KR20160137749A - Sequence Characterized Amplified Region Molecular Marker Related to WMV and ZYMV resistance Characteristic in Squash and use thereof - Google Patents

Abstract

The present invention provides a method for detecting or amplifying a marker comprising the sequence of a polynucleotide consisting of 5 to 444 consecutive bases represented by SEQ ID NO: 1, or a WMAR and a ZYMV resistant zucchini-identifying SCAR marker comprising a complementary polynucleotide sequence thereof, A kit comprising the composition, a kit for identifying WMV and ZYMV resistant zucchini comprising the composition, and a method for discriminating WMV and ZYMV resistant zucchini. Using the SCAR marker comprising the sequence of the polynucleotide consisting of 5 to 444 consecutive bases or the complementary polynucleotide sequence thereof represented by SEQ ID NO: 1 of the present invention, it is possible to easily obtain the amber for WMV and ZYMV It is possible to judge the resistance.

Description

[0002] SCAR markers for discrimination of watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) resistant zucchini and their uses [0002]

The invention pumpkin (Cucurbita spp . ) ≪ / RTI > for quality screening. More particularly, the present invention relates to a method of screening for SCM markers for squash quality selection for a trait with resistance to Watermelon Mosaic Virus (WMV) and Zucchini Yellow Mosaic Virus (ZYMV), detecting or amplifying the SCAR markers A kit comprising the composition, and a method for identifying WMV and ZYMV resistant amber.

Pumpkin ( Cucurbita spp . ) Is a major vegetable crop that occupies the third largest vegetable and vegetable area in the world and the second largest planted area in Korea. Amber is cultivated and cultivated in most farms by facility cultivation, which causes continuous damages of crops, loss of yields and deterioration of quality when pest damage occurs. Among the main causes of damaging pumpkins are WMV (Watermelon Mosaic Virus) and ZYMV (Zucchini Yellow Mosaic Virus). WMV and ZYMV are viruses that infect many pests and crops, causing 30% of domestic pumpkin farmers to be selected as the biggest problem. There have been efforts to control WMV and ZYMV because of the serious damage, but until now there is no other way besides chemical control. Chemical control methods can cause ecosystem degradation problems such as the residual problem of treated medicines, environmental pollution problems, the possibility of emergence of new pathogens with strong resistance, and there is a problem that additional cost and labor are required in the treatment process.

As a solution to this problem, cultivation of resistant strains has been demanded, and breeders have pursued breeding by using the traits of phenotypes as markers to select and cultivate resistant strains. However, this method has a disadvantage in that it is uncertain and limited to observe the accurate genotype because various environmental factors act as variables until a phenotype appears.

To overcome these drawbacks, research on molecular breeding, which introduces DNA molecule markers associated with resistance traits into sarcoma, and practical applications are continuing. Because molecular markers are made from plant DNA, they enable accurate genotyping without disturbing environmental factors, allowing selection at the beginning of breeding, and comparing with phenotypic traits . As such versatility is verified by many crops, SSR (Simple Sequence Data) based on Sequence Data of plant genome from AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA) and RFLP (Restriction Fragment Length Length Polymorphism) Sequence Repeat) and SNP (Single Nucleotide Polymorphism).

Among them, the RAPD marker is a technique that can be developed when there is no genome information of the target plant. It is a method of analyzing the polymorphism by amplifying whole plant DNA using a short primer of 10mer length. The polymorphism refers to a case where two or more alleles exist in a single gene locus, and genetic differences between individuals are determined by these polymorphisms. The RAPD markers are simple to test and quick to confirm the results, while there has been a problem of reproducibility due to short primers. Sequence Characterized Amplified Region (SCAR) markers have been developed to compensate for this. The SCAR analyzes the nucleotide sequence of a polymorphic marker resulting from an experiment such as RAPD and sequencer and rewrites it into a 20-mer molecular marker including a primer site, thereby solving the problem of reproducibility of RAPD It is a technique that can be solved.

Recently, there have been widely used methods of sorting varieties for specific plants using the above marker systems due to the development of molecular biology. However, the use of molecular genetic markers, especially SCAR markers, on the amber has shown resistance to both WMV and ZYMV Can not be identified.

Indeed, prior art J. AMER . SOC . HORT . SCI . 134 (5): 529-534. 2009 , mapping of genes associated with ZYMV-FL resistance and eIF4E genes in watermelon and eIF4E-CAPS markers were found to be useful in the breding program using MAS and ZYMV-FL resistance in watermelon Discloses the preparation of ZYRP-SCAR markers using PI 595203, but the above-mentioned prior art uses only the known ZYMV-FL resistance gene PI 595203 and confirmed its resistance only to ZYMV-FL Only.

Further, Prior Art Theor Appl In Genet (2004) 109: 707-712 , the ZYMV virus affects peptides and plants, and the primer sequences for amplifying genetic regions in cucumber and melon are YCZ-SCAR-1, YCZ- Discloses a primer of a marker, but does not disclose whether it is capable of discriminating resistance to WMV from amber and primers thereof.

Under these circumstances, the present inventors have made extensive efforts to develop a SCAR marker capable of easily discriminating the resistance of amber to WMV and ZYMV within a short time, and as a result, it has been found that a RAPD marker showing a specific polymorphism between resistant individuals and susceptible individuals The SCAR molecular marker was developed and confirmed that the SCAR can be easily diagnosed in the case of confirming the SCAR through the PCR primer, that WMV and ZYMV resistance of amber can be easily diagnosed.

One object of the present invention is to provide a SCAR marker for discriminating WMV and ZYMV resistant amber including a sequence of 5 to 444 consecutive nucleotides represented by SEQ ID NO: 1 or a sequence complementary to the above sequence.

It is another object of the present invention to provide a composition for discriminating WMV and ZYMV resistant amber which comprises an agent capable of detecting or amplifying the SCAR marker.

It is another object of the present invention to provide a kit for discriminating WMV and ZYMV resistant amber including the above composition.

It is another object of the present invention to provide a method for discriminating WMV and ZYMV resistant amber.

In order to breed pumpkin resistant to WMV and ZYMV, the present inventors collected pumpkin resources having resistance and susceptibility to two viruses, harvested leaves therefrom, isolated DNA of the harvested leaves, analyzed RAPD, The specific amplification sites to be converted to SCAR were identified by comparing and comparing the resistant phenotype and the susceptible phenotype of the amphibian species. The DNA was extracted from the amplified region and the nucleotide sequence was confirmed, and the WMV, ZYMV, , And it was confirmed that the presence of WMV and ZYMV resistance in amber could be easily diagnosed when the SCAR was identified through PCR primers.

Therefore, in one embodiment of the present invention for achieving the above object, the present invention provides a polynucleotide consisting of 5 to 444 consecutive bases represented by SEQ ID NO: 1, or a watermelon mosaic virus comprising the complementary polynucleotide thereof Watermelon Mosaic Virus (WMV), and Zucchini Yellow Mosaic Virus (ZYMV) resistant squash markers.

As used herein, the term "polynucleotide" refers to a polymer in which several deoxyribonucleotides or ribonucleotides are present in a single-stranded or double-stranded form and, unless specifically stated otherwise, Nucleotide analogs.

As used herein, the term "SCAR marker" is an abbreviation of Sequence Characterized Amplified Region Marker. The sequence of a polymorphic marker, which is the result of an experiment such as RAPD, is analyzed by sequencing and a primer site This is a technique that can solve the reproducibility problem of RAPD by rewriting with a 20-mer molecular marker included.

As used herein, the term "marker" means a molecule quantitatively or qualitatively related to the presence of a biological phenomenon, and the marker of the present invention comprises a polynucleotide consisting of 5 to 444 consecutive bases, But is not limited thereto. Specifically, the polynucleotide may be all or a part of the polynucleotide consisting of SEQ ID NO: 1, wherein the sequence of SEQ ID NO: 1 includes a base sequence in which one to several bases are deleted, substituted or added, And a base sequence that can hybridize under specific conditions.

The complementary polynucleotide may be a nucleotide sequence complementary to a polynucleotide sequence composed of 5 to 444 consecutive bases, wherein 1 to several bases are deleted, substituted or added in the complementary nucleotide sequence And includes a base sequence that can hybridize under certain conditions with the sequence.

As used herein, the term "complementary" when used in connection with a nucleic acid refers to a nucleic acid having a polarity in one direction containing a polynucleotide sequence that binds to bases of a polynucleotide of another nucleic acid whose bases have opposite polarities . For example, a nucleic acid having a sequence GCAU in the 5 'to 3' direction is complementary to a nucleic acid having a sequence CGTA in the 3 'to 5' direction. As used herein, the term complementary is used to include substantially complementary nucleic acids. Substantially complementary means that not all nucleotides are paired with the nucleotides of another nucleic acid, but nevertheless the two nucleic acids have sequences capable of forming stable hybrids under certain conditions.

As used herein, the term "hybridization" is used to mean the formation of a stable double structure between substantially complementary nucleotide sequences.

As used herein, the term "WMV " is abbreviation of WMV (Watermelon Mosaic Virus), which means watermelon mosaic virus. Plants infected with the watermelon mosaic virus have mosaic symptoms on the leaves and appear to have soot on the skin. In the past, the virus was a problem in the US and Europe. However, since the plant movement between countries has become frequent, the virus has recently developed in Korea, causing a decrease in production and a decrease in quality.

The term "ZYMV" is abbreviated as ZYMV (Zucchini Yellow Mosaic Virus), which means zucchini yellow mosaic virus. The virus belongs to the potyrivus family. Plants infected with the virus have wrinkles around the lateral nervous system, pale green mosaic lesions between the lateral and lateral nerves, and other parts turn yellow. In fruit, it is accompanied by malformations and mosaic lesions.

According to one embodiment of the present invention, an F1 line having a resistance phenotype is grown using four individuals of the SP213 line identified with the WMV and ZYMV resistance phenotype and the SP203 line identified with the susceptibility phenotype, F2 line individuals in which the lines were separated by self-crossing were used as a control group of the resistant phenotype. The selected SP213 line, SP203 line, F1 and F2 individuals were sown to extract leaves, DNA was extracted, RAPD analysis was performed on the separated DNA, PCR was performed, and electrophoresis was performed. As a result, (Fig. 1). As shown in Fig. In addition, the OPF19 band was extracted and sequenced to confirm the specific sequence of SEQ ID NO: 1, and a SCAR primer containing the OPF19 primer portion was prepared (FIG. 2). PCR analysis with the above primers confirmed the possibility of using the SCAR marker as a molecular marker for selection of WMV and ZYMV resistance system, and it was confirmed that a distinct band was found in some of the resistance phenotype SP213, F1 line and F2 line 3). Therefore, by amplifying the genotype of the SCAR marker of the present invention, performing PCR analysis, and reading the genotype of the SCAR marker, it is expected to be able to discriminate WMV and ZYMV resistant zucchini. It is the first to be identified.

According to another aspect of the present invention, there is provided a composition for discriminating WMV and ZYMV resistant amber including a preparation capable of detecting or amplifying said SCAR marker.

The marker is as described above.

The term "agent capable of detecting or amplifying a SCAR marker" means a composition capable of discriminating WMV and ZYMV resistant zucchini by amplifying the polymorphic site of the WMV and ZYMV resistance gene as described above But is not limited to, a primer capable of specifically amplifying a polynucleotide of the SCAR marker.

The primers used for the SCAR marker amplification can be amplified using appropriate conditions in suitable buffers (for example, four different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) and template-directed DNA Stranded oligonucleotide which can serve as a starting point of synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the SCAR marker but should be sufficiently complementary to hybridise with the SCAR marker and specifically include the polynucleotide sequence consisting of SEQ ID NOS: 2 and 3, But is not limited to.

As used herein, the term "primer" refers to a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, Quot; refers to a short sequence functioning as a starting point for < / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. At this time, the PCR conditions, the lengths of the sense and antisense primers can be modified based on those known in the art.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The suitable length of the primer is determined by the characteristics of the primer to be used, but it can be generally used as a length of 15 to 30 bp. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

The primer pair consisting of SEQ ID NO: 2 and SEQ ID NO: 3 can be preferably a continuous nucleotide defined by the nucleotide sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 3, and can be a nucleotide sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 3 But may include a base sequence which includes a base sequence in which one to several bases are deleted, substituted or added in the sequence, and which can hybridize under the specific conditions with the above sequence.

According to another aspect of the present invention, there is provided a kit for discriminating WMV and ZYMV resistant amber comprising the above composition.

The kit of the present invention can discriminate WMV and ZYMV resistant zucchini by confirming whether or not the genotype amplification of the SCAR marker, which is a marker for distinguishing WMV and ZYMV resistance, is confirmed.

The kit of the present invention may comprise a composition comprising an agent capable of detecting or amplifying the marker to identify WMV and ZYMV resistant zucchini.

The kit of the present invention can be used for identifying the level of expression of the marker to discriminate between WMV and ZYMV resistant amber.

The kit of the present invention may be an RT-PCR kit, a real-time RT-PCR kit, a real-time QRT-PCR kit, or a microarray chip kit.

In the present specification, RT-PCR means PCR using cDNA, which is DNA made by reverse transcription, as a template. Here, RT (Reverse Transcription) targets RNAs whose cell mass varies depending on environmental changes or specific conditions for analysis of gene expression patterns. Single-stranded RNA is more unstable than double-stranded DNA Reverse transcription (RT) is the process of reversing the DNA into a more stable DNA due to the limitation that the RNA itself can not be amplified in vitro.

In this specification, real-time RT-PCR refers to real-time RT-PCR, in which a thermal cycler and a spectrophotometric fluorescence spectrometer are matched, and the amount of target DNA can be analyzed by monitoring the generation process of the PCR amplification product in real time . Real-time RT-PCR does not require electrophoresis for confirmation of PCR amplification products, and it has excellent rapidity and quantification ability to quantify more precisely by comparing amplification products that occur exponentially with amplification.

In this specification, real-time QRT-PCR is a quantitative real-time PCR, which is an improvement over the well-known PCR technique. Actually existing PCR amplification can amplify a very small amount of sample in a large amount and has a simple process. However, since it amplifies and amplifies nucleic acid exponentially, it is difficult to estimate the amount of originally existing nucleic acid . To overcome these limitations, a qRT-PCR technique was developed to quantitatively detect the amount of amplified nucleic acid. This technique fluoresces the amplified nucleic acid material and continuously detects the fluorescence. Theoretically, since all PCRs are amplified at a magnification of 2 ⁿ (n = cycles) in the case of amplification, if the mathematical regression is performed with amplification values measured in real time, the amount of the initial template nucleic acid existing in the sample according to the number of cycles is inversely estimated I can do it.

In the present specification, a microarray chip means that very small DNA fragments are integrated on a solid surface and are used for simultaneously measuring the expression level of a large amount of genes or for genotyping various parts of the genome.

According to another aspect of the present invention, there is provided a method for detecting a protein comprising the steps of: (a) amplifying a polynucleotide consisting of SEQ ID NO: 1 from DNA or RNA of a sample separated from an individual; And (b) determining the resistance of WMV and ZYMV by identifying the amplified product of step (a).

The sequence of 5 to 444 consecutive nucleotides represented by SEQ ID NO: 1 is as described above.

The sample of step (a) can be isolated from any tissue of amber, and specifically can be isolated from the main stem of the early stage of development. The method for extracting DNA or RNA from the sample in step (a) is not particularly limited, and a technique known in the art or a commercially available DNA or RNA extraction kit can be used. For example, a phenol chloroform extraction method and a protease K treatment method can be used. DNA or RNA extraction kits are available from Qiagen, Inc and Stratagene. When the RNA is extracted and used, cDNA can be prepared by reverse transcription.

The sequence of 5 to 444 consecutive nucleotides represented by SEQ ID NO: 1 can be identified in the amplified product from the separated DNA or RNA. Any method known in the art can be used. For example, Can be determined to be resistant to WMV and ZYMV when confirmed by amplified products.

The present invention provides a method for detecting or amplifying a marker comprising the sequence of a polynucleotide consisting of 5 to 444 consecutive bases represented by SEQ ID NO: 1, or a WMAR and a ZYMV resistant zucchini-identifying SCAR marker comprising a complementary polynucleotide sequence thereof, A kit comprising the composition, a kit for identifying WMV and ZYMV resistant zucchini comprising the composition, and a method for discriminating WMV and ZYMV resistant zucchini. Using the SCAR marker comprising the sequence of the polynucleotide consisting of 5 to 444 consecutive bases or the complementary polynucleotide sequence thereof represented by SEQ ID NO: 1 of the present invention, it is possible to easily obtain the amber for WMV and ZYMV It is possible to judge the resistance.

FIG. 1 shows PCR amplification results and polymorphic sites using OPF19 random primers in RAPD analysis using amber WMV and ZYMV resistant phenotype SP213 individuals and susceptibility phenotype SP203 individuals, F1 and F2 individuals.
FIG. 2 shows a design of a primer using the SCAR site (SEQ ID NO: 1) obtained through RAPD analysis using the amber WMV and ZYMV resistant phenotype SP213 individuals and the susceptibility phenotype SP203 individuals, F1 and F2 individuals and the SCAR site.
FIG. 3 shows the result of PCR using zucchini WMV and ZYMV resistant phenotype SP213 individuals and susceptibility phenotype SP203 individuals, F1 and F2 individuals using SCAR primers prepared for development of amber WMV and ZYMV resistant trait-related molecular markers.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One. WMV  And ZYMV  Obtain resistance-resistant phenotype and susceptibility phenotype

The present inventors sowed seeds of WMV and ZYMV resistance-resistant pumpkin seeds and susceptible phenotypic amphibian seeds provided by the farming company Hongik Bio, and collected leaves at the time when three or more leaflets were developed.

The present inventors used four individuals of the SP213 lines identified as WMV and ZYMV resistant phenotypes and three individuals of the SP203 line identified as susceptibility phenotypes, and breed F1 lines having a resistance phenotype by crossing SP213 line and SP203 line . Individuals of the F2 line, which had been separated by self crossing of the cultivated F1 line, were used as a control group of the resistant phenotype.

Example 2. DNA Extraction and RAPD Screening of Selected Amber Entities

The selected WMV and ZYMV resistant phenotype SP213 individuals, susceptive phenotype SP203 individuals and F1 and F2 individuals were sown in the greenhouse and the leaves were harvested at the time when more than three leaflets were developed.

The state and concentration of the extracted DNA were confirmed by a spectrophotometer (UV-Vis Spectrophotometer, NanoDrop ND-100) (Table 1).

Name Conc. (Ng / ul) A260 / A280 Total vol. (Ul) Total quantity SP 213-1 3228 1.82 40 129ug SP 213-2 963 1.87 40 38ug SP 213-3 1720 1.88 40 68ug SP 213-4 2211 1.84 40 88ug SP 203-1 3481 1.83 40 139ug SP 203-2 2294 1.85 40 91ug SP 203-3 2462 1.89 40 98ug F ₁ -1 2230 1.83 40 89ug F ₁ -2 2754 1.84 40 110ug F ₁ -3 3701 1.79 40 148ug F ₁ -4 3584 1.81 40 143ug F ₁ -5 3332 1.82 40 133ug F ₁ -6 3347 1.80 40 133ug F ₂ -1 2354 1.87 40 94ug F ₂ -2 2911 1.85 40 116ug F ₂ -3 3015 1.84 40 120ug F ₂ -4 1762 1.75 40 70ug F ₂ -5 2438 1.78 40 97ug F ₂ -6 1608 1.82 40 64ug F ₂ -7 2809 1.76 40 112ug F ₂ -8 2391 1.81 40 95ug F ₂ -9 2491 1.78 40 99ug F ₂ -10 1224 1.81 40 48ug F ₂ -11 3246 1.77 40 129ug F ₂ -12 2892 1.79 40 115ug

OPP, OPP, and OPAN random primer set (iNtRON Maxime PCR Premix (i-taq) were used to confirm WMV and ZYMV resistant traits by using the isolated DNA. OPERON), 0.67 μl each, and 1 μl of DNA per individual. RAPD analysis was performed using a PCR machine at 94 ° C. for 3 minutes, denaturation at 94 ° C. for 1 minute, annealing at 37 ° C. for 1 minute, Followed by 40 cycles of extension at 72 ° C for 1 minute. The results were confirmed by electrophoresis on 1.5% agarose gel, and a random primer OPF19, a specific band that is specifically polymorphic to resistant traits, was found (FIG. 1).

Example  3: Specific band extraction and sequencing of resistant individuals SCAR  Marker detection primer  design

After extracting a band of OPF19 showing a specific polymorphism in only a part of it, sequencing was performed to identify a specific sequence of SEQ ID NO: 1 including OPF primer site. Then, a SCAR primer capable of detecting the nucleotide sequence of SEQ ID NO: 1 was designed using Oligo Perfect ( ^TM) software to amplify the nucleotide sequence of SEQ ID NO: 1 (Fig. 2). The primers were named VirSq-F19-F (SEQ ID NO: 2) and VirSq-F19-R (SEQ ID NO: 3), which are capable of binding to the nucleotide sequences shown in yellow and orange in the nucleotide sequence of SEQ ID NO: Table 2).

primer Base sequence VirSq-F19-F
(SEQ ID NO: 2) 5'-CCT CTA GAC CAA CTA TAA ATT AGA TAG-3 ' VirSq-F19-R
(SEQ ID NO: 3) 5'-CCT CTA GAC CCA CAG TAG GTT-3 '

Example  4: SCAR Marker  Verification

For the verification of WMV and ZYMV resistant trait-related SCAR molecular markers, 3 susceptibility phenotypes SP203, 6 F1 lines and 12 F2 lines were used, including the resistant phenotype SP213 4 individuals shown in Table 1 used for RAPD analysis.

PCR analysis was carried out with the primers of SEQ ID NOS: 2 and 3 for 25 individuals shown in Table 1 to verify the possibility of using the SCAR marker developed in Example 3 as a molecular marker for selection of WMV and ZYMV resistance system .

PCR was performed using iNtRON Maxime PCR PreMix (i-taq) with 1ul of 10p primer and 1ul of individual DNA. The PCR was carried out at 95 ° C for 1 minute and then at 95 ° C Thirty cycles of denaturation at 55 ° C for 30 seconds and annealing at 72 ° C for 30 seconds were performed.

As a result, distinct bands were confirmed in some of the resistance phenotype SP213, F1 line and F2 line, and no band was observed in the rest of the susceptibility phenotype SP203 and F2 lines (FIG. 3).

From this, it was confirmed that the SCAR amplified region was a gene region significantly associated with the WMV and ZYMV resistant traits of amber. In order to select the WMV and ZYMV resistant strains of amber by a simple PCR method, primers of SEQ ID NO: 2 to SEQ ID NO: 3 Can be utilized effectively.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Sequence Characterized Amplified Region Molecular Marker Related          to WMV and ZYMV resistance Characteristic in Squash and use          the <130> KPA150334-KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 444 <212> DNA <213> Artificial Sequence <220> <223> SCAR marker <400> 1 cctctagacc aagtataaat tagataggct tcttatatat aaaagttata tattaatggt 60 aaaaaaaaat atatcgaga tgtgaatgct cgtgattcac tcaatatagt 120 gatggatatc atggctgaac tacaaggaag attttctaga attagtccac ttcccattat 180 tcgacaataa aaatttacca cgtgtaatta aaagagttat tgaaagatga ctaaaacaag 240 agtgcaccgt gtaattaaag gaaccaaatg ttattaagat acctgtagac tcttattgca 300 accttaacct agtaggcatc catgggcttt tctttaagct ctatgaagtt caggtctaac 360 tttatagtta ttcgagtttc taggtaagcc tcgaagtttt gagtgtttct tataaccatc 420 acaaacctac tgtgggtcta gagg 444 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VirSq-F19-F <400> 2 cctctagacc aactataaat tagatag 27 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VirSq-F19-R <400> 3 cctctagacc cacagtaggt t 21

Claims (6)

A Watermelon Mosaic Virus (WMV) and a Zucchini Yellow Mosaic Virus (WMV) comprising a sequence of a polynucleotide consisting of 5 to 444 consecutive bases represented by SEQ ID NO: 1 or a complementary polynucleotide thereof, , ZYMV) SCAR marker for discriminating resistant pumpkin.
A composition for the determination of WMV and ZYMV resistant amber which comprises an agent capable of detecting or amplifying the marker of claim 1.
3. The composition according to claim 2, wherein the preparation is a primer represented by the polynucleotide consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
A kit for identifying WMV and ZYMV resistant amber comprising the composition of claim 2 or 3.
5. The kit according to claim 4, wherein the kit is an RT-PCR kit, a real-time RT-PCR kit, a real-time QRT-PCR kit or a microarray chip kit.
(a) amplifying the polynucleotide consisting of SEQ ID NO: 1 from the DNA or RNA of the sample separated from the individual; And
(b) determining the resistance of WMV and ZYMV by identifying the amplified product of step (a)
Determination of WMV and ZYMV resistant amber.

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