifically hybridize in stringent conditions with the sequence of the nucleic acid marker molecule of the specified second QTL.

In another embodiment, the JV the property according to the invention has a phase detection in the specified plant presence of these QTL on the same chromosome carried out with the use of hybridization techniques in situor amplification techniquesin situ. In turn, these methods appropriately using probes and primers to identify a molecular marker for these QTL or those that specifically hybridize in stringent conditions with the sequence of the nucleic acid marker molecule for a given QTL.

In aspects of the invention molecular markers are preferably SNP markers mutations insert or AFLP markers, more preferably, the markers selected from the group consisting of the markers listed in tables 2-4, and the markers of the reference literature, specially in the present document.

In another aspect, the present invention relates to a method of producing a plant of the formCucumis sativusat the plants of which is shown resistance to closterovirus cucumbers and mildew of cucumbers, which includes stages:

a) selecting a first plant of a cucumber, which includes chromosomal region that confers resistance to closterovirus cucumber, by detecting in the genome of the specified plants the presence of at least one marker linked to the QTL, which imparts resistance to closterovirus cucumbers, shown by markers E16/M50-244, E16/M50-188 and E11/M48-251;

b) selecting a second plant of cucumber, which includes at least one chromosomal region that gives ustoichivosti to powdery mildew, by detecting in the genome of the specified plants the presence of at least one marker associated with the first QTL, which imparts resistance to powdery mildew, the following tokens:

- marker single nucleotide polymorphism 39T→G in SEQ ID NO:1,

- marker single nucleotide polymorphism 29G→A in SEQ ID NO:2, and

markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001; or

by detecting in the genome of the specified plants the presence of at least one token associated with the second QTL, which imparts resistance to powdery mildew, the following tokens:

- marker single nucleotide polymorphism 193C→T in SEQ ID NO:3,

the insertion mutation 5'-AATTT-3' at position 221 in SEQ ID NO:4, and

markers E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200;

(C) crossing these plants from stage a) and stage (b) to obtain F1 seeds;

d) growing a certain number of F1 seeds in the F1 plants, create the next population of descendants of these F1 plants by crossing or selfing and

e) selecting from among the following plants are descendants of plants that includes at least one marker linked to the QTL, which imparts resistance to closterovirus cucumber, as defined in stage a), and at least one marker linked to the QTL, which imparts resistance to powdery mildew of cucumber, as defined in stage b).

Specialist in D. the authorized area is clear, that said, the following population of offspring must be of sufficient size that allows you to detect the presence of plants specified in the next population of descendants who have had at least two acts of homologous recombination on the same chromosome. In General, the population of approximately 1000 plants should be sufficient. Advantage equipment selection with the proposed marker is that in a population of this size can easily make a selection on the presence of interesting genotypes, which may not be identified phenotypically.

In a preferred embodiment of the method according to the present invention qualifying second cucumber plants at the stage b) comprises selecting a second plant of cucumber, with only one of these, the first or the second QTL, which imparts resistance to powdery mildew, cucumber, and where the method additionally includes the stage:

f) selection of the third cucumber plants having other than the first or second QTL, which imparts resistance to powdery mildew of cucumber (i.e. the QTL, which is not present in the specified second cucumber plant);

g) crossing F1 plants obtained in stage e), with the specified third plant of cucumber for further plant-descendants and

h) selection of the number of plants descendants of plants that includes QTL, giving sustainable is here to closterovirus cucumber, as defined in stage a), and both QTL, which imparts resistance to powdery mildew of cucumber, as defined in stage b).

In a preferred embodiment, choose plants that include both the locus ofpm-handpm-lgiving resistance to powdery mildew, and in which the locus, which imparts resistance to closterovirus characterized by QTL-1, is located between locipm-handpm-lgiving resistance to powdery mildew.

Thus, in another preferred embodiment, a method of producing a plant of the formCucumis sativusthat have shown resistance to closterovirus and to powdery mildew, cucumber, qualifying plants of cucumber, which contains a chromosomal region that confers resistance to powdery mildew of cucumber, as defined in the stages b), e), f) or h), includes:

detection of the presence in the genome of the specified plants at least one token associated with the first QTL, which imparts resistance to powdery mildew of cucumbers, shown SNP marker T→G in SEQ ID NO:1, SNP marker 29G→A in SEQ ID NO:2 and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001; and

- detecting in the genome of the specified plants the presence of at least one token associated with the second QTL, which imparts resistance to powdery mildew of cucumbers, shown SNP marker 193C→T in SEQ ID NO:3, the insertion mutation 5'-AATTT-3' at position 221 in SEQ ID NO: markers E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.

In another preferred embodiment, a method of producing a plant of the formCucumis sativus, which has shown resistance to closterovirus and to powdery mildew, cucumber, at least one of stages a), b), e), f) or h) includes a step of obtaining a sample of genomic DNA from the indicated plant and detecting in a specified sample of genomic DNA indicated at least one token.

In an alternative preferred embodiment, stage (e) includes detecting in the genome of the plant at least one token associated with QTL-1, and at least one marker associated withpm-handpm-lpreferably, detecting in the genome of plants and on one chromosome at least one token associated with QTL-1, at least one token associated withpm-hand at least one marker associated withpm-lpreferably, the content of the markers selected from the group of markers listed in tables 2, 3 and 4.

In another preferred embodiment, stage (e) carry out the method according to the invention for the selection of plant speciesCucumis sativusdouble resistance, as described above.

It should be understood that the method described in stage (C)to (e), in which at least two acts of homologous recombination in one chromium is ome in a single interbreeding with the production of F1 dual resistance, can be implemented with multiple generations such, so offer (at least two) of the act homologous recombination and, thus, the formation of plants with double resistance is implemented in F2, F3, F4, F5, or in any subsequent generation. Such variations are within the scope of the present invention and can be easily carried out by a specialist in this field.

In an alternative method of obtaining plantsCucumis sativus,which is resistant to closterovirus and mildew of cucumber, the method may include stages:

a) selecting a first plant speciesCucumis sativuswith resistance to closterovirus and to powdery mildew, cucumber implementation of the method according to the invention;

b) inbreeding specified plants with obtaining plant lines, homozygous for the specified QTL;

(C) crossing these plants stage a) and stage b) to produce F1 seeds;

d) growing seeds F1 in F1 plants.

In another aspect the invention relates to plants obtained by the methods according to the invention, or their parts.

The present invention makes possible very rapid screening of plant descendants to select plants that are able to double the resistance. For example, among the plants of the descendants you can find plants that have at least onepm-hmarkers, at least one of thepm-lmarkers and what about the at least one QTL-1 markers. These plants may have acquired the desired introgression. Screening of additional markers may increase the confidence level of the prediction that plant-descendant has double the resistance. After the discovery of plants with at least one marker in each of the three QTL plant can be selected and used in breeding programs or for additional experiments to determine the characteristics.

The advantage of the plants of the present invention, in which the signs are located on the same chromosome, is that they provide better opportunities for a more free movement of genes that control both forms of resistance, in other (hybrid) plants using conventional breeding, although if the signs are located on separate chromosomes, it would be much harder to get hybrids containing a combination of both characteristics by applying the techniques of conventional breeding.

Description of the DRAWINGS

In figures 1-6 shows a possible scenario of recombination, in which resistance to powdery mildew (pm) and closterovirus (QTL-1) two separate plants together in one plant-derived. Note that different parents listed as having multiple loci stability, can themselves be the result of crossing or selfing. So about what atom, the plant, having both QTL,pm-landpm-hcan be obtained by crossing plants with one QTL.

The figure 7 shows the location of different genes for resistance to powdery mildew (pm) fromCucumis sativusNPI and resistance genes closterovirus (QTL-1)C.sativusKhira in one group cohesion. Herein Pm-leaf is equivalent topm-land Pm hypo is equivalent topm-h.

The figure 8 shows the DNA sequence in the region and SNP markers inserts that are shown with the corresponding SEQ ID tables 2 and 3.

DETAILED description of the INVENTION

Definitions

The term "cucumber", used herein, refers to the plant, or its parts, of the formCucumis sativus,including, without limitation, plants, usually referred to as cucumber (Cucumber), American gherkins (American gherkin), cassabanana (Cassabanana), Zieleniec (Cuke), gherkins (Gherkin), greenhouse cucumber (Hothouse cucumber, cucumber-lemon (Lemon cucumber, cucumber Mandera (Mandera cucumber, gherkins (Pickling cucumber), snake cucumber (Serpent cucumber)salad, cucumber (Slicing cucumber), snake cucumber Snake cucumber and cucumber West Indies (West Indian gherkin).

Used herein, the term "plant part" refers to part of the plant, including single cells and tissue, such as plant cells that are intact in plants, clumps of cells and culture the notches, of which can be regenerated plants. Examples of plant parts include, without limitation, isolated cells and tissues of the pollen, semjazachatki, leaves, embryos, roots, root tips, anthers, flowers, fruits, and lateral shoots; as well as pollen, semjazachatki, leaves, embryos, roots, shoots roots, anthers, flowers, stems, shoots, shoots, roots, shoots, seeds, protoplasts, and Callie etc.

The term "closterovirus"used herein refers to a virus family Closteriviridae, including, but not limited to, viruses, commonly called virus disease yellow stunting cucumber (CYSDV), virus infectious jaundice salad (LIYV), virus pseudoalhagi beet (BPYV; also known under the synonyms virus chlorotic leaf spot of cucumber (CCSV), jaundice virus cucumber, jaundice virus melon or virus paludosa strawberry), preferably, BPYV and CYSDV, most preferably, CYSDV. Preferably, in respect of a cucumber, the term "closterovirus" refers to the genus particularly important for plants of cucumbers, ieCriniviideae.

The term "powdery mildew", used herein, refers to a fungal disease caused in cucumber (Cucumis sativus L.) fungusSphaerotheca fuliginea(also known asPodosphaera xanthiiandS.cucurbitae), and/or fungusErysiphe cichoracearum(also swetnam as Golovinomyces cichoracearum), and/or fungusLeveillula taurica(also known asOidiopsis taurica, Erysiphe taurica, Ovulariopsis cynarea, Leveillula solanacearum).

The term "QTL" is used herein in its accepted in this field the value of the chromosomal region containing alleles (for example, in the form of genes or regulatory sequences)that are associated with the expression of constantly presents (quantitative) of the phenotypic trait.

The term "QTL resistance to disease" refers to a region located on a specific chromosome, which is associated with at least one of the genes that encodes resistance, or at least with the regulatory region, i.e. a region of a chromosome that controls the expression of one or more genes associated with resistance. For the consideration of QTL associated with resistance in this document use a shorter equivalent of "locus, sustainability". QTL can be established by identifying their genetic location in the genome of specific sampleCucumis sativus,using one or more molecular genetic markers. One or more markers, in turn, identify a specific locus. Distances between loci is usually measured by frequency of crossing over between the loci on the same chromosome. The more distant are two Loka is a, the higher the probability of crossing over between them. Conversely, if two loci are close to each other, the crossover between them less likely. Typically, one centimorgan (function cards Kosambi (cm)is approximately equivalent to 1% recombination between loci (markers) (Lui, 1997). When QTL can be shown multiple markers, the genetic distance between the end point markers indicates the size of the QTL.

The term "chromosome" is used herein in its accepted in this field the value of self-replicating genetic structure in a cell nucleus that contains the cell's DNA and carrier in its nucleotide sequence the linear array of genes.

Used herein, the term "coupling" refers to all genes or genetic traits that are located on the same chromosome. Within the group of coupling those loci that are close enough to each other, will form the clutch in genetic crosses. Because the probability of crossing-over increases with the physical distance of the genes on the chromosome, genes whose location is considerably removed from each other within the group clutch may not show any coupling in the direct genetic tests. The term "clutch" is used mainly in relation to genetically the fir loci have been linked behavior in genetic systems, where chromosomal distribution have not yet been made. Thus, in the present context, the term "clutch" is a synonym (physical object) of the chromosome.

Used herein, the term "allele (alleles)" means any one or more of the alternative forms of a gene and belongs at least to one trait or characteristic. In a diploid cell has two alleles of this gene occupy corresponding loci on a pair of homologous chromosomes. Since the present invention relates to a QTL, i.e. genomic regions, which may include one or more genes or regulatory sequences, in some cases more accurate to speak of "haplotype" (i.e. allele chromosomal segment) instead of the allele, but in such cases it should be understood that the term "allele" includes the term "haplotype".

Herein "gene" refers to a unit of heredity, consisting of a sequence of DNA that occupies a specific location on the chromosome and which contains the genetic instruction for a specific characteristic or trait of an organism.

In this document "locus" refers to the position of this gene or regulatory sequence on the chromosome of this species.

"Th is ologica recombination" is a currency ("crossover") DNA fragments between two DNA molecules or chromatid pairs of chromosomes in the region of identical nucleotide sequences. Under the "act recombination" herein should be understood meiotic crossing over.

Used herein, the term "molecular marker" refers to the indicator, which is used in the methods of visualization of differences in the characteristics of the sequences of nucleic acids. Examples of such indicators are markers of length polymorphism fragments (RFLP)markers length polymorphism of amplified fragments (AFLP), single nucleotide polymorphisms (SNP), insertion mutation, microsatellite markers (SSR)markers amplified region is characterized by the sequence (SCAR)markers cleaved amplified polymorphic sequence (CAPS) or isozyme markers or combinations of markers described herein, which define specific genetic or chromosomal location. "Molecular marker linked to a QTL", as defined herein, may, therefore, refers to the SNP, insertion mutations, as well as to more conventional AFLP markers, or any other type of marker used in this field. In the context of AFLP markers mentioned in this document, the markers indicate the specific DNA sequence of cucumber, flanked by two AFLP-primers these primers consist of a Central law is the size of E and M, the corresponding sites of the restriction enzymes EcoRI and MseI, (Vos et al., 1995; Bai et al. 2003), followed by 2 or 3 additional selective base as an indicator, each accompanied by a two-digit code that identifies selective nucleotides, which increases Central primer" (codes are listed in table 1).

E16/M50-244 is a token obtained using amplification primers EcoRI + CC and MseI + CAT, to obtain a fragment, with a total length of 244 BP Length fragment may depend on the method used for detection of the fragment, and represents an approximation of its true length plus or minus a few reasons. When defining marker, as provided herein, must be made with reference to the position on the chromosome of the marker relative to other markers in the map module. Thus, the marker E16/M50-244 determined by its sequence of primers and using its length as the product of amplification and using its position relative E14/M59-F-200 and/or E23/M38-M003 or, as provided herein, using its position relative to other markers, as shown in the numbered list, with the corresponding distance in cM in the matrix in table 4. However, it should be taken into account that the interbreeding of races, the events can result in the loss of certain markers, so the lack of a specific marker does not exclude the presence of a genetic element that confers resistance to disease and is associated with the specified token.

Table 1
Codes primers that are typically used in AFLP analyses and are used in this document (source: Keygene, Wageningen, The Netherlands)
Code primerIncreaseCode primerIncreaseCode primerIncreaseCode primerIncrease
01A32AAC53CCG74GGT
0233AAG54CCT75GTA
03G34 AAT55CGA76GTC
04T35ASA56CGC77GTG
11AA36ACC57CGG78GTT
12AC37ACG58CGT79TAA
13AG38ACT59HUNDRED80TAG
14AT39AGA60CTC81 TAG
15CA40AGC61CTG82TAT
16CC41AGG62CTT83TCA
17CG42AGT63GAA84TCC
18CT43ATA64GAC85TCG
19GA44ATS65GAG86TCT
20GC45 ATG66GAT87TGA
21GG46ATT67GCA88TGC
22GT47CAA68GCC89TGG
23THE48CAC69GCG90TGT
24TC49CAG70GCT91TTA
25TG50CAT71GGA92 TTC
26TT51CCA72GGC93TTG
31AAA52CCC73GGG94TTT

The term "specific DNA sequence of the cucumber" means a polynucleotide sequence with homology to the nucleotide sequence of more than 80%, preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, even more preferably more than 97%, most preferably more than 99%, with the genome sequence of the formCucumis sativus,which shows the greatest similarity with her, preferably, in the case markers QTL-1, part of the DNA sequence of the sample cucumber PI 250147 flanking markers for the QTL-1.

The term "homology of the nucleotide sequence"used herein denotes the homology between the two polynucleotide. Polynucleotide are "homologous" sequence, if the sequence of nucleotides in the two is posledovatelnostyakh is the same, if it is aligned for maximum correspondence. Comparison of sequences of two or more polynucleotides usually carried out by comparing the two parts of the sequence in the comparison window to establish and compare the similarity of the local regions in the sequences. The comparison window is usually from 20 to 200 neighboring nucleotides. "Percent homology sequences" polynucleotide, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100% homology sequences can be established by comparing two optimally aligned sequences in the comparison window, where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of two sequences. Percentage calculated using: (a) determine the number of positions in which the same base nucleic acid is both sequences, and counting the number of matching positions; (b) dividing the number of matched positions by the total number of positions in the comparison window, and (C) multiplying the result by 100 to obtain the percent homology sequences. Optimal alignment of sequences for comparison may be conducted using the computer the computer programs known algorithms or by visual inspection. Easily accessible algorithms of sequence comparison and multiple sequence alignment are respectively of ' the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990; Altschul et al, 1997) and "ClustalW", available on the Internet. Other suitable programs include, but are not limited to, GAP, BestFit, PlotSimilarity and FASTA in the software package Wisconsin Genetics (Genetics Computer Group (GCG), Madison, WI, USA) (Devereux et al., 1984).

The term "pm-h"used herein refers to the estimated gene of resistance to powdery mildew, expressed in the hypocotyl, preferably determined by the presence of specific SNP listed in table 2.

Table 2
SNP showing full connection with the phenotype of resistance to powdery mildew, shown herein as QTLpm-h.
SNP#DesignationImmune n.Susceptible n.The position of the nucleotide (SEQ ID NO)
SNP139T>GGT39 (SEQ ID NO:1)
SNP2AG29 (SEQ ID NO:2)

The term "pm-l"used herein refers to the estimated gene of resistance to powdery mildew pm-leaf, preferably defined by the presence of specific mutations listed in table 3.

Table 3
Mutations showing full connection with the phenotype of resistance to powdery mildew, shown herein as QTLpm-l.
MutationDesignationImmune n.Susceptible n.The position of the nucleotide (SEQ ID NO)
SNP3S>TT193 (SEQ ID NO:3)
Insertl221→AATTTAATTT-221-225 (SEQ ID NO:4)

The term "QTL-1" refers to a genomic region associated with resistance to closterovirus defined by markers E16/M50-244, E16/M50-188 and/or E11/M48-251, described in detail between the national patent WO 02/22836, described in this context as a reference.

The term "plant-child" refers to any plant produced as offspring when vegetative or sexual reproduction from one or more parent plants or their descendants. For example, the plant-child relationship can be obtained by cloning or samoobrazovaniem parent plants or by crossing two parent plants and covers self-pollination as well as F1 or F2 or more remote generation.

F1 is a descendant of the first generation from the parents, at least one of which is used for the first time as a donor characteristic, while the offspring of second generation (F2) or subsequent generation (F3, F4, etc.) represents the instances obtained by self-pollination of F1, F2, etc. Therefore, F1 can be (and usually is) a hybrid obtained by crossing two homozygous parents, while F2 can be (and usually is) the offspring resulting from self-pollination of these F1 hybrids.

Used herein, the term "heterozygote" means this is a genetic condition, when different alleles reside at corresponding loci on homologous chromosomes.

Used herein, the term "heterozygote" means so the e is a genetic condition, when identical alleles reside at corresponding loci on homologous chromosomes.

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

Used herein, the term "inbred" means, essentially homozygous individual or line.

Used herein the term "introgression", "introgressive" and "introgressive" refers to both natural and artificial process by which genomic regions of the same species, variety or cultivar is transferred into the genome of another species, variety or cultivar by crossingover these species. The process is optional, can be added back crossover with recurrent parent.

"Genetic engineering", "transformation" and "genetic modification" are used herein as synonyms move isolated and cloned genes in DNA, usually in the chromosomal DNA or genome of another organism.

Used herein, the term "population" means a genetically heterogeneous collection of plants that have a common genetic origin is giving.

Used herein, the terms "class" and "cultivar" means a group of similar plants that by structural or genetic characteristics and/or productivity can be distinct from other varieties of the same species.

The terms "sustainable" and "sustainability" include partial and complete resistance to infection. Sensitive plant can either be unstable or have low levels of resistance to infection. The term is used to include such separately identifiable forms of resistance as "full strength", "immunity", "average strength", "partial resistance", "hypersensitivity" and "tolerance".

"Full strength" refers to the complete inability of the disease to develop after infection, and it can be either the result of the failure of the disease to penetrate into the cell (not the initial infection), or may be the result of the inability of the agent to multiply in the cell and infect further cells (no subliminal infection, no distribution).

The term "sensitive" is used herein in relation to a plant that does not have resistance to disease, leading to the defeat of the plant disease resulting in the symptoms of the disease. The term "sensitive" is respectively equivalent to the term "Neue is sustainable".

The term "hybrid" in the context of nucleic acids refers to double-stranded molecule of nucleic acid, or duplex, formed by hydrogen bonding between complementary nucleotide bases.

The terms "hybreed" or "denaturiruet" refers to the process by which a single chain nucleic acids sequences form two helical segments through hydrogen bonding between complementary bases.

The term "probe" refers to single-stranded oligonucleotide sequence, which forms a hydrogen-linked duplex with a complementary sequence in the analyte mistaway nucleic acid sequences or cDNA derived.

The term "primer"used herein refers to an oligonucleotide that is capable of renaturation of target amplification and allows you to join DNA polymerase, being a point of initiation of DNA synthesis when placed in conditions in which the induced synthesis of the product, lengthening due to the primer, i.e. in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at suitable temperatures and pH. For maximum efficiency in amplification primer (amplification) is preferably single-stranded. Preferably, the primer is a small town in which to place oligodeoxyribonucleotide. The primer must be sufficiently long to start the synthesis of elongated segments in the presence of a polymerization agent. The specific lengths of the primers will depend on many factors, including temperature and composition (the content of A/T and G/C) primer. A pair of bi-directional primers consists of one forward and one reverse primer, usually used for DNA amplification, such as PCR amplification. It should be understood that the term "primer"used herein may refer to more than one primer, particularly in the case where there is some ambiguity in the information terminal sequences (sequences) mesiniaga region for amplification. Therefore, the term "primer" includes a group of oligonucleotide primers containing sequences representing possible variations in the sequence of, or consists of nucleotides that allow typical connection base pairs. Oligonucleotide primers can be obtained in any suitable way. Methods for producing oligonucleotides specific sequences are well known in this field and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. The methods of chemical synthesis may include, for example, phosphodi or phosphocreatine the manual, diethylphosphoramidite way and the way on a solid substrate, are described, for example, in international patent WO 4458066. Primers, if desired, can be labeled include funds, defined, for example, by a spectroscope, fluorescence, photochemically, biochemical, immunochemical or chemical means. Dependent matrix elongation of the oligonucleotide primer (primer) is catalyzed by polimerizuet agent in the presence of appropriate amounts of the four deoxyribonucleotides (dATP, dGTP, dCTP and dTTP, i.e. dNTP) or their equivalents in the reaction environment, which includes the appropriate salts, metal cations, and a buffer system pH. Suitable polimerizuet agents include enzymes that catalyze the synthesis of DNA-dependent primer and matrix. Known DNA polymerases include, for example, DNA polymerase I, E.coli or piece maple, T4 DNA polymerase and Taq DNA polymerase. Reaction conditions catalysis of the synthesis of DNA data DNA polymerase known in the field. The products of synthesis are double molecules consisting of chains of the matrix and chain elongation of the primer, which include the given sequence. These products, in turn, serve as a matrix of another replication cycle, the chain elongation of the primer of the first cycle sanatoriums with its complementary PR is amerom; the synthesis provides a short product that communicates on both 5'- and 3'-ends of the primer sequences or their additions. Repeated cycles of denaturation, renaturation of primer and extension result in the exponential accumulation of the target region defined by the primers.

To obtain the desired number of polynucleotide containing mechaniy region of nucleic acid, conduct a sufficient number of cycles. Desired number may vary and is determined by the function of the polynucleotide product. The PCR method is described in detail in the guidelines and a well-known specialist in this field.

After amplification by PCR miseriya polynucleotide can be determined by hybridization with polynucleotide probe which forms a stable hybrid with such mistaway sequence, under conditions of hybridization and washing conditions from hard to moderately hard. If you expect that the probes are essentially complementary (i.e., about 99% or more) mistaway sequence, then use the hard conditions. If you wait a mismatch, for example, if you expect variants of strains with the result that the probe is completely complementary, the rigidity of hybridization can be reduced. However, choose conditions which exclude non-specific/random linking. Conditions, which is haunted affect hybridization and which select against non-specific binding, known in this field and are described, for example, in Sambrook and Russell, 2001. In General, whiter low salt concentration and higher temperature increase the rigidity of the hybridization conditions.

The phrase "stringent hybridization conditions" refers to conditions under which polynucleotide will hybridisierung with his mistaway sequence, typically in a complex mixture of nucleic acids, but essentially no other sequences. Hard conditions depend on the sequence and will vary under different circumstances. A detailed description of the hybridization of nucleic acids can be found in Tijssen, 1993. As a rule, choose the hard conditions that 5-10°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength pH. Tmis the temperature (under defined ionic strength, pH and concentration of nucleic acids)at which 50% of the probes complementary to the target, hybridized with mistaway sequence at equilibrium (as miseriya sequence presents in excess, in Tm50% of the probes is in the equilibrium position).

Stringent conditions are those conditions under which the salt concentration is less than 1.0 M of sodium ions, generally the concentration of the sodium ion (or other salts) is from about 0.01 to 1.0 M at pH ot,0 to 8.3, and the temperature is at least 30°C for short probes (e.g., 10 to 50 nucleotides) and at least 60°C for long probes (e.g., more than 50 nucleotides). Stringent conditions can be achieved by the addition of destabilizing agents such as formamide.

For selective or specific hybridization, a positive signal is equal at least two times, preferably 10 times background hybridization. Rough tough hybridization are often the following conditions: 50% formamide, 5×SSC and 1% SDS, incubating at 42°C, or 5×SSC, 1% SDS, incubating at 65°C, with wash in 0.2×SSC and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for weak amplification, although annealing temperature may vary between about 32°C and 48°C, depending on the length of the primer. Additional recommendations for selection of the parameters of hybridization are provided in numerous references (e.g., Ausubel, et al., 1999).

Description of the preferred embodiments

Obtaining plants with dual resistance

The aim of breeding programs in agriculture and horticulture is increased plant productivity by improving their genetic composition. In essence, this improvement increases with increase in the frequency of favourable alleles of genes that affect not only the matter of the interest features of productivity. Line of wild plants provide a rich resource of genetic and phenotypic diversity. In agricultural or horticultural practice traditionally use the specified diversity by selecting lines of wild plants or his descendants to the desired genotypic or potential phenotypic properties, crossing his line, having additional desired genotypic or potential phenotypic properties, and selection among plants of the descendants of those plants that show (increased frequency) desired genotypic or potential phenotypic properties.

Increasing the understanding and use of the Mendelian laws of heredity in combination with the tools of molecular genetics has facilitated the selection process in the last century. For example, became available methods of selecting plants with the desired genotypic or potential phenotypic properties, based on testing specified plants for the presence of quantitative trait loci (QTL), i.e. the presence of the chromosomal region containing the alleles associated with the expression of continuously presents (quantitative) of the phenotypic trait. Usually QTL is characterized by one or more markers that are statistically associated with quantitative diversity in fenotipicheskaya, and, essentially, it is a synonym of the gene. The QTL mapping allows to identify the loci candidates, influencing the expression of the trait of interest. In plant breeding, QTL mapping allows assisted selection marker (MAS), i.e. the selection of plants having favorable alleles by detecting in plant markers associated with QTL.

One of the biggest challenges of breeding programmes of cultivated plants is the presence of negative genetic correlations between different traits. For example, the case of a negative genetic correlation between reproductive capacity and productivity in various lines of plants that are resistant to diseases. The explanation appears if show that introgression of DNA from the genome of one plant lines to another can interfere with or influence the expression of key reproductive characteristics. Similarly, try to introgressive genetic sequence, sustainability, from one plant to another can remove signs of resilience, already existing in the recipient line.

Knowledge of the inheritance of different traits allow, for example, breeding lines, homozygous for the QTL associated with resistance to disease. Application of knowledge about the genetic origin and location of the desired sign of the breeding program increases the accuracy of the predicted result of selection and speed up the selection compared to traditional breeding programmes. For example, the fact that the genetic basis of the desired trait inheritance is associated with a different characteristic, helps to improve the homogeneity of these two traits in the offspring, because the parent heterozygote for the desired alleles will send them to the most offspring that will appear in the form of weak segregation in the progeny.

As mentioned earlier, the authors of the present invention researched plants that were resistant to closterovirus and mildew. Such plants were not known previously. Attempts to introgressive DNA lines resistant to closterovirus, in the line of plants that already have resistance to powdery mildew, or Vice versa previously had no success.

In addition, the inventors found that plants with dual resistance genes resistance to closterovirus and powdery mildew are closely related, that, essentially, they are both inherited as a single unit. This genetic configuration so closely related genes are sometimes referred to as alleles in-phase coupling (in cis). In such cases it is usually supposed that genes are located on the same chromosome. In fact, if make reference to the presence of genes or QTL on the same chromosome, it means that they are in-phase coupling.

The inventors found that plants with dual resistance is awn resistance genes to closterovirus and mildew represented in the group of clutch 4. According Horejsi et al. (2000), the clutch 4 Cucumis sativus L also includes genes for resistance to downy mildew, (dm) resistance. Next LG 4 can be described (or) one or a combination of the following RFLP markers (defined using a software package INTMAP, Keygene, Wageningen, The Netherlands; the position on the map in cM in parentheses): CsC032a/El (25.9); CsP357/H3 (31.7); CsC588/H3 (34.2); CsC477H3 (35.3); CsC694/E5 (38.5); CsP347/H3 (38.5); CsC365/El (41); CsC386/El (41); CsC230/El (41.7); CsP064/El (45.5).

The exact chromosome number of cucumber, which are QTLl resistance to closterovirus andpm-landpm-hresistance to powdery mildew has not been identified.

Despite this, the chromosome can be identified by reference to the group clutch (LG 4), where these and other genomic regions. The term "group cohesion", used herein, refers to the physical unit of the genome, which are alleles, sustainability, and which has the same hierarchical level as the chromosome.

The consequences of the discovery that the genes for resistance to closterovirus pm and are located on the same chromosome, have a double meaning.

First, for breeding purposes it is very convenient to have genes located on the same chromosome, more preferably, even on the closest segment of the chromosome, because they will jointly be transmitted to the plants-then the am. As a result of physical connection between genes responsible for resistance to powdery mildew, and genes responsible for resistance to closterovirus, plants are descendants of crosses in which the plant of the present invention with dual use sustainability as a parent, provide low frequency segregation in plants descendants.

Secondly, the knowledge can facilitate plant breeding, because they give a possible explanation why it is so difficult to combine two things in one line of plants. Not bound by any theory, believe that the specific position of the QTL responsible for resistance to closterovirus, and breeding for this QTL prevented the formation of the desired introgressions or at least allowed the emergence of the very few desired introgressions. QTL associated with resistance to powdery mildew, and QTL resistance to closterovirus were located in such close proximity that the introgression of QTL characteristic of the second resistance (for example, closterovirus) in the plant line that already has a first resistance (e.g. powdery mildew), could lead to a (partial) loss of the first sign of resistance. In cases where the plant line has both QTL of resistance to powdery mildew (pm-landpm-h), the QTL introgression of resistance to closterovirus can occur only what about in a very small region of the genome, without "damaging" the genetic information that underlies resistance to powdery mildew.

Thus, in addition to the discovery that genes of different stability were on the same chromosome, it was found that, if both alleles of resistance to powdery mildew were presented in plants with dual resistance, QTL associated with resistance to closterovirus (QTL-1), was located between the two QTL associated with resistance to PM (pm-handpm-l).

This discovery may additionally, at least partially, to explain the observed low frequency of recombination in the desired genotype. In addition, it suggests at least three possible scenarios of how these plants can be created, allowing multiple reproducible methods for their preparation.

The first method could include the introgression of loci, which imparts resistance to closterovirus (referred to in this document as QTL-1) plants of the first line of interest, resistant to powdery mildew plant the second line of interest and carrying two loci, which imparts resistance to PM,pm-handpm-l. If this results in the heredity of the second line of interest, the method could include the introgression of QTL-I in the genomic region between the two loci, which imparts resistance to PM,pm-hand pm-l, that is to result in a set of genomic DNA sequences with the configuration ofpm-l- QTL-1 -pm-hin the plant-descendant with heredity second line of interest (see figure 1). On this basis suggest that only the act of double homologous recombination (i.e. one act for each crossover) in a specific region of one chromosome can result in a phenotype with dual resistance to closterovirus and PM in the heredity of the second line of interest.

Making the right introgression in plants-the descendants can be tested using specific markers QTL-1,pm-1andpm-h.

In the second method it is possible to combine signs of resilience in a different genetic environment, for example, in the first line of interest. This method can include the introgression of both loci, which imparts resistance to PM,pm-handpm-lfrom plants of the second line of interest into the plant resistant to closterovirus first line of interest, which is the locus QTL-I. In this case, the method could include the introgression of both loci,pm-handpm-lregardless, on both sides of the locus QTL-1, obtaining the result set of the genetic sequence of the DNA having the configuration ofpm-l- QTL-1 -pm-hin the plant-descendant with the heredity of the first line of interest (smot and figure 2). Based on this configuration, predict that requires at least two acts of homologous recombination (i.e. four of the act of crossing over) in specific regions of the same chromosome to obtain the phenotype of the double resistance to closterovirus and PM. Making the right introgression in plants-the descendants again can be tested using specific markers QTL-1,pm-1andpm-h.

The above can explain why the introgression of CYSDV sustainability, for example, from wild-type, for example, in PM-sustainable, commercially valuable line of cucumber is a very rare event. You can also explain why the introgression of both loci PM sustainability, for example, from the wild, for example, in resistant closterovirus, commercially valuable line of cucumber is an even more rare event.

Recombination is the exchange of information between two homologous chromosomes during meiosis. In recombinant plant DNA, which source is on a special place in the chromosome is exchanged for the DNA of another plant (i.e. maternal to paternal or Vice versa). In the double recombinant such exchange occurred twice, for example, on both sides of the gene/locus. To change only the desired material and as much as possible to save an important source of information on the chromosome, it is usually necessary to undertake the tee four crossover (see above). The usual way of detecting such a double recombinant represents a mass screening of the population of F2 plants. This population should be of sufficient size to detect rare (low frequency) double recombinants. The frequency of double recombination is the product of the frequencies of single recombinations. For example, recombinant in the area of 10 cM can be detected with a frequency of 10%, and double recombinants can be detected by frequency 10% × 10% = 1% (1 centimorgan defined as 1% recombinant offspring in test crosses).

One of the workarounds for this problem of low frequency of occurrence is conducting parallel recombination". Essentially, this means that after two acts of recombination in two different plants, conduct combined acts in the same plant recombination by simply crossing and selection in the resulting F2. The result is a reduced number of plants for screening.

Therefore, the third method of obtaining plants with dual resistance could include the introgression of one of the two loci, which imparts resistance to PM,pm-horpm-lfrom the first line of interest into the plant resistant to closterovirus second line of interest, which is the locus QTL-1, which results in the set of sequences of genomic DNA, there is a corresponding configuration pm-l- QTL-1 or QTL-1 -pm-hand introgression remaining two loci, which imparts resistance to PM,pm-horpm-lfrom the third line of interest in other plant resistant to closterovirus second line of interest, which is the locus QTL-1 to obtain the result set sequence of genomic DNA that has a configuration QTL-1 -pm-horpm-l- QTL-1 (see figure 3). Then crossed plants descendants containing introgression from each individual recombination, to obtain a sequence of genomic DNA having the configuration ofpm-l- QTL-1 -pm-h(see figure 3). Making the right introgression in different plants descendants again can be tested using specific markers QTL-1, pm-l and pm-h, on the basis of which the selected plants for further crosses. Other methods can be obtained by the person skilled in the art from figures 4-6.

The way to limit the number of plants for screening can be carried out, for example, as follows: to cross two individuals, one containing both alleles PM resistance (pm-landpm-h), and one containing the allele resistance QTL, QTL-1. Select plants with 1 crossover between one of pm alleles and QTL-1 as follows: if the genetic distance between the two end points of two pm-alleles is 10 cM and QTL is located approximately 5 cM between QTL-1 any of the pm-1 or pm-h, then, the recombination between QTL-1 and any pm-locus occurs with a frequency of 5%. In this regard, spend the first selection of 200 plants descendants of crosses, of which 10 will be recombinants of type A and 10 - recombinants type B. Snoopys identified in this way recombinants and filter 10 plants nursery homozygotes using markers flanking both loci, QTL-1 and pm-locus. Spend 10 crosses between homozygotes (A × B (10 homozygotes And were crossed with 10 homozygotes (B), and in the resulting F2 descendants filter 10 plants to find 1 double recombinant. In the end receive 10 independent double recombinants. The number of plants in the screening of only 500, while in the ordinary way would be screened 4000 plants to get in the 10 independent double recombinants (a distance of 5 cM between QTL-1 and any pm-locus with the frequency of double recombinants is equal to 0,05 x 0,05=0,25%). The present invention provides improved model selection using markers (MAS). Therefore, the invention relates to methods for breeding plants and to methods of plant breeding, in particular of a cucumber, especially of cultivated plants of cucumbers, as a manufacturer of plants for use in breeding programs or cultivated plants of cucumber for the floor of the treatment desired genotypic or potential phenotypic properties, in particular associated with obtaining valuable fruit of the cucumber, also referred to herein commercially valuable plants. In this document cultivated plant called the plant, which was specially selected or derived from a plant that was specially selected agricultural or horticultural practice to achieve the desired genotypic or potential phenotypic properties, in particular a plant obtained by inbreeding.

Plants with double resistance according to the present invention may have, for example, the genotype AABBcc, AABbcc, AaBBcc or AaBbcc, where "A" represents the genotype of sustainability, based on the dominant locus ofpm-land "a" is its corresponding allele instability; "B" represents the genotype of sustainability, based on the dominant locus QTL-1, and b is its corresponding allele instability; and "c" represents the genotype of sustainability, based on a recessive locus ofpm-h. Thus, plants with double strength in the present invention include homozygous plants and hybrid plants. These genotypes can be obtained by implementation of the following crosses (the genotype of the parental gametes): ABc X ABc; ABc×Ab; ABc x aBc; ABc x abc, and Abc x aBc. Thus, it is seen that the plant, giving the gamete abc (a plant that is not your condition is stable, excluding the pm-h), however, can be used to obtain plants (hybrid) according to the invention with dual resistance according to the invention. In a particularly preferred variant of the invention, the plant according to the invention with double resistance is inbred plant is homozygous for the alleles of sustainability. Because the locus ofpm-his recessive, it is impossible to control in F1 or BC1 using myexperiment, as occurs when using a sensitive parent to obtain F1/BC1 (as adopted in breeding). Therefore, a special advantage is that making the right introgression in plants-the descendants can be observed using QTL-specific markers, as provided herein. By using methods MAS or MAB specialist in this area, therefore, has ways of selection of plants.

Thus, the present invention also relates to methods selection of plant speciesCucumis sativuswith resistance against closterovirus cucumber powdery mildew of cucumber, including the discovery in the specified plant availability on one chromosome QTL-1, described herein, and at least one QTL,pm-landpm-hdescribed in this document. In the preferred method according to the invention for the selection of such plants the way VC is uchet:

a) obtaining a sample of genomic DNA from a plant of cucumber;

(b) detecting in the specified sample of genomic DNA at least one molecular marker linked to a QTL selected from the group consisting of QTL-1,pm-landpm-hmore preferably, detection of at least two molecular markers of the specified group, where one marker identifies resistance to closterovirus and another marker identifies resistance to powdery mildew.

Stage a sample of genomic DNA from a plant of a cucumber can be carried out by standard methods of DNA extraction are well known in this field.

Stage detection of molecular marker (stage b) may include in the preferred embodiment, application of a set of bidirectional markers that were used in the AFLP method for producing the product of amplification, which, as was proved later, is a suitable marker for the QTL. This set of primers is known as the primers that define AFLP marker or marker-specific primers. "Bidirectional" means that the orientation of the primers is such that one primer is functioning as the direct and the other functions as a reverse primer in the amplification reaction of nucleic acids.

Alternative stage detection of molecular marker (stage b) may include others who d preferred embodiment, the application of the probe nucleic acid with the main sequence, which is, basically, a complementary nucleic acid sequence that defines the specified molecular marker, and a probe nucleic acid that specifically's hybrid under stringent conditions with a nucleic acid sequence that defines the specified molecular marker. A suitable probe nucleic acid can be, for example, a single strand of the amplification product corresponding to the token.

Stage detection of molecular marker (stage b) may also include the implementation of the amplification reaction of nucleic acid at the said genomic DNA to detect one or more QTL. This can be done by conducting a PCR reaction using a set of marker-specific primers. In a preferred embodiment, the specified stage b) includes applying at least one set of primers that define AFLP marker for a given QTL, or set of primers that specifically hybridize under stringent conditions with the nucleic acid sequence AFLP marker for a given QTL.

Phase detection of the amplified DNA fragment having the predicted length or predicted sequence of the nucleic acid stage d)is preferably carried out so that amplificatory the DNA fragment has a length, to the which corresponds to (plus or minus a few reasons, for example, approximately the length of one, two, or three bases) the expected length based on a similar reaction with the same primers with DNA from a plant, which first discovered the marker, or a nucleic acid sequence that corresponds to (has a homology of more than 80%, preferably more than 90%, more preferably more than 95%, even more preferably 97%, still more preferably more than 99%) expected sequence based on the sequence of a marker linked to the QTL in the plant, which first discovered the specified token. The person skilled in the art it is known that the markers that are absent in resistant plants, whereas they were present in the susceptible parents (the so-called TRANS-markers), can also be effective in experiments on the detection of resistance in plants-descendants, although testing for the absence of a marker to detect the presence specificheskogo characteristic is not optimal.

Phase detection of the amplified DNA fragment with the predicted length or predicted sequence of the nucleic acid can be carried out by standard methods gel electrophoresis or using automated DNA sequencers. There is no need to describe these methods in this document, since it is well-known specialist in this field.

For detection in plants the presence of two QTL on the same chromosome can also be used in methods of staining chromosomes. In such methods, at least the first QTL and at least a second QTL can be detected in a single chromosome using in situ hybridization or PCR techniques in situ. These techniques can also be used to calculate the position of the QTL-1 onpm-landpm-hin this chromosome, and they are known in this field (for example,videJiang et al., 1995). The fact that the two QTL are represented on a single chromosome, it is more convenient to confirm, showing that they are in-phase coupling, i.e., that the signs show less segregation compared to genes located on separate chromosomes.

Molecular markers and QTL

Molecular markers are used to visualize differences in the sequences of nucleic acids. Such a visualization is possible thanks to the techniques of hybridization DNA-DNA after cleavage with restriction enzyme (RFLP) and/or techniques using polymerase chain reaction (for example, STS, microsatellites, AFLP). Any differences between the two parental genotypes will be selected for mapping populations (e.g., BC1F2; see figure 2), based on crossbreeding data of the parental genotypes. You can compare the segregation of various markers in chislet frequency of recombination. The frequency of recombination of molecular markers on different chromosomes are usually 50%. The recombination frequency between markers located on the same chromosome, depends on the distance between them. Low recombination frequency corresponds to a small genetic distance between markers on a chromosome. Comparison of all frequencies of recombination will result in the most logical order of molecular markers on the chromosome. This is the most logical order can be shown on the map clutch (Paterson, 1996). A group of adjacent or contiguous markers on the map clutch, which is associated with increased levels of resistance to disease, for example, with a decrease in incidence after infectious contact with the agent of the disease and/or a decrease in the growth rate of damage after the establishment of infection, pinpoints the location of QTL associated with resistance to this disease.

The markers identified herein, can be used in various aspects of the invention, as will now be illustrated. Aspects of the invention is not limited to the use of markers that are described herein. The authors of the present invention emphasize that aspects can be used markers, not disclosed explicitly herein or not yet identified. the excellent from the "gene" genetic unit, where phenotypic expression depends on a number of factors that cannot be predicted, genetic unit "QTL" refers to a region of the genome, which directly relates to phenotypic, quantifiable basis. Thus, if the genes themselves are not connected or not connected with the cultivation of plants, the QTL is directly used in the breeding of plants. The authors of the present invention showed that the QTL described in this document, to give stability to closterovirus and mildew should have a specific position relative to each other in the genome of plants descendants. The inventors have made the discovery on the basis of the observation that the presence of a continuous chain of genetic markers belonging to different QTL on the same chromosome in the genome of the cucumber, correlated with the presence of both phenotypic signs of stability, and the inventors have shown that the genomic organization can be inherited according to the ordinary laws of inheritance Mendel.

QTL described in this document are located on the same chromosome or group cohesion, and their position is best characterized by several other randomly selected markers. In the present work were used markers length polymorphism of amplified fragments (FLP), single nucleotide polimorfismo (SNP) markers mutation inserts, though could also be used markers length polymorphism restriction fragments (RFLP), microsatellite markers (e.g., SSR)markers amplified region is characterized by nucleotide sequence (SCAR)markers cleaved amplified polymorphic sequence (GAPS) or isozyme markers or combinations of the above. In General, QTL can cover the area in a few million bases. Therefore, providing information about the full sequence of the QTL is virtually impossible, but also unnecessary, because the way first discovered QTL due to the observed correlation between the presence of a continuous chain of genetic markers and the presence of a certain phenotypic trait allows you to find in the population of plant-descendants of those plants that have the genetic potential for the manifestation of a particular phenotypic trait. The present invention relates to a limited list of markers, thereby providing for breeding programmes effective property QTL. The marker is specific for a particular line varieties. Thus, a specific characteristic associated with a particular token. The markers presented in this paper not only shows the arrangement is their QTL, they are also due to the presence of specific phenotypic trait in a plant. It is important to note that continuous genetic markers that show the location of the QTL in the genome, are, in principle, arbitrary or unlimited. In General, the location of the QTL was determined by an unbroken chain of markers that demonstrate a statistical correlation to the phenotypic trait. As soon as the marker is detected outside the chain (i.e. a token that has (LOD-score), the rate of detection limit below a certain critical level, indicating that the marker is so remote that recombination in the area between marker and QTL is so common that the presence of the marker does not correlate statistically significantly with the presence of phenotype), then set the boundaries of the QTL. Thus, it is also possible to show the location of the QTL using other markers in this specified region.

Next, it is important to note that continuous genomic markers can also be used to show the presence of QTL (and thus phenotype) in a separate plant, i.e. they can be used in the procedures of selection using markers (MAS). In principle, the amount of potentially suitable markers is limited, but can be very large, and a specialist in this region the STI can easily identify additional markers, in addition to the markers listed in this document. In proceedings of the MAS can be used any token that is associated with QTL, for example, falls inside the physical boundaries of the genomic region covered with the marker with the targets set in the limit of detection LOD above a certain critical level, indicating that between marker and QTL recombination does not occur or is very small recombination in crosses; and any token in the nonrandom distribution of QTL; and markers that are really causal mutations within the QTL. This means that the markers referred to in this document as associated with QTL, such as AFLP markers E16/M50-244, E16/M50-188 and E11/M48-251 for QTL-1, are merely examples of markers suitable for use in MAS procedures. In addition, when the QTL, or its specific part, bringing a sign, introgression in a different genetic environment (i.e. in the genome of plants of other species), some markers may no longer be detected in the offspring, although the sign it represented, indicating that these markers are outside the genomic region, which is a specific part of the QTL, giving the sign, only the original parent line, and indicating that the new genetic environment has a different genomic organization.

These mark the market, the absence of which indicates the successful introduction of the genetic element in the offspring, called "Transmarine" and they can be just as comfortable in MAS procedures in the framework of the present invention.

After the identification of QTL effect QTL (resistance) can be confirmed, for example, by assessing sustainability in BC2S1the offspring, released on QTL study. Sustainability assessment can be carried out using Biotest on sustainability, well-known in this area for closterovirus and powdery mildew of cucumbers. For example, can be used to isolate CYSDV, which is stored with an access number PV-0592/EWSN_6 in the collection of the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), and experimental infection can be caused, for example, by passing through the tobacco whitefly (Bemisia tabaci) (B biotype). In addition, can be carried out (field) tests in natural infection conditions for evaluation of resistance to CYSDV or other closterovirus. Resistance to powdery mildew can be estimated using artificial infection of plant materials. The markers provided by the present invention, can be used to detect the presence of one or more QTL according to the invention in a plant of a cucumber, presumably resistant to mildew and/or closterovirus, and thus the nutrient, can be used in methods that include the breeding of using markers and plant breeding cucumber, resistant to closterovirus and powdery mildew. Preferably, detection of the presence of a QTL of the invention performs at least one of the markers for QTL described in this document. Therefore, in another aspect, the present invention relates to a method for detecting the presence of QTL resistance to powdery mildew and/or closterovirus, comprising detecting the presence of nucleic acid sequence specified QTL in a plant of a cucumber, presumably resistant to mildew and/or closterovirus, the presence of which can be found using the specified token.

The nucleotide sequence of the QTL of the present invention may be installed, for example, by determining the nucleotide sequence of one or more markers associated with QTL, and designing internal primers for the indicated marker sequences, which can then be used to further define the sequence QTL outside of these marker sequences. For example, the nucleotide sequence of AFLP markers from tables 2, 3 and 4 can be obtained by release of these markers from the electrophoretic gel, used to define the presence of these markers in the genome of the test plants, and determining the nucleotide sequences of these markers, for example, using dideoxy-methods, circuits, well known in this field.

In embodiments, the implementation of these methods for detecting the presence of a QTL in a plant of a cucumber, presumably resistant to mildew and/or closterovirus, the method may also include the stage of granting the oligonucleotide or polynucleotide capable hybridisierung in stringent conditions of hybridization with the nucleic acid sequence of a marker associated with the specified QTL, preferably selected from the markers of tables 2, 3 and 4; the interaction of the indicated oligonucleotide or polynucleotide with treated genomic nucleic acid plants cucumber, presumably resistant to mildew and/or closterovirus, and determine the presence of specific hybridization of the indicated oligonucleotide or polynucleotide with the specified processed genomic nucleic acid.

Preferably, the specified method is carried out on the sample of nucleic acid derived from the specified plants cucumber, presumably resistant to mildew and/or closterovirus, but can also be used hybridization methodsin-situ.An alternative and more preferred embodiment, if it is determined Amu is echidna sequence QTL, the person skilled in the art can create specific hybridization probes or oligonucleotides capable of hybridizing in stringent conditions with the nucleic acid sequence specified QTL, and may use a specific hybridization probes in the methods of detecting the presence of a QTL of the invention in a plant of a cucumber, presumably resistant to mildew and/or closterovirus.

Marker main locus of the gene of resistance to closterovirus can be obtained by selective amplification of DNA fragments cucumber from sustainable species and sensitive species, originating from the parent varieties:

in which these fragments were subjected to phase processing enzymes (e.g., EcoRI and MseI) followed by joining to attach them to the ends of the adapters additional fragments for primers, which have at its end one or more specific nucleotides, where one or more primers of the pair of primers is labeled for detection;

- separation of amplification products by electrophoresis in a gel under denaturing conditions and

- comparison of profiles gel electrophoresis obtained with mixtures of fragments derived from sustainable offspring, and mixtures originating from sensitive progeny, with fragments originating from p is witelski varieties, to identify polymorphic bands that are genetically linked to a locus of resistance. After this identification should the investigation stage, in which there is a control on all individuals and calculate the rate of genetic recombination between the marker and the locus of resistance.

In principle, a separate AFLP markers used in this document, are assigned to the token code. This code refers to two primers, optionally in combination with a number which indicates the length of the amplification product of primers in a given sample (also see description for table 1 above). Thus, the token identifies a fragment of single-stranded or double-stranded DNA obtained by conducting the reaction amplification genomic DNA cucumber, which, if the specified sample results in a fragment of a specified length. In addition, the token contains a 5'-3' direction, a sequence consisting of the sequence of the first primer, the specific DNA sequence of the cucumber and the sequence of the second primer and its complement. Thus, the specific DNA sequence of the cucumber is flanked by the two primers. The term "specific DNA sequence of the cucumber" refers to the nucleotide sequence of the region, flanked by appropriate primers, which is a sequence amplified from a sample of cucumber Khira PI250147, as described in international patent application WO 02/22836 using these primers, or a sequence having homology with the specified sequence at least 90%, preferably 95% and most preferably at least 98%.

Obtaining cucumber plants resistant to powdery mildew and closterovirus, transgenic methods.

According to another aspect of the present invention the sequence of a nucleic acid (preferably DNA)that contains at least QTL-1 and one, preferably bothpm-landpm-hor parts thereof, sustainability, can be used to produce plants of the cucumber of the present invention with dual resistance. In this aspect, the invention provides for the use of QTL described herein, or portions thereof, sustainability, to obtain plants of cucumber with dual resistance, which includes the introduction of a nucleic acid sequence containing the specified QTL, in a suitable plant-recipient. As indicated, the sequence of the nucleic acid can be obtained from a suitable plant-donor-resistant closterovirus and/or powdery mildew.

A suitable source of locus of resistance to closterovirus, oboznachennomu herein as QTL-1, is cucumber Landrace Kira, PI 250147 originating from Pakistan. The number PM of resistant cultivars of cucumber along with their commercial sources listed, for example, http://cuke.hort.ncsu.edu/cucurbit/cuke/cukemain.html. PM resistant plants can be obtained, for example, by T. C. Wehner, the curator genetics cucumbers in (Cucurbit Genetics Cooperative) (CGC), Department of horticulture, University of the state of North Carolina, Raleigh, NC 27695-7609 USA. The source of both pm loci described in this document is NPI, which was obtained by crossing Natsufushinari (PI 279465) with PI 200815. Samples can be obtained, for example, from the Centre for Genetic Resources, the Netherlands (CGN), Wageningen, the Netherlands. Multiple databases plants are publicly available to assist in selecting suitable repository collections, such as the ECP/GR Cucurbits Database, hosted by the Center for conservation and breeding of agriculture (Center for the Conservation and Breeding of Agrodiversity) (COMAV) in Valencia, Spain, or the Germplasm Resources Information Network (GRIN), placed the USDA''s National Germplasm Resources Laboratory, Beltsville, Maryland. Other plants of cucumbers, have shown resistance or closterovirus or mildew, can also be used as a donor plant resistance, since the present invention describes how this material can be identified.

Once the sequence of nucleic acids, which contain what it QTL resistance to closterovirus or mildew or part of them, sustainability identified in a suitable donor plant, it can be transferred to a suitable plant-recipient in any way. For example, the nucleic acid sequence may be transferred by crossing plants of the donor-resistant closterovirus or mildew, sensitive plant-recipient (i.e. introgression), transformation, fusion of protoplasts, by a doubled haploid technique or by embryo rescue or any other system of transfer of nucleic acid, optionally with subsequent selection of plants descendants containing QTL, and have shown resistance. For transgenic methods of transferring a nucleic acid sequence containing QTL resistance to closterovirus or mildew, or portion thereof, sustainability, can be selected from the specified plants-donor using methods known in this field, and, thus, an isolated sequence of nucleic acid can be transferred into the plant-recipient transgenic ways, for example by means of a vector in the gamete or in any other suitable transmitting element, such as ballistic particle coated with the specified nucleic acid sequence.

Usually the transformation of plants includes the creation of age of the ora expression, which will work in the plant cells. In the present invention, such a vector includes QTL resistance to closterovirus or mildew or part of sustainability, the vector may contain a gene that gives resistance to closterovirus or mildew, which is under control of or operatively linked to a regulatory element such as a promoter. The expression vector may contain one or more functionally related combinations of gene/regulatory element, provided that at least one of the genes contained in the combinations, encodes resistance to closterovirus or mildew. Vector (vectors) can be in the form of a plasmid and can be used separately or in combination with other plasmids, to provide transgenic plants that are resistant to closterovirus and powdery mildew, using transformation methods known in this field, such as system transformationAgrobacterium.

The expression vectors can include at least one marker gene functionally linked to a regulatory element (such as a promoter) that allows you to select transformed cells containing the marker, using negative selection (inhibition of growth of cells that do not contain breeding marker gene) or positive selection (with what rininger product encoded by the marker gene). Many widely used breeding marker genes for plant transformation are known in this field and include, for example, genes that encode enzymes that neutralize metabolic selective chemical agent which may be an antibiotic or herbicide, or genes that encode the modified target which is insensitive to the inhibitor. In this area there are several ways of positive selection, such as selection on mannose. An alternative for the production of plants without these marker genes can be used markerless transformation techniques which are known in this field.

One of the methods for introducing the expression vector into plants is based on a natural system transformationAgrobacterium(see, for example, Horsch et al., 1985).A.tumefaciensandA.rhizogenesare plant pathogenic soil bacteria that are genetically transform plant cells. Plasmid Ti and RiA.tumefaciensandA.rhizogenesaccordingly, carry genes responsible for genetic transformation of plants (see, for example, Kado, 1991). Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant cells with theAgrobacterium tumefaciens(Horsch et al., 1985). DescribeAgrobacteriumve is Torno systems and methods Agrobacterium-mediated gene transfer provided by Gruber and Crosby, 1993, and Moloney et al., 1989. See also U.S. patent No. 5591616. General descriptions of plant expression vectors and reporter genes and transformation protocols and descriptionsAgrobacteriumvector systems and methodsAgrobacterium-mediated gene transfer can be found in Gruber and Crosby, 1993. General methods of culturing plant tissues are provided, for example, Miki et al., 1993 and Phillips, et al., 1988. A suitable guide to molecular cloning techniques and appropriate expression vectors is Sambrook and Russell, 2001.

Another method for introducing the expression vector into plants is based on the transformation-mediated microkeratome, where DNA is transferred on the surface of micronarrative. The expression vector is introduced into plant tissues biolistics device that accelerates microarray up to speed from 300 to 600 m/sec, which is sufficient for penetration (see Sanford et al., 1987, 1993; Sanford, 1988, 1990; Klein et al., 1988, 1992). Another method of introducing DNA into plant represents the sonication of target cells (see Zhang et al., 1991). An alternative to the introduction of expression vectors used fusion of liposomes or spheroplasts (see, for example, Deshayes et al., 1985 and Christou et al., 1987). Also reported direct introduction of DNA into protoplasts using deposition CaCl2polyvin the business of alcohol or poly-L-ornithine (see, for example, Hain et al., 1985 and Draper et al., 1982). Electroporation of protoplasts and whole cells and tissues has also been described (D Halluin et al., 1992 and Laursen et al., 1994).

Other well-known techniques such as the use of BAC, in which part of the genome of the cucumber is introduced into a bacterial artificial chromosome (BAC), i.e. the vectors used to clone DNA fragments (100 - 300 TPN the size of the insert; average, 150 TPN) in cellsEscherichia colibased on natural plasmids P-factor, discovered in the bacteriumE. coli. (Zhao and Stodolsky, 2004), can be used, for example, in combination with the system BIBAC (Hamilton, 1997) to obtain transgenic plants.

After transformation of target tissues of cucumber expression of the above-described selective marker genes allows selective breeding of transformed cells, tissues and/or plants, using regeneration methods and selection, well-known in this field.

Receipt of a cucumber, resistant to closterovirus and powdery mildew, necroshine ways

In an alternative embodiment, to obtain the cucumber plants that are resistant to closterovirus and mildew, can be used fusion of protoplasts for transfer of nucleic acids from a plant-donor plant-recipient. Fusion of protoplasts is a spontaneous or evoked compound such as somatic hybridization, two or more protoplasts (cells, whose walls are removed by treatment with enzymes) to create a single two - or multinucleated cells. Hybrid cell, which can be obtained even with species that do not interbreed in nature, grown in tissue culture in a hybrid plant, demonstrating the desired combination of characteristics. In more detail, the first protoplast can be obtained from a plant of cucumber or another plant that exhibits resistance to infection closterovirus or mildew. The second protoplast can be obtained from the second cucumber or plants of another variety, preferably, the lines of cucumber, which contains commercially valuable characteristics, without limitation, such as resistance to disease, resistance to insects, valuable fruit characteristics, etc. and Then the protoplasts connect using normal procedures to connect the protoplasts, which are known in this field.

Alternatively, the salvation of the embryo can be used to transfer nucleic acid that includes one or more QTL described in this document, from a plant-donor plant-recipient. The salvation of the embryo can be used as a procedure for isolation of embryos from crosses in which plants are unable to produce viable seeds. In Dan the om process opylene ovary or immature seed plants are grown in tissue culture to create new plants (Pierik, 1999).

The present invention also relates to a method for obtaining a plant of cucumber, resistant to closterovirus and powdery mildew, which includes stages of the method of detecting the presence of a quantitative trait locus (QTL)associated with resistance to closterovirus or mildew in the donor plant cucumber according to the above invention, and transferring nucleic acid sequence comprising at least one QTL detected in this way, or part thereof, which imparts resistance to closterovirus or mildew from specified plants-donor in the recipient plant cucumber sensitive closterovirus or mildew. The transfer of the specified nucleic acid sequence may be carried out by any method previously described in this document.

A preferred variant of the method includes migrating through introgression of the specified nucleic acid sequence of a donor plant of cucumber, resistant to closterovirus or mildew in the recipient plant cucumber sensitive closterovirus or mildew, by crossing these plants. The transfer may be performed using conventional cultivation techniques. Preferably QTL introgression in commercial varieties on ursov using selection using markers (MAS) or breeding using marker (MAB). MAS and MAB include one or more molecular markers for identification and selection of such plants descendants that contain one or more genes that encode a desired trait. In this case, this identification and selection based on breeding QTL of the present invention or related markers. Also MAS can be used to create near-isogenic lines (NIL), containing the QTL of interest, allowing a more detailed study of each effect QTL and MAS is also an effective way to create populations of inbred lines back-crossing (BIL) (see, e.g., Nesbitt et al., 2001; van Berloo et al., 2001). Plants of cucumber, created according to this variant implementation, can get most of its characteristics from the recipient plant and to obtain resistance to closterovirus and mildew from a plant of the donor.

As briefly discussed above, conventional techniques of cultivation can be used to introgressive nucleic acid sequence that encodes resistance to closterovirus or mildew in the recipient plant cucumber sensitive closterovirus and mildew. In one way, which is referred to as purebred breeding, donor plant of cucumber, which has shown resistance to Kloss is reovirus or mildew, and includes the nucleic acid sequence encoding the resistance to closterovirus and powdery mildew, and hybrid recipient plant cucumber sensitive closterovirus or mildew, which preferably shows a commercially desirable characteristics, without restrictions such as resistance to disease, resistance to insects, valuable fruit characteristics, etc. of the Obtained population of plants (representing hybrids F1then snoopys and planted the seeds (seeds F2). The F2 plants grown from seeds of F2, then selected for resistance to closterovirus and powdery mildew. The population can be examined in several different ways.

First, the population can be surveyed using conventional screening of the disease. This screening of the disease known in this field. Preferably, use a quantitative Biotest. Secondly, it can be selection using markers with the use of one or more of the above described molecular markers to identify those descendants that contain the nucleic acid sequence for resistance to closterovirus or mildew. Can be used other ways, referred to above methods for detecting the presence of QTL. In addition,selection using markers can be used to confirm the results, obtained in quantitative biotests, and therefore, some methods may also be used in combination.

Inbred plant lines cucumber, resistant to closterovirus and mildew, can be created using the techniques of recurrent selection and back-crossing, selfing, and/or dihaploids or any other equipment used to create the parent lines. In the method of recurrent selection and back-crossing resistance to closterovirus and powdery mildew can be introgression in recipientname plant target (the recurrent parent) by crossing the recurrent parent with the first plant-donor, which is different from the recurrent parent and is called in this document "arecurrently parent". Recurrency parent is a plant that is unstable or has a low level of resistance to closterovirus and mildew and possesses commercially desirable characteristics, such as, without limitation, (optional) resistance to disease, resistance to insects, valuable fruit characteristics, etc. Arecurrently parent has resistance to closterovirus and mildew and is comprised of a sequence of nucleic acid, which encodes resistance to closterovirus and powdery mildew. Precorrin the first parent can be any plant varieties or inbred lines, which is cross-fertilized with the recurrent parent. Progeny derived from crosses between the recurrent parent and arecurrently parent, crossed back crosses with the recurrent parent. Then in the resulting populations of plants spend screened for desirable characteristics, this screening can be performed in different ways. For example, selection in a population can be made using sorts on phenotypic pathology or quantitative biotests known in this field. Alternatively, instead of using biotests can be performed selection using markers (MAS) using one or more previously described molecular markers, hybridization probes or polynucleotides to identify the specified seed, which contains a nucleic acid sequence encoding the resistance to closterovirus and powdery mildew. Also MAS can be used to confirm the quantitative results of biotests. And in this case, the recessive nature of thepm-hindicates that this gene could not be detected in the F1or BC1populations with phenotypic sorts, such as biotests on sustainability. Therefore, the markers described herein are ultimately suitable for on the ora of the respective plants descendants using genotypic scan.

After screening the hybrid F1 plants that exhibit the phenotype of resistance to closterovirus and mildew or, more preferably, the genotype and, therefore, comprise the desired nucleic acid sequence, coding for resistance to closterovirus and mildew, then select and teach backcross with recurrent parent for several generations, to increase inbreast plants of cucumber. This process can be carried out within two to five generations. In principle, the offspring, the resulting process of crossing the recurrent parent with arecurrently parent-resistant closterovirus and mildew, is heterozygous for one or more genes that encode resistance to closterovirus and powdery mildew.

It should be remembered that, for example, when the line of plants introgression both locus ofpm-landpm-hthen the locus ofpm-his recessive, and in plants-the descendants of the resistant phenotype will appear only in conditions in which there may be formed homozygouspm-hplants.

Generally, the method of introducing a desired trait into hybrid cucumber variety involves the following stages:

(a) crossing inbred cucumber-parent with another cucumber plant that includes one or more of the same is emich signs, to obtain plants of the F1 offspring, in which the desired trait selected from the group consisting of resistance closterovirus and resistance to powdery mildew;

(b) the selection of these plants of the F1 offspring that have the desired trait to obtain selected plants of the F1 offspring, preferably using molecular markers described herein;

(c) selected backcross progeny plants with the specified inbred parent plant of cucumber for getting plants descendants of back-crossing;

(d) the selection of plants-descendants of back-crossing that have the desired trait and morphological and physiological characteristics of this inbred parent plants of cucumber, where the specified selection enables selection of genomic DNA and testing of the specified DNA in the presence of at least one molecular marker for the QTL-1,pm-land/orpm-hpreferably described herein;

(e) repeating steps (c) and (d) two or more times in succession to obtain plants of the descendants of the third or more back-crossing;

(f) optionally selfing of the selected offspring back-crossing to establish homozygous plants;

(g) crossing at least one offspring back-crossing or self-pollinated plants the deposits with the other inbred parent plants of cucumber (preferably plant which is pm hypo stable, that is homozygous for thepm-hto create hybrid cucumber varieties with the desired trait and all of the morphological and physiological characteristics of hybrid cucumber varieties when they are grown in the same environmental conditions.

As shown, the offspring of the last back-crossing can samobility to provide homozygous offspring of pure bred (inbred) in terms of resistance to closterovirus and mildew. Thus, the result of recurrent selection of back-crossing and selfing is the creation of lines that are genetically uniform genes associated with resistance to closterovirus and mildew, as well as other genes associated with traits of commercial interest.

Plants and seeds of cucumber, resistant to closterovirus and powdery mildew

The goal of plant breeding is to combine in a single variety or hybrid variety of desirable traits. For commercial crops such traits may include resistance to diseases and insects, the ability to tolerate heat and drought, accelerated maturation, greater yield, and better agronomic quality. The uniformity of the characteristics of plants, such as sprouting and the formation of grass, / min net is ü growth maturity and plant height, can also be of great value.

Commercial culture is diluted using techniques that take advantage of the vegetative method of pollination. The plant is self-pollinating, if pollen from one flower is transferred to the same or another flower of the same plant. The plant is Sib-pollinating, when used for pollination individuals of the same family or line. The plant is cross-pollinated when pollen of a flower comes on a different plant from a different family or line.

Plants that sampylalle and selected type for many generations become homozygous in almost all gene loci and produce a uniform population of offspring rearing of homozygotes. Crossing two different lines of homozygotes gives a uniform population of hybrid plants that may be heterozygous for many gene loci. Crossing two plants, each of which is heterozygous at multiple gene loci will give the population of heterozygous plants that differ genetically, and it will not be uniform.

The development of hybrid varieties of cucumbers in the breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) samobile the e selected plants from the breeding crosses for several generations of breeding crosses for a series of inbred lines, which individually transmit the trait to offspring and are highly uniform; and (3) crossing the selected inbred lines with unrelated inbred lines to obtain the hybrid progeny (F1). After a sufficient amount of inbreeding subsequent FILIAL generation will only serve to increase seed created inbred. Preferably, the inbred line should contain the homozygous alleles of about 95% or more loci.

An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a specific pair of inbreeders, will always be the same. Once identified inbreeders, which has created the perfect hybrid, you can create a constant supply of hybrid seeds using data from inbred parents, and then the hybrid plants of the cucumber can be grown from the stock of hybrid seeds.

Plant cucumber-resistant closterovirus and mildew, or part thereof, obtained by the method of the invention is an aspect according to the present invention.

Another aspect of the present invention relates to a plant of cucumber, resistant to closterovirus and mildew, or part thereof, including QTL in any configuration, as described previously, where at least one of these QTL nahoditsya in its natural genetic environment. The cucumber plants of the present invention, resistant to closterovirus and powdery mildew can be any genetic type, such as inbred, hybrid, haploid, diploid or transgenic. Further, plants of the present invention can be heterozygous or homozygous for signs of resilience, preferably homozygous. Although QTL of the present invention and parts thereof, sustainability, can be transfected into any plant to provide a plant that is resistant to closterovirus and powdery mildew, the methods and plants according to the invention preferably are in the formCucumis sativus.

Inbred lines cucumber, resistant to closterovirus and powdery mildew, described herein, can be used in additional crosses to create hybrid plants that are resistant to closterovirus and powdery mildew. For example, the first inbred cucumber plant of the invention is resistant to closterovirus and mildew, can be crossed with a second inbred cucumber plant, bearing commercially valuable traits, such as, but without limitation, resistance to disease, resistance to insects, desirable fruit characteristics, etc. of the Specified second inbred cucumber line may or may not be resistant to closterovirus and powdery mildew. Predpochtite the flax this line is homozygous for the pm-h, to this recessive trait expressively in hybrid plants descendants.

Another aspect of the present invention relates to a method of producing seeds that can be grown plants resistant to closterovirus and powdery mildew. In one embodiment, the method includes the stage of granting of cucumber plants of the invention are resistant to closterovirus and powdery mildew, crossing the specified plants resistant to closterovirus and powdery mildew, with another cucumber plant, and collecting the seeds resulting from a specified crossing that when planting give plants resistant to closterovirus and powdery mildew.

In another embodiment, the method includes the stage of granting of cucumber plants of the invention are resistant to closterovirus and powdery mildew, crossing the specified plants resistant to closterovirus and powdery mildew, cucumber plant, and collecting the seeds resulting from a specified crossing, restore these seeds in plants, selection of plants resistant to closterovirus and powdery mildew, any of the methods described herein, self-pollination of selected plants for a sufficient number of generations to obtain plants which set allele, which confers resistance to closterovirus and powdery is th dew, backcross plants with obtaining, thus, the cucumber plants having desirable phenotypic traits for a sufficient number of generations to obtain the cucumber plants that are resistant to closterovirus and mildew and have desirable phenotypic traits, and collecting the seeds produced by the plants, resulting from a recent back-crossing, which when planting give the cucumber plants that are resistant to closterovirus and powdery mildew.

Table 4 shows the format of a matrix in the presence or absence of plants with dual resistance known markers that indicate the origin or from pm-resistant parent, or parent-resistant closterovirus (shown as a sustainable presence in the parent). Next, the matrix shows the origin and the relative position of AFLP markers associated with lines of plants with double (PM and closterovirus) resistance. Markers, indicated by the termspm-landpm-hnote the Central position of two corresponding loci of resistance to powdery mildew, referred to in this document. The position of the QTL locus-l has not been established, while using position markers associated with closterovirus, it was possible to determine that the locus was located between the two locipm. From this table what s clear that pool Pool nrs. 1-6 received shorter introgression of parent-resistant closterovirus than got pool Pool nrs. 10-15. The explanation of the matrix is as follows: marker E23/M40-M003 (56.9 cM), for example, was presented in pm-resistant parent and is also represented in the progeny with dual resistance, while the marker E11/M80-M003 (79.1 cM) was presented in the parent-resistant closterovirus, but not in the offspring with dual resistance. Thus, the marker E23/M40-M003 PM is associated with a marker whose presence indicates the presence ofpm-lassociated genetic region in the tested plant lines, while the marker E11/M80-M003, on the other hand, shows a genetic region adjacent to the QTL-l locus associated with resistance to closterovirus, but that is not important for the expression of pm-hypo (pm-h) stability.

Table 4

Indicators of AFLP markers in plants of the invention with dual resistance

+ = is present in the homozygous (lane high intensity)

- = absent

+/- = is present in the form of heterozygotes (band low intensity)

D = average presence (lane medium intensity)

= index token (and the presence of both [+] and absence [-] are informative) as a parent, stable the CSOs to RM

= index marker as a parent, resistant to closterovirus

= the presence of the marker in the parent lines are not installed.

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1. The plant, the cat who PoE is resistant to closterovirus cucumbers and mildew, moreover, this plant is a plant of the species Cucumis sativus, this plant contains one chromosome at least one chromosomal region that confers resistance to closterovirus, and at least one chromosomal region that confers resistance to powdery mildew,
where the specified at least one chromosomal region that confers resistance to closterovirus associated with at least one marker selected from the group consisting of markers e/M50-244, e/M50-188 and E11/M48-251, and
where the specified at least one region, which confers resistance to powdery mildew, associated with at least one marker selected from the group consisting of:
marker single nucleotide polymorphism T→ G in SEQ ID NO:1,
marker single nucleotide polymorphism 29G→ in SEQ ID NO:2,
marker single nucleotide polymorphism S→ T in SEQ ID NO:3,
the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and
markers E16/M50-F-194, E11/M48-F-251, e/M38-M, e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

2. The plant according to claim 1, where the specified plant includes at least two chromosomal region, which give resistance to powdery mildew, where the first of these at least two regions connected by at least one marker selected from the group consisting of:
marker adenucio odnogo polymorphism T→ G in SEQ ID NO:1,
marker single nucleotide polymorphism 29G→ in SEQ ID NO:2, and
markers E16/M50-F-194, E11/M48-F-251, e/M38-M,
and where the second of these at least two regions connected by at least one marker selected from the group consisting of:
marker single nucleotide polymorphism S→ T in SEQ ID NO:3,
the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and
markers e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

3. The plant according to claim 2, where the specified at least one region, which confers resistance to closterovirus placed between at least two areas that give resistance to powdery mildew, as defined in section 2.

4. Part of the cucumber plants according to any one of claims 1 to 3, where a specified part of the plant contains at least one chromosomal region that confers resistance to CYSDV, and at least one chromosomal region that confers resistance to powdery mildew, as defined in claim 1.

5. Part of the plant according to claim 4, where a specified part of a plant selected from the pollen of semjazachatki, leaves, roots, root tips, anthers, flowers, fruits, stems, shoots, grafting cuttings, root sprouts, seeds, protoplasts and calli.

6. The F1 seed obtained by crossing plants of the cucumber according to any one of claims 1 to 3 with a second cucumber plant or a plant of another variety, prefer the Ino from a number of cucumbers, which contain desirable from a commercial point of view specifications, where specified seed contains: at least one marker of resistance to closterovirus selected from the group consisting of markers e/M50-244, e/M50-188 and E11/M48-251 and at least one marker of resistance to powdery mildew selected from the group consisting of:
marker single nucleotide polymorphism T→ G in SEQ ID NO:1,
marker single nucleotide polymorphism 29G→ in SEQ ID NO:2,
marker single nucleotide polymorphism S→ T in SEQ ID NO:3,
the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and
markers E16/M50-F-194, E11/M48-F-251, e/M38-M, e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

7. The seed according to claim 6, where the specified second cucumber plant is sensitive to closterovirus, and where the specified at least one marker of resistance to powdery mildew presented in the form of homozygotes.

8. The seed according to claim 6 or 7, where the aforementioned second cucumber plant is an inbred plant.

9. A hybrid plant obtained by growing the seed according to any one of claims 4 to 8.

10. Part of the hybrid plant of claim 9, where a specified part of the plant contains at least one chromosomal region that confers resistance to CYSDV, and at least one chromosomal region that confers resistance to powdery mildew, as defined in claim 1.

11. Some races the program of claim 10, where a specified part of a plant selected from the pollen of semjazachatki, leaves, roots, root tips, anthers, flowers, fruits, stems, shoots, shoots, root sprouts, seeds, protoplasts and calli.

12. The method of selection of the plant that is resistant to closterovirus cucumbers and mildew of cucumbers, where this plant is a plant of the species Cucumis sativus, this method enables the analysis to determine the token, where the specified analysis involves the following stages:
a) detecting in the genome of the specified plants at least one marker associated with resistance to closterovirus selected from the group consisting of markers e/M50-244, e/M50-188 and E11/M48-251, and
(b) detecting in the genome of the specified plants at least one marker associated with resistance to powdery mildew selected from the group consisting of:
marker single nucleotide polymorphism T→ G in SEQ ID NO:1,
marker single nucleotide polymorphism 29G→ in SEQ ID NO:2,
marker single nucleotide polymorphism S→ T in SEQ ID NO:3,
the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and
markers E16/M50-F-194, E11/M48-F-251, e/M38-M, e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

13. The method according to item 12, which stage includes:
detection in the specified genome of the plant at least one token associated with the first QTL, giving stability to unistall mildew of cucumbers, shown SNP marker T→ G in SEQ ID NO:1, SNP marker 29G→ in SEQ ID NO:2 and markers E16/M50-F-194, E11/M48-F-251, e/M38-M; and
detection in the specified genome of the plant at least one token associated with the second QTL, which imparts resistance to powdery mildew of cucumbers, shown SNP marker S→ T in SEQ ID NO:3, the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and markers e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

14. The method according to item 12, where the method includes:
a) obtaining a sample of genomic DNA from the specified plants;
b) detection of the specified at least one marker associated with resistance to closterovirus, and the specified at least one marker associated with resistance to powdery mildew, in a specified sample of genomic DNA from the indicated plants.

15. The method according to item 13, where the method includes:
a) obtaining a sample of genomic DNA from the specified plants;
b) detection of the specified at least one marker associated with resistance to closterovirus, and the specified at least one marker associated with resistance to powdery mildew, in a specified sample of genomic DNA from the indicated plants.

16. The method according to 14, in which the specified stage (b) includes using at least one primer set, to determine the specified token(s), or the use of at least one the Honda nucleic acid sequence of bases which is essentially complementary to the sequence of the nucleic acid, determining the marker, and a probe for nucleic acid that specifically's hybrid in stringent conditions with the sequence of the nucleic acid, determining the marker(s).

17. The method according to any of p-16, which further determines that these markers are located on the same chromosome, showing that they are in the phase of the clutch, for example, demonstrating reduced segregation between markers in the experiments breeding, when they are compared with non-linked markers.

18. The plant selected by the method according to any of PP-17.

19. A method of producing a plant of the species Cucumis sativus, which plants showing resistance to closterovirus cucumbers and mildew of cucumbers, which includes stages:
a) selecting a first plant of a cucumber, which contains a chromosomal region that confers resistance to closterovirus cucumber, by detecting in the genome of the specified plants at least one marker linked to the QTL, which imparts resistance to closterovirus cucumbers identified markers e/M50-244, e/M50-188, and E11/M48-251;
b) selecting a second plant of cucumber, which contains at least one chromosomal region that confers resistance to powdery mildew, by detecting in GE is ome of the specified plants at least one marker, associated with the first QTL, which imparts resistance to powdery mildew, the following tokens:
marker single nucleotide polymorphism T→ G in SEQ ID NO:1,
marker single nucleotide polymorphism 29G→ in SEQ ID NO:2, and
markers E16/M50-F-194, E11/M48-F-251, e/M38-M; or
by detecting in the genome of the specified plants at least one token associated with the second QTL, which imparts resistance to powdery mildew, the following tokens:
marker single nucleotide polymorphism S→ T in SEQ ID NO:3,
the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and
markers e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200;
c) crossing these plants stage a) and stage b) to produce F1 seeds;
d) growing a certain number of seeds F1 to F1 plants, creating the following population of descendants of these F1 plants by crossing or selfing, and
e) selecting from among the following plants-seeds of plants, which contains at least one marker linked to the QTL, which imparts resistance to closterovirus cucumber, as defined in stage a), and at least one marker linked to the QTL, which imparts resistance to powdery mildew of cucumber, as defined in stage b).

20. The method according to claim 19, where the stage of selection of the second cucumber plants at the stage b) comprises selecting a second plant of cucumber with one of the specified who's first or second QTL, giving resistance to powdery mildew, cucumber, and where the method additionally includes the stage:
f) selection of the third cucumber plants having other than the first or second QTL, which imparts resistance to powdery mildew of cucumber (i.e. the QTL, which is not present in the specified second cucumber plant);
g) crossing F1 plants obtained in stage e) with the specified third plant of cucumber with obtaining subsequent plant generations, and
h) selection of the number of plants descendants of plants that contains QTL, which imparts resistance to closterovirus cucumber, as defined in stage a), and both QTL, which imparts resistance to powdery mildew of cucumber, as defined in stage b).

21. The method according to claim 19, in which qualifying cucumber plants containing chromosomal region that confers resistance to powdery mildew of cucumbers, as defined in the stages b), e), f) or h), includes
detecting in the genome of the specified plants at least one token associated with the first QTL, which imparts resistance to powdery mildew of cucumber, shown SNP marker T→ G in SEQ ID NO:1, SNP marker 29G→ in SEQ ID NO:2 and markers E16/M50-F-194, E11/M48-F-251, e/M38-M; and
detecting in the genome of the specified plants at least one token associated with the second QTL, which imparts resistance to powdery mildew of cucumbers, shown SNP marker S→ T in SEQ ID NO:3, what utala insert 5'-AATT-3' at position 221 in SEQ ID NO:4, and markers e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

22. The method according to claim 20, in which qualifying cucumber plants containing chromosomal region that confers resistance to powdery mildew of cucumbers, as defined in the stages b), e), f) or h), includes
detecting in the genome of the specified plants at least one token associated with the first QTL, which imparts resistance to powdery mildew of cucumber, shown SNP marker T→ G in SEQ ID NO:1, SNP marker 29G→ in SEQ ID NO:2 and markers E16/M50-F-194, E11/M48-F-251, e/M38-M; and
detecting in the genome of the specified plants at least one token associated with the second QTL, which imparts resistance to powdery mildew of cucumbers, shown SNP marker S→ T in SEQ ID NO:3, the insertion mutation 5'-AATT-3' at position 221 in SEQ ID NO:4, and markers e/M40-M, e/M46-M, e/M46-M, E12/M-M, E/M-M, E14/M59-F-134 and E14/M59-F-200.

23. The method according to any of PP-22, where at least one of stages a), b), e), f) or h)includes a step of obtaining a sample of genomic DNA from the indicated plant and detecting in a specified sample of genomic DNA at least one of the specified token.

24. A method of producing a plant of the species Cucumis sativus, which has shown resistance to closterovirus cucumber and mildew of cucumber, which includes stages:
a) the selection of plants of the species Cucumis sativus, which contains a chromosomal region, the cat is that impart resistance against closterovirus cucumber powdery mildew of cucumber by the method of any of p-17;
b) inbreeding specified plants with obtaining homozygous lines plants of these QTL;
c) crossing these plants stage a) and stage b) to produce F1 seeds and (d) the cultivation of these F1 seeds to plant the offspring of F1.

25. Plant or part thereof, obtained by the method according to any of PP-24.



 

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FIELD: medicine.

SUBSTANCE: live attenuated Pasteurella multocida bacterium is modified by introduing a mutation into the gene Orf-15. The mutation represents insertion and/or deletion in the gene Orf-15. As a result of modification, the bacterium is not able to express the functional protein Orf-15 that leads to its attenuation. Also, disclosed is the use of such bacterium for preparing a related vaccine for human or animal protection against Pasteurella multocida bacteria infection or pathogenic effects of the infection.

EFFECT: increased efficacy of applying the composition.

16 cl, 1 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: genetic predisposition is determined by analysing polymorphism of genes ACTN3 (R577X), CNB (5I/5D), AMPD1 (C34T) through a multiplex polymerase chain reaction. For this purpose, a set of primers is used, wherein for the gene ACTN3 (R577X) the primes used are ACTGCTGCCCTTTCTGTTGCCT-3' and S'-CTGCAGGTGGCACTGACCATA3', for the gene CNB (5I/5D) the primers used are 5'-GGAGTTTAAAAGCCAGCCAGTCATACTA-3' and 5'-TGGAAGATCACACCATTTGATTAGCAGT-3', for the geneAMPD1 (C34T) the primers used are S'-GCAATCTACATGTGTCTACCCCAAAG-3' and 5'-CACTGCTGAAAAATAGCCATGTTTCTG-3'. Further, 3 pairs of primers with average melting point of 58-59°C are merged in a single test tube at standard conditions. The reaction product is analysed through analysis of polymorphism of the length of restriction fragments.

EFFECT: invention provides a simple, cheap, specific method which enables to identify functionally significant polymorphous gene loci, grow the number of analysed genes for further introduction into any standard clinical laboratory.

2 cl, 2 dwg, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: present invention can be used during DNA diagnostics in medicine, veterinary, sanitary and epidemiological analysis and in criminalistics to identify criminals. The method of amplifying specific nucleic acid fragments employs thermally stable DNA polymerase having chain-displacement activity. Also, the present method employs direct and reverse primers in form of head-to-tail arranged tandem recurrent sequences of the main primer. The present invention enables to amplify specific DNA or RNA fragments with increasing multiplication factor for each cycle.

EFFECT: shorter reaction time and high sensitivity of the reaction.

4 cl, 8 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: transformed soya bean seeds contain a nucleic acid sequence identical to a fragment of soya bean intron FAD2-1A and a nucleic acid identical to a fragment of soya bean FADB gene. Seeds of the transformed plant, as well as oil and flour obtained therefrom contain oleic acid in amount ranging from approximately 42 wt % to approximately 85 wt % of the total content of fatty acids. Content of saturated fatty acids is less than 8 wt % of the total amount of fatty acids.

EFFECT: higher fatty acid content.

28, 37 dwg, 26 tbl, 31 ex

FIELD: agriculture.

SUBSTANCE: sunflower seeds have been genetically modified with the help of two cycles of artificial induction of mutations, in each case with further identification of mutant species, which bore required criterion. Modified seeds are characterised by the fact that 26-80% of tocopherols are present in them in the form of delta-tocopherols. This level of delta-tocopherols is determined by genotype of seeds modified by this criterion and is independent on conditions of cultivation. From seeds they grow genetically modified plants of sunflower, which produce seeds with high levels of delta-tocopherol as a result of self-fertilisation.

EFFECT: oil is extracted from seeds with high concentration of delta-tocopherol.

8 cl, 1 tbl

FIELD: agriculture.

SUBSTANCE: genetic modification of wheat in the form of mutation in gene SBEIIa with reduction of level of its activity.

EFFECT: grain with high content of amylose in its starch; wheat with reduced level of SBEIIb-activity with grain with non-wrinkled phenotype, with relatively high content of amylose.

62 cl, 28 dwg, 12 tbl, 16 ex

FIELD: agriculture.

SUBSTANCE: into plant or its part it is introduced nucleic acid, which encodes product, capable to support plant of family Solanaceae and its posterity by stability against injury by oomycetes fungus Phytophthora infestans.

EFFECT: ability to potato and tomato kinds steady against buck eye rot.

28 cl, 26 dwg, 4 tbl, 7 ex

FIELD: agriculture; biotechnology.

SUBSTANCE: to identify MON863 maize transformants, diagnosis is carried out of the presence of DNA-sequence, built in the gene of the maize, through transformation of the recombinant structure, containing the Cry3Bb gene and genome sequences, flanking the insertion site.

EFFECT: maize plants, regenerated from MON863 transformants, are resistant to beetle insects.

20 cl, 2 dwg, 2 ex

FIELD: agriculture.

SUBSTANCE: plant seeds are treated with ethylmethanesulphonate during 16 hours, then dried or greensprouted. The resulting plants M1 are self-pollinated and their seeds are obtained, which are used for growing the M2 wheat plants. The M2 plants are treated with imazamox, and plants with higher resistance to that as compared to the wild type plants are selected. The other version implies the transformation of plants with an expression vector containing one or more nucleic acids IMI.

EFFECT: higher resistance of plants to imidazolinone and possibility to use them when controlling weeds using herbicide.

44 cl, 8 dwg, 4 ex

FIELD: agriculture.

SUBSTANCE: plant seeds are treated with ethylmethanesulphonate during 6 hours, then dried or greensprouted. The resulting plants M1 are self-pollinated and their seeds are obtained, which are used for growing the M2 wheat plants. The M2 plants are treated with imazamox, and plants with higher resistance to that as compared to the wild type plants are selected. The other version implies the transformation of plants with an expression vector containing one or more nucleic acids IMI. The said nucleic acids are preferably located in different genomes or obtained from different genomes.

EFFECT: resulting plants have higher resistance to imidazolinone and may be used when controlling weeds using herbicide.

47 cl, 13 dwg, 5 ex

FIELD: molecular biology, genetic engineering, biochemistry.

SUBSTANCE: invention relates to polynucleotides optimized for expression in plants and encoding processing enzymes. Polynucleotides encode mesophilic, thermophilic or hyperthermophilic enzymes that are activated under conditions suitable for interaction with a necessary substrate. Incorporation of these polynucleotides into the plant genome results to preparing "self-processing" transgenic plants wherein their parts, for example, grain, fruit or seed express one or more indicated enzymes and have the varied composition. Autoprocessing plants can be used, for example, for preparing foodstuffs eliciting improved taste.

EFFECT: improved and valuable biological properties of plants.

29 cl, 23 dwg, 6 tbl, 41 ex

FIELD: agriculture.

SUBSTANCE: barley plant including mutation in gene SBE11a, or transgene which codes activity inhibitor of SBE11a, has low level of activity of enzyme SBE11a. Starch produced from grain of such plant may have amylase content of at least 40% (weight/weight). Also, barley of such kind may have lowered levels of activity of SBE11b. Barley grain may have non-rugose phenotype despite damaged amylopectin synthesis path.

EFFECT: lower levels of activity of branching enzyme and increased amylase content of starch-containing products.

46 cl, 19 dwg, 7 tbl, 11 ex

FIELD: plant gene engineering and food processing industry.

SUBSTANCE: glutamate-glyoxilate-aminotransferase activity is inhibited in plant by disruption of gene functionality encoding thereof. Due to inhibition of said enzyme activity glutamate levels in modified plant is higher in contrast to corresponding wild-type plants cultivated under the same conditions, including seeds thereof. Seeds and other plant parts are useful in foodstuff productions.

EFFECT: new method for elevation of glutamate content in plants.

31 cl, 12 dwg, 3 tbl, 2 ex

FIELD: agriculture.

SUBSTANCE: plant material is treated with mutagen or is transformed with a polynucleotide structure, containing a sequence of nucleic acid coding mutant polypeptide of large subunit of acetohydroxyacidsynthase (AHASL).

EFFECT: plants acquire tolerance to wide range of herbicides.

74 cl, 2 dwg, 6 tbl, 4 ex

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