CN109777883B - Specific primer of codominant molecular marker of tomato yellow leaf curl virus Ty-2 resistance gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a specific primer of a codominant SCAR molecular marker of a tomato yellow leaf curl virus resistance gene Ty-2 and application thereof. The nucleotide sequence of the resistance gene Ty-2 is shown in SEQ ID No. 1. The SCAR marker of the Ty-2 gene screened by the invention can be effectively used for marker-assisted selection of the Ty-2 gene, and the molecular marker is a molecular marker of a tomato yellow leaf curl virus resistance gene Ty-2. The marker improves the accuracy of selection, shortens the breeding period and lays a foundation for the functional research of the Ty-2 gene.

Description

Specific primer of codominant molecular marker of tomato yellow leaf curl virus Ty-2 resistance gene and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a specific primer of a codominant SCAR molecular marker AW910 of a tomato yellow leaf curl virus Ty-2 resistance gene and application thereof.

Background

Tomato Yellow Leaf Curl Virus (TYLCV) belongs to the Geminiviridae (Geminiviridae) genus phaseolus viridae (Begomovirus), is transmitted mainly by Bemisia tabaci (Bemisia tabaci), and poses serious and even devastating hazards to Tomato production. The existing method for preventing and controlling the virus is to use gauze for physical isolation, but the method needs to consume a large amount of pesticides and invest a large amount of funds.

The breeding of disease-resistant variety is the most effective method for preventing and treating the disease. Research shows that the wild tomato has the gene resisting TYLCV, and the disease-resistant gene Ty-2 shows higher resistance to TYLCV in Vietnam, India, Israel, mainland of China and other places, especially Asian regions. Ty-2 is a single dominant gene from solanum lycopersicum, originally located on a fragment of approximately 19cM of the long arm of chromosome 11, and was further located in the 6.5cM region in 2009. In view of this, there is a need to more finely locate Ty-2 and develop molecular markers for this gene to improve tomato breeding efficiency. However, researches show that a 200kb genetic inversion structure exists on a chromosome fragment where a target gene is located in the tomato with hairy tomato and cultivated tomato, and the genetic inversion structure seriously inhibits the recombination exchange of the chromosome fragment, so that the fine localization and cloning efficiency of the gene Ty-2 are influenced.

Disclosure of Invention

In order to solve the problems of large investment and unsatisfactory effect of preventing and controlling the tomato yellow leaf curl virus in the prior art, the invention provides a co-dominant SCAR molecular marker of a Ty-2 gene, a specific primer and application thereof by finely positioning a resistance gene Ty-2 of the tomato yellow leaf curl virus.

The invention aims to provide a codominant SCAR molecular marker of a tomato yellow leaf curl virus Ty-2 resistance gene.

It is still another object of the present invention to provide a primer set for the above molecular marker.

It is still another object of the present invention to provide a vector for the above molecular marker.

It is still another object of the present invention to provide the use of the above molecular marker.

According to the embodiment of the invention, the SCAR molecular marker AW910 of the tomato yellow leaf curl virus Ty-2 resistance gene has a nucleotide sequence shown in SEQ ID No. 1:

ATGGGACATCTAGCTCTAATGTACCCAAAGATCGGAAGAGGACATGACCATGGTTGTCTACGCTTCAACTCAGTTTACCCAGTATTGTTGGTTATCGATCATTCTTGTTTGCGAATTACTGCTAGCATTCAACAGATTTCAGAAATGGAGGTTGTCGTTGCAATTGGTGATGTTTTTCTCTCTTCAGCTTTCGATGTTCTCATTGATAGGCTTGCTCCTGAGGGTGATCTTCTCAAAATGTTTCGGAAGCGTAAGAATGATGTTGAGCTCTTAAAGAAGCTGAAATTGACTTTGCTTGGTCTTCAAGCTGTGCTAACTGATGCAGAGAATAAGCAGGCATCAAATCAATTTGTGAGAGAGTGGCTTAATGAGCTTCGACTTGCTGTAGACTCTGCTGAAAACTTGATTGAACAAGTCAATTATGAAGCTTTGAAGCTTAAGGTGGAAGGTAAGCATCAAAATCTTGCAGAAAAAATATTGAAACATTGTAGATTTTGCAACGTGTGCTTGAGTGATCATTCTTTTCTTAACATAAACGAAAAGTTGAAAAAGACTATTGAAATATTGGAGGTGTTGGAAAACCAAATTGGTCGCCTTGGCTTACAGAAACATTTTGGTTTGACTAAACAAGAAACTAGAACACCTTCAACTTCTTTGGTTGATGAATCTGATATCTTTGGTAGGCAGAAGGAAATAAAGGATTTGATTGACCTTTTATTGTCTGAAGATGCAAGTGGACGAAACCTGACTGTAGTTCCCATTGTTGGAATGGGCGGTGCAGGTAAGACAACACTAGCTAAAAAGGTTTACAATGATGCCAAGGTGAAGAACCATTTTGGTTTGAAAGCTTGGTATTGTGTTTCTGAGGCATATGATGCTTTGAGAATCACGAAAGGATTACTTCAAGAAATTGGCTCATTTGAGCCAAAGGATGACGGTAATTTTAATAAGCTACAAGTCAAATTGAAGGAAAGCCTGAAGGGAAAGAAGTTCCTTATTGTCCTCGATGATGTGTGGAATGATGACTATAATGAGTGGGATAACCTGAGAAATGTTTTTGTACAAGGAGAAATGGGAAGTAAGATCATTGTAACTACACGTAAGGAGAGTGTTGCTCAGACGATGTGTGTTGATCGTTGCACAATCACCGTGGGGAATCTGTCTAGTGAAGACTCTTGGTCTTTATTCAAACGACATTCACTAGAAAATAGGGATCATCCGGAACTTGAAAAGATTGGGAAAAAAATTGCAAACAAGTGCAAAGGGTTGCCTCTAGCTCTAAAGGCACTTGCTGGTGTTTTACGCGGCAAATCAGAGGTGGATGAGTGGAGAAACATTTTAAGGAGTGAAATATGGGATCAACAAAGTTGTTGGAATGGTATATTACCATCGTTGATGTTGAGCTACAATGATCTTCCTCCACATTTGAAGCGATGTTTTGCTTTTTGTGCGATATATCCCAAAGATTATGAATTCCGCAAAGACCAAGTTATTTACCTGTGGATTGCTAATGGTCTTGTAAAGCAGTTTTGTTTAGGTAATAAATACTTTGATGAGTTGAGATCAAGATCGTTGTTTGAAAGAGTCCCAGAATCAGAATGGAAATCGGAGGGATTCGTAATGCACGACCTTATCAATGATTTGGCCCAAACTGCATCTTCAAAACATTGTATTAGATTTGAAGAGAACGAAGGATCTGCTGTTATGTTAGAACAAAGTCGGCACATGTCCTATTCCATGGGAGAAAAAGGTGAATTTGAGAATTTGAAACAACTCTTCGAATCAGAGCAGCTGAGGACATTGCTTCCAACCAATAACAATTACTTTTCTAAACTAAGCAAGAGGGTGTTGCATAACATACTGCCAAGACTGACATCCTTAAGGGCATTGTCATTGTCATGTTATAGGATTAAGGAGTTGCCAAATGACTTGTTTATCAAATTAAAGATCCTCAGATTTTTGGACCTTTCTCGGACAGAGATTGAAAAGTTGCCAGATTCCATTTGTGTATTATATAACTTGGAGACACTTCTCTTGTCATCTTGTAAATATCTTGTGGAGCTACCGCTGCAGATGGAGAAGTTGATCAACTTGCGTCATCTTGACTTAAGCAACACTTCTCGCTTGAAGATGCCGCTACATCTGAGCAAGTTGAAAAGTCTCCAAATGCTAGTGGGAGCCAAGTTTCTTGTAGGTGGTAGCGGTGGTTTTAGAATGGAAGATTTGGGTGAAGTACATAACTTGTATGGATCTCTATCAGTTTTAGAGTTGCAAAATGTAGTTGATAGAAGGGAAGCTTTGAAGGCAAATATAAAGGAAAAGGAACATGTTGAGGAGTTGTATTTGCAGTGGAGTGAAGGTAGTTCTGCTTACGATTCTCAAACTGAAAGAGAGATACTTGATGAGCTACACCCACACACAAATATAAAACAATTAGAAATCAGGGGATATAGAGGGGCAAAATTTTCAAATTGGGTAGCTGATCCTTTGTTTCTTAAGCTTGTGAAATTGTCTCTTAGTGACTGCAAGGACTGTGATTCCCTGCCAGCACTAGGACAGCTTTGTTGTTTAAAATTCCTTTACATTAGAGGGATGCATGGAATAAGAGAAGTGACGGAAGAATTCTATGGCAGTTCCCTATACTCCAAAAAGCCTTTTAACTCTCTTGAGAAGCTTGAATTTAGAGATATGCGGGAGTGGAAGCAATGGCACGTACTAGGAAGCGGGGAGTTCCCTAAACTTGAGAAGGTTCTAATTCAAAATTGTCCCGAATTGACAGGGAAGTTTCCTGAAAATCTTTGTTCTCTGACTGAATTGAAAATTTCAGGATGTCCTCAACTCAATTTGGATAGATCCCAATTTGAGGGAATGAAGCAGATTGAGGTATTATTTATTAATAATTGCAACTCTGTTACCTCCTTACCTTTTAGCATACTTCCCAGTTCCTTGAAGATAATATCCATACGTAATTGTGAGAAATTGAAATTGGAGGAGGCAGTTGGTTATAGTAACATGTTTCTCGAGGAATTGCGACTGCAAGAATGTGATTGTATAGATGATATGTCACCTGAGTTTCTCCCAAGAGCACGTGAATTGAGTCTAGAGCGTTGCCACAACCTTACTAGGTTTTTGATTCCTACTGCCACTGAAAGTCTCACTATTCAGAATTGTGAGAATGTTGAAATACTTTCGGTGGCAGTTGACTCTGCGATGACATATTTGGAGATTCGGAATTGTGAGAAGCTGCAAGAAATACATCTCTTCAATTTACAAGTCCTTAAGATGAGTAATTGCAAGAAAGTAGTGAACAGTCGAAAGGAGTGGTGTTTACAGAGACTCACTGAGTTAGAGATCATACATGATGGCAGTGATGAAGAGATCCAACATTGGCAGTTGCCTTCCTCTATCACAAGACTTACCATAAAGAATCTGAAAACATTAAGCAGCCAAGTTCTGAAAAGCCTCACCTCTCTTCAATATCTACATGTTGAGGGTAATATACCTTCGTCACTGGTGGAACAAGGTGGGCTTCCCTCCTCTCTTTCTGAGCTATATTTAGGATACAATGATGAGCTCCATTCACTACATCTTTCCCACCTCACTTCACTTATATGTCTACACATCTCCCATTGCCATAATCTCCAATCACTCTCCGAATCAGCACTACCCTCCTCCCTCTCCGAGCTGACTATCTTCTATTGCCCTAAGCTCCAATCACTTTCCGAATCAGCACTGCCCTTCTCCCTCTCTCAGCTGACCATCTCTTATTGTGATAATCTCCAATCACTCTCCGAATCAGCACTGCCCTCCTCCCTCTCTCAGCTGACCATCATCAATTGCCGTAATCTCCAATCACTCTCCGAATCAGCACTGCCCTCCTCCCTCTCTCAGCTGACCATCATCAATTGCCGTAATCTCCAATCACTCTCCGAATCAGCACTGCCCTCCTCCCTCTCTCAGCTGACCATCATCGATTGCCGTAATCTCCAATCCCTTCCAGCAAAAGAGATGCCCTCTTCCCTCTCTATATTACATATTAGCAGATGCCCATTGCTCAAACCACTACTAGAATTTGACAAAGGGAAATACTGGCAAGATATTGCTCAAATTCCCATCTTATACATCAATGATGAATGGCTGTGA

the amino acid sequence of the protein coded by the Ty-2 resistance gene is shown as SEQ ID No. 2:

MGHLALMYPKIGRGHDHGCLRFNSVYPVLLVIDHSCLRITASIQQISEMEVVVAIGDVFLSSAFDVLIDRLAPEGDLLKMFRKRKNDVELLKKLKLTLLGLQAVLTDAENKQASNQFVREWLNELRLAVDSAENLIEQVNYEALKLKVEGKHQNLAEKILKHCRFCNVCLSDHSFLNINEKLKKTIEILEVLENQIGRLGLQKHFGLTKQETRTPSTSLVDESDIFGRQKEIKDLIDLLLSEDASGRNLTVVPIVGMGGAGKTTLAKKVYNDAKVKNHFGLKAWYCVSEAYDALRITKGLLQEIGSFEPKDDGNFNKLQVKLKESLKGKKFLIVLDDVWNDDYNEWDNLRNVFVQGEMGSKIIVTTRKESVAQTMCVDRCTITVGNLSSEDSWSLFKRHSLENRDHPELEKIGKKIANKCKGLPLALKALAGVLRGKSEVDEWRNILRSEIWDQQSCWNGILPSLMLSYNDLPPHLKRCFAFCAIYPKDYEFRKDQVIYLWIANGLVKQFCLGNKYFDELRSRSLFERVPESEWKSEGFVMHDLINDLAQTASSKHCIRFEENEGSAVMLEQSRHMSYSMGEKGEFENLKQLFESEQLRTLLPTNNNYFSKLSKRVLHNILPRLTSLRALSLSCYRIKELPNDLFIKLKILRFLDLSRTEIEKLPDSICVLYNLETLLLSSCKYLVELPLQMEKLINLRHLDLSNTSRLKMPLHLSKLKSLQMLVGAKFLVGGSGGFRMEDLGEVHNLYGSLSVLELQNVVDRREALKANIKEKEHVEELYLQWSEGSSAYDSQTEREILDELHPHTNIKQLEIRGYRGAKFSNWVADPLFLKLVKLSLSDCKDCDSLPALGQLCCLKFLYIRGMHGIREVTEEFYGSSLYSKKPFNSLEKLEFRDMREWKQWHVLGSGEFPKLEKVLIQNCPELTGKFPENLCSLTELKISGCPQLNLDRSQFEGMKQIEVLFINNCNSVTSLPFSILPSSLKIISIRNCEKLKLEEAVGYSNMFLEELRLQECDCIDDMSPEFLPRARELSLERCHNLTRFLIPTATESLTIQNCENVEILSVAVDSAMTYLEIRNCEKLQEIHLFNLQVLKMSNCKKVVNSRKEWCLQRLTELEIIHDGSDEEIQHWQLPSSITRLTIKNLKTLSSQVLKSLTSLQYLHVEGNIPSSLVEQGGLPSSLSELYLGYNDELHSLHLSHLTSLICLHISHCHNLQSLSESALPSSLSELTIFYCPKLQSLSESALPFSLSQLTISYCDNLQSLSESALPSSLSQLTIINCRNLQSLSESALPSSLSQLTIINCRNLQSLSESALPSSLSQLTIIDCRNLQSLPAKEMPSSLSILHISRCPLLKPLLEFDKGKYWQDIAQIPILYINDEWL

the primer pair of the SCAR molecular marker AW910 for amplifying the resistance gene of the tomato yellow leaf curl virus Ty-2 according to the specific embodiment of the invention has the following sequence:

AW910F:5’-AGAAGGTTAACGCGCTAAATTA-3’

AW910R:5’-AAGCCAAGAAGTTTGAAAACAC-3’。

according to the method for detecting the resistance of the tomato yellow leaf curl virus, which is a specific spectral band capable of identifying the genotypes of the male parent, the female parent and the heterozygote of the male parent and the female parent simultaneously by using the tomato genome DNA of the anti-tomato yellow leaf curl virus as a template through PCR amplification, the specific spectral band comprises a plant spectral band carrying a resistance gene and a plant spectral band not carrying the resistance gene, and the plant spectral band carrying the resistance gene is a molecular marker of a gene with a nucleotide sequence shown as SEQ ID No. 1.

The invention also provides application of the molecular marker in resistance gene detection or marker-assisted breeding of tomato yellow leaf curl viruses.

The invention has the beneficial effects that:

(1) the specific primer marked by the SCAR of the tomato yellow leaf curl virus resistance gene Ty-2 has the advantages of strong specificity, high stability, simple and quick screening method operation, low requirements on quality of detection equipment and a primer template, small using amount of test reagents, high speed, low cost, suitability for large batch, high flux and automation, and suitability for molecular breeding in modern agriculture;

(2) the invention lays important molecular genetics information for the gene function research of the tomato yellow leaf curl virus resistance gene Ty-2;

(3) the molecular marker specific primer of the tomato yellow leaf curl virus resistance gene Ty-2 is applied to breeding work, so that the economic loss of tomato yellow leaf curl virus to tomato production is greatly reduced, the pesticide consumption is reduced, the production cost is reduced, and the molecular marker specific primer has great application potential and higher economic value.

(4) The SCAR marker of the Ty-2 gene is screened out by the invention, and can be effectively used for marker-assisted selection of the Ty-2 gene, the marker improves the accuracy of selection, and the breeding period is shortened.

Drawings

FIG. 1 is a genetic analysis of 9000F 2 individuals derived from crossing a resistance gene with a susceptible material using molecular markers;

FIG. 2 shows the results of detection of AW910 marker in tomato breeding populations of the F2 generation after crossing of the resistance gene TY-2 with the susceptible parent 18-2;

FIG. 3 shows the detection results of the co-dominant SCAR molecular marker of Ty-2 on susceptible parent P1, susceptible parent P2, susceptible parent P3 and disease-resistant parent material Ty-2;

FIG. 4 shows the results of molecular marker selection of F2 population derived from the hybridization of backbone 18g1471 with TY2 gene-containing material H24 using marker AW 910.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 Fine localization and cloning of tomato yellow leaf curl Virus disease resistance Gene Ty-2

Materials used for fine mapping of the Ty-2 resistance gene and screening of the Ty-2 gene co-dominant SCAR marker: the disease-resistant homozygote (Ty-2/Ty-2) and the susceptible homozygote (Ty-2/Ty-2) are selected from Ty-2 (disease-resistant material, H24), S.habr.g1.1257(P1) (susceptible material), S.habr.g1.1560(P2) (susceptible material) and S.habr.g1.1290(P3) (susceptible material), and the disease-resistant material (H24) containing Ty-2 gene has 9000 single strains in total with corresponding F2 generation groups after hybridization of susceptible hairy tomato material S.1257, S.1606 and S.1290.

And (3) inoculation identification: see methods of Griffiths and Scott (2001). After 2-3 seedlings are in the true leaf period or the cuttings take roots, the seedlings are placed in a growth room with the tobacco whiteflies with the toxicity, the tobacco whiteflies with the toxicity are killed after two weeks, then the seedlings are transplanted into a greenhouse or a field, and the disease condition results are observed.

Extracting genome DNA from leaf of 2-3 week seedling. The PCR amplification system included 0.4. mu.M each of the forward and reverse primers, 5.0. mu.L of 2 XStart FastPfu Mix, 15ng of DNA template, and 10.0. mu.L of sterile water. Carrying out PCR, wherein the reaction program is pre-changed for 5min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 30-60s, and 37 cycles; extending for 5-10min at 72 ℃. For SCAR marks, the enzyme digestion system with the total volume of 10 mu L contains 4uL of PCR products and 1.5 units of corresponding enzymes, and the reaction lasts for 30-60 min. The PCR or restriction products were separated on 2.0% agarose gel with EB, and developed under UV light.

And determining primers with polymorphism among the parental, disease-resistant and disease-susceptible pools according to the PCR amplification result, and verifying the polymorphic primers through the recombinant single plant population and the progeny recombinant inbred line thereof. Positioning and verifying the disease-resistant gene by using the positioning information of the primer; then, the fine localization of the disease resistance gene was further analyzed.

According to the related sequence of Tomato genome, 252 pairs of primers are designed, and at the same time, 18 pairs of known marker primers on a Tomato genome-EXPEN genetic map are synthesized, genotype verification is carried out on P1, P2, P3 and a disease-resistant parent material complex, and 64 polymorphic markers are obtained by screening, wherein the polymorphic markers comprise 16 SCAR markers and 48 CAPS markers. And selecting 28 markers uniformly distributed on the chromosome to construct a high-density genetic map of the target region, wherein the average genetic distance between the markers is 0.05 cM. 9000F 2 populations hybridized with TY2 and susceptible parents are taken as materials, individuals recombined between flanking molecular markers C2_ At1g07960 and T0302 are screened, the offspring of the recombined individuals are subjected to genotype and bemisia tabaci inoculation identification, and as a result, the Ty-2 gene is finely positioned between the molecular markers C2At2g28520 and C2At2g32930, and the physical distance is about 350kb, which is shown in figure 1.

The Ty-2 gene codominant SCAR marker verifies the used group materials: the F2 generation tomato colony material of transferred Ty-2 gene is used for verification, the colony identification result is shown in figure 2, the length of the nucleotide sequence of the characteristic band of the plant containing TY2 homozygous resistance gene is 480bp, 15 positive single plants are detected in a common way, the length of the nucleotide sequence of the characteristic band of the negative plant without TY2 gene is about 700bp, the heterozygous single plant simultaneously contains 480bp and 700bp characteristic bands, the heterozygous single plant is F2 generation tomato colony material, and the F2 generation tomato colony material is obtained by hybridizing the H24 containing TY-2 gene material with a parent 1802.

And (3) field natural inoculation identification: and planting the verification material group in a corridor base of a tomato planting area with serious TYLCV diseases in autumn, and observing the disease condition result.

9000 individuals of the obtained 3F 2 populations are subjected to recombinant individual screening by using molecular markers TY21, CLEN, UF07960, AW52090, cL2, P1-19, UP15, M1, 51663, 32930 and TMA17, 576 recombinant individuals are screened out IN total, genetic analysis is carried out on the screened recombinant individuals by using the molecular markers distributed IN a chromosome fragment IN which the TY2 gene is located, and IN combination with field disease resistance identification, the Ty2 gene is further positioned between cL2 and TMA17 at a physical distance of 59kbp, fine positioning work is completed, 17 new candidate genes are arranged IN a target fragment, and then the genes, the Ty-2 gene and an expected protein sequence are cloned, namely SEQ IN No.1 and SEQ ID No. 2.

PCR amplification and screening are carried out on the disease-resistant parent Ty-2, the disease-susceptible parent P1, P2 and P3, a primer capable of amplifying polymorphism between parents is obtained, and an SCAR primer which has the best reliability and stability and does not need enzyme digestion is selected as a selective marker AW910 of the tomato yellow leaf curl virus gene Ty-2 from electrophoresis analysis and disease resistance identification results of a recombinant single-plant progeny segregation population. The amplification result of the primer is shown in FIG. 2, and the specific primer sequences are as follows:

AW910F:5’-AGAAGGTTAACGCGCTAAATTA-3’

AW910R:5’-AAGCCAAGAAGTTTGAAAACAC-3’。

example 2 molecular marker-assisted selection of the Ty-2 Gene Using AW910 marker

Molecular marker assisted selection of F2 population material obtained after crossing TY-2 gene-containing material with parents 1802 using AW910 markers. The results show that: among the 72 individuals, 15 positive individuals were detected by marker AW910 (480 bp in nucleotide sequence was positive), as shown in FIG. 3, and the numbers were 4, 11, 16, 18, 26, 28, 33, 35, 42, 45, 48, 52, 54, 68 and 70, respectively; detecting 21 negative individuals (the nucleotide sequence length is 700bp, which is negative), and the numbers are respectively No. 5, 9, 10, 14, 12, 21, 23, 25, 29, 31, 34, 37, 44, 46, 47, 49, 55, 59, 60, 61 and 66 individuals; 36 heterozygous individuals are detected, the numbers of the individuals are respectively No.1, 2, 3, 6, 7, 8, 13, 14, 15, 17, 19, 20, 22, 24, 27, 30, 32, 36, 38, 39, 40, 41, 43, 50, 51, 56, 58, 62, 63, 64, 65, 67, 69, 71 and 72, the individuals detected as positive and heterozygous are identified as resistant by disease resistance in the field, the individuals detected as negative are identified as susceptible by disease resistance in the field, and the codominant SCAR molecular marker marked as Ty-2 is proved.

The F2 group which is derived from the hybridization of backbone 18g1471 and a material H24 containing TY2 gene is subjected to molecular marker selection by utilizing a marker AW910, the result is that the nucleotide sequence of a characteristic band of a plant containing TY2 homozygous resistance gene is 480bp, the nucleotide sequence of a characteristic band of a negative plant without TY2 gene is about 700bp, a heterozygous single plant simultaneously contains two characteristic bands of 480bp and 700bp, the selection efficiency of the molecular marker and the field identification result coincidence rate are higher than 5%, and the result is shown in figure 4.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> vegetable and flower institute of Chinese academy of agricultural sciences

Specific primer of codominant molecular marker of tomato yellow leaf curl virus Ty-2 resistance gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4143

<212> DNA

<213> hairy tomato (L.hirsutum)

<400> 1

atgggacatc tagctctaat gtacccaaag atcggaagag gacatgacca tggttgtcta 60

cgcttcaact cagtttaccc agtattgttg gttatcgatc attcttgttt gcgaattact 120

gctagcattc aacagatttc agaaatggag gttgtcgttg caattggtga tgtttttctc 180

tcttcagctt tcgatgttct cattgatagg cttgctcctg agggtgatct tctcaaaatg 240

tttcggaagc gtaagaatga tgttgagctc ttaaagaagc tgaaattgac tttgcttggt 300

cttcaagctg tgctaactga tgcagagaat aagcaggcat caaatcaatt tgtgagagag 360

tggcttaatg agcttcgact tgctgtagac tctgctgaaa acttgattga acaagtcaat 420

tatgaagctt tgaagcttaa ggtggaaggt aagcatcaaa atcttgcaga aaaaatattg 480

aaacattgta gattttgcaa cgtgtgcttg agtgatcatt cttttcttaa cataaacgaa 540

aagttgaaaa agactattga aatattggag gtgttggaaa accaaattgg tcgccttggc 600

ttacagaaac attttggttt gactaaacaa gaaactagaa caccttcaac ttctttggtt 660

gatgaatctg atatctttgg taggcagaag gaaataaagg atttgattga ccttttattg 720

tctgaagatg caagtggacg aaacctgact gtagttccca ttgttggaat gggcggtgca 780

ggtaagacaa cactagctaa aaaggtttac aatgatgcca aggtgaagaa ccattttggt 840

ttgaaagctt ggtattgtgt ttctgaggca tatgatgctt tgagaatcac gaaaggatta 900

cttcaagaaa ttggctcatt tgagccaaag gatgacggta attttaataa gctacaagtc 960

aaattgaagg aaagcctgaa gggaaagaag ttccttattg tcctcgatga tgtgtggaat 1020

gatgactata atgagtggga taacctgaga aatgtttttg tacaaggaga aatgggaagt 1080

aagatcattg taactacacg taaggagagt gttgctcaga cgatgtgtgt tgatcgttgc 1140

acaatcaccg tggggaatct gtctagtgaa gactcttggt ctttattcaa acgacattca 1200

ctagaaaata gggatcatcc ggaacttgaa aagattggga aaaaaattgc aaacaagtgc 1260

aaagggttgc ctctagctct aaaggcactt gctggtgttt tacgcggcaa atcagaggtg 1320

gatgagtgga gaaacatttt aaggagtgaa atatgggatc aacaaagttg ttggaatggt 1380

atattaccat cgttgatgtt gagctacaat gatcttcctc cacatttgaa gcgatgtttt 1440

gctttttgtg cgatatatcc caaagattat gaattccgca aagaccaagt tatttacctg 1500

tggattgcta atggtcttgt aaagcagttt tgtttaggta ataaatactt tgatgagttg 1560

agatcaagat cgttgtttga aagagtccca gaatcagaat ggaaatcgga gggattcgta 1620

atgcacgacc ttatcaatga tttggcccaa actgcatctt caaaacattg tattagattt 1680

gaagagaacg aaggatctgc tgttatgtta gaacaaagtc ggcacatgtc ctattccatg 1740

ggagaaaaag gtgaatttga gaatttgaaa caactcttcg aatcagagca gctgaggaca 1800

ttgcttccaa ccaataacaa ttacttttct aaactaagca agagggtgtt gcataacata 1860

ctgccaagac tgacatcctt aagggcattg tcattgtcat gttataggat taaggagttg 1920

ccaaatgact tgtttatcaa attaaagatc ctcagatttt tggacctttc tcggacagag 1980

attgaaaagt tgccagattc catttgtgta ttatataact tggagacact tctcttgtca 2040

tcttgtaaat atcttgtgga gctaccgctg cagatggaga agttgatcaa cttgcgtcat 2100

cttgacttaa gcaacacttc tcgcttgaag atgccgctac atctgagcaa gttgaaaagt 2160

ctccaaatgc tagtgggagc caagtttctt gtaggtggta gcggtggttt tagaatggaa 2220

gatttgggtg aagtacataa cttgtatgga tctctatcag ttttagagtt gcaaaatgta 2280

gttgatagaa gggaagcttt gaaggcaaat ataaaggaaa aggaacatgt tgaggagttg 2340

tatttgcagt ggagtgaagg tagttctgct tacgattctc aaactgaaag agagatactt 2400

gatgagctac acccacacac aaatataaaa caattagaaa tcaggggata tagaggggca 2460

aaattttcaa attgggtagc tgatcctttg tttcttaagc ttgtgaaatt gtctcttagt 2520

gactgcaagg actgtgattc cctgccagca ctaggacagc tttgttgttt aaaattcctt 2580

tacattagag ggatgcatgg aataagagaa gtgacggaag aattctatgg cagttcccta 2640

tactccaaaa agccttttaa ctctcttgag aagcttgaat ttagagatat gcgggagtgg 2700

aagcaatggc acgtactagg aagcggggag ttccctaaac ttgagaaggt tctaattcaa 2760

aattgtcccg aattgacagg gaagtttcct gaaaatcttt gttctctgac tgaattgaaa 2820

atttcaggat gtcctcaact caatttggat agatcccaat ttgagggaat gaagcagatt 2880

gaggtattat ttattaataa ttgcaactct gttacctcct taccttttag catacttccc 2940

agttccttga agataatatc catacgtaat tgtgagaaat tgaaattgga ggaggcagtt 3000

ggttatagta acatgtttct cgaggaattg cgactgcaag aatgtgattg tatagatgat 3060

atgtcacctg agtttctccc aagagcacgt gaattgagtc tagagcgttg ccacaacctt 3120

actaggtttt tgattcctac tgccactgaa agtctcacta ttcagaattg tgagaatgtt 3180

gaaatacttt cggtggcagt tgactctgcg atgacatatt tggagattcg gaattgtgag 3240

aagctgcaag aaatacatct cttcaattta caagtcctta agatgagtaa ttgcaagaaa 3300

gtagtgaaca gtcgaaagga gtggtgttta cagagactca ctgagttaga gatcatacat 3360

gatggcagtg atgaagagat ccaacattgg cagttgcctt cctctatcac aagacttacc 3420

ataaagaatc tgaaaacatt aagcagccaa gttctgaaaa gcctcacctc tcttcaatat 3480

ctacatgttg agggtaatat accttcgtca ctggtggaac aaggtgggct tccctcctct 3540

ctttctgagc tatatttagg atacaatgat gagctccatt cactacatct ttcccacctc 3600

acttcactta tatgtctaca catctcccat tgccataatc tccaatcact ctccgaatca 3660

gcactaccct cctccctctc cgagctgact atcttctatt gccctaagct ccaatcactt 3720

tccgaatcag cactgccctt ctccctctct cagctgacca tctcttattg tgataatctc 3780

caatcactct ccgaatcagc actgccctcc tccctctctc agctgaccat catcaattgc 3840

cgtaatctcc aatcactctc cgaatcagca ctgccctcct ccctctctca gctgaccatc 3900

atcaattgcc gtaatctcca atcactctcc gaatcagcac tgccctcctc cctctctcag 3960

ctgaccatca tcgattgccg taatctccaa tcccttccag caaaagagat gccctcttcc 4020

ctctctatat tacatattag cagatgccca ttgctcaaac cactactaga atttgacaaa 4080

gggaaatact ggcaagatat tgctcaaatt cccatcttat acatcaatga tgaatggctg 4140

tga 4143

<210> 2

<211> 1380

<212> PRT

<213> hairy tomato (L.hirsutum)

<400> 2

Met Gly His Leu Ala Leu Met Tyr Pro Lys Ile Gly Arg Gly His Asp

1 5 10 15

His Gly Cys Leu Arg Phe Asn Ser Val Tyr Pro Val Leu Leu Val Ile

20 25 30

Asp His Ser Cys Leu Arg Ile Thr Ala Ser Ile Gln Gln Ile Ser Glu

35 40 45

Met Glu Val Val Val Ala Ile Gly Asp Val Phe Leu Ser Ser Ala Phe

50 55 60

Asp Val Leu Ile Asp Arg Leu Ala Pro Glu Gly Asp Leu Leu Lys Met

65 70 75 80

Phe Arg Lys Arg Lys Asn Asp Val Glu Leu Leu Lys Lys Leu Lys Leu

85 90 95

Thr Leu Leu Gly Leu Gln Ala Val Leu Thr Asp Ala Glu Asn Lys Gln

100 105 110

Ala Ser Asn Gln Phe Val Arg Glu Trp Leu Asn Glu Leu Arg Leu Ala

115 120 125

Val Asp Ser Ala Glu Asn Leu Ile Glu Gln Val Asn Tyr Glu Ala Leu

130 135 140

Lys Leu Lys Val Glu Gly Lys His Gln Asn Leu Ala Glu Lys Ile Leu

145 150 155 160

Lys His Cys Arg Phe Cys Asn Val Cys Leu Ser Asp His Ser Phe Leu

165 170 175

Asn Ile Asn Glu Lys Leu Lys Lys Thr Ile Glu Ile Leu Glu Val Leu

180 185 190

Glu Asn Gln Ile Gly Arg Leu Gly Leu Gln Lys His Phe Gly Leu Thr

195 200 205

Lys Gln Glu Thr Arg Thr Pro Ser Thr Ser Leu Val Asp Glu Ser Asp

210 215 220

Ile Phe Gly Arg Gln Lys Glu Ile Lys Asp Leu Ile Asp Leu Leu Leu

225 230 235 240

Ser Glu Asp Ala Ser Gly Arg Asn Leu Thr Val Val Pro Ile Val Gly

245 250 255

Met Gly Gly Ala Gly Lys Thr Thr Leu Ala Lys Lys Val Tyr Asn Asp

260 265 270

Ala Lys Val Lys Asn His Phe Gly Leu Lys Ala Trp Tyr Cys Val Ser

275 280 285

Glu Ala Tyr Asp Ala Leu Arg Ile Thr Lys Gly Leu Leu Gln Glu Ile

290 295 300

Gly Ser Phe Glu Pro Lys Asp Asp Gly Asn Phe Asn Lys Leu Gln Val

305 310 315 320

Lys Leu Lys Glu Ser Leu Lys Gly Lys Lys Phe Leu Ile Val Leu Asp

325 330 335

Asp Val Trp Asn Asp Asp Tyr Asn Glu Trp Asp Asn Leu Arg Asn Val

340 345 350

Phe Val Gln Gly Glu Met Gly Ser Lys Ile Ile Val Thr Thr Arg Lys

355 360 365

Glu Ser Val Ala Gln Thr Met Cys Val Asp Arg Cys Thr Ile Thr Val

370 375 380

Gly Asn Leu Ser Ser Glu Asp Ser Trp Ser Leu Phe Lys Arg His Ser

385 390 395 400

Leu Glu Asn Arg Asp His Pro Glu Leu Glu Lys Ile Gly Lys Lys Ile

405 410 415

Ala Asn Lys Cys Lys Gly Leu Pro Leu Ala Leu Lys Ala Leu Ala Gly

420 425 430

Val Leu Arg Gly Lys Ser Glu Val Asp Glu Trp Arg Asn Ile Leu Arg

435 440 445

Ser Glu Ile Trp Asp Gln Gln Ser Cys Trp Asn Gly Ile Leu Pro Ser

450 455 460

Leu Met Leu Ser Tyr Asn Asp Leu Pro Pro His Leu Lys Arg Cys Phe

465 470 475 480

Ala Phe Cys Ala Ile Tyr Pro Lys Asp Tyr Glu Phe Arg Lys Asp Gln

485 490 495

Val Ile Tyr Leu Trp Ile Ala Asn Gly Leu Val Lys Gln Phe Cys Leu

500 505 510

Gly Asn Lys Tyr Phe Asp Glu Leu Arg Ser Arg Ser Leu Phe Glu Arg

515 520 525

Val Pro Glu Ser Glu Trp Lys Ser Glu Gly Phe Val Met His Asp Leu

530 535 540

Ile Asn Asp Leu Ala Gln Thr Ala Ser Ser Lys His Cys Ile Arg Phe

545 550 555 560

Glu Glu Asn Glu Gly Ser Ala Val Met Leu Glu Gln Ser Arg His Met

565 570 575

Ser Tyr Ser Met Gly Glu Lys Gly Glu Phe Glu Asn Leu Lys Gln Leu

580 585 590

Phe Glu Ser Glu Gln Leu Arg Thr Leu Leu Pro Thr Asn Asn Asn Tyr

595 600 605

Phe Ser Lys Leu Ser Lys Arg Val Leu His Asn Ile Leu Pro Arg Leu

610 615 620

Thr Ser Leu Arg Ala Leu Ser Leu Ser Cys Tyr Arg Ile Lys Glu Leu

625 630 635 640

Pro Asn Asp Leu Phe Ile Lys Leu Lys Ile Leu Arg Phe Leu Asp Leu

645 650 655

Ser Arg Thr Glu Ile Glu Lys Leu Pro Asp Ser Ile Cys Val Leu Tyr

660 665 670

Asn Leu Glu Thr Leu Leu Leu Ser Ser Cys Lys Tyr Leu Val Glu Leu

675 680 685

Pro Leu Gln Met Glu Lys Leu Ile Asn Leu Arg His Leu Asp Leu Ser

690 695 700

Asn Thr Ser Arg Leu Lys Met Pro Leu His Leu Ser Lys Leu Lys Ser

705 710 715 720

Leu Gln Met Leu Val Gly Ala Lys Phe Leu Val Gly Gly Ser Gly Gly

725 730 735

Phe Arg Met Glu Asp Leu Gly Glu Val His Asn Leu Tyr Gly Ser Leu

740 745 750

Ser Val Leu Glu Leu Gln Asn Val Val Asp Arg Arg Glu Ala Leu Lys

755 760 765

Ala Asn Ile Lys Glu Lys Glu His Val Glu Glu Leu Tyr Leu Gln Trp

770 775 780

Ser Glu Gly Ser Ser Ala Tyr Asp Ser Gln Thr Glu Arg Glu Ile Leu

785 790 795 800

Asp Glu Leu His Pro His Thr Asn Ile Lys Gln Leu Glu Ile Arg Gly

805 810 815

Tyr Arg Gly Ala Lys Phe Ser Asn Trp Val Ala Asp Pro Leu Phe Leu

820 825 830

Lys Leu Val Lys Leu Ser Leu Ser Asp Cys Lys Asp Cys Asp Ser Leu

835 840 845

Pro Ala Leu Gly Gln Leu Cys Cys Leu Lys Phe Leu Tyr Ile Arg Gly

850 855 860

Met His Gly Ile Arg Glu Val Thr Glu Glu Phe Tyr Gly Ser Ser Leu

865 870 875 880

Tyr Ser Lys Lys Pro Phe Asn Ser Leu Glu Lys Leu Glu Phe Arg Asp

885 890 895

Met Arg Glu Trp Lys Gln Trp His Val Leu Gly Ser Gly Glu Phe Pro

900 905 910

Lys Leu Glu Lys Val Leu Ile Gln Asn Cys Pro Glu Leu Thr Gly Lys

915 920 925

Phe Pro Glu Asn Leu Cys Ser Leu Thr Glu Leu Lys Ile Ser Gly Cys

930 935 940

Pro Gln Leu Asn Leu Asp Arg Ser Gln Phe Glu Gly Met Lys Gln Ile

945 950 955 960

Glu Val Leu Phe Ile Asn Asn Cys Asn Ser Val Thr Ser Leu Pro Phe

965 970 975

Ser Ile Leu Pro Ser Ser Leu Lys Ile Ile Ser Ile Arg Asn Cys Glu

980 985 990

Lys Leu Lys Leu Glu Glu Ala Val Gly Tyr Ser Asn Met Phe Leu Glu

995 1000 1005

Glu Leu Arg Leu Gln Glu Cys Asp Cys Ile Asp Asp Met Ser Pro Glu

1010 1015 1020

Phe Leu Pro Arg Ala Arg Glu Leu Ser Leu Glu Arg Cys His Asn Leu

1025 1030 1035 1040

Thr Arg Phe Leu Ile Pro Thr Ala Thr Glu Ser Leu Thr Ile Gln Asn

1045 1050 1055

Cys Glu Asn Val Glu Ile Leu Ser Val Ala Val Asp Ser Ala Met Thr

1060 1065 1070

Tyr Leu Glu Ile Arg Asn Cys Glu Lys Leu Gln Glu Ile His Leu Phe

1075 1080 1085

Asn Leu Gln Val Leu Lys Met Ser Asn Cys Lys Lys Val Val Asn Ser

1090 1095 1100

Arg Lys Glu Trp Cys Leu Gln Arg Leu Thr Glu Leu Glu Ile Ile His

1105 1110 1115 1120

Asp Gly Ser Asp Glu Glu Ile Gln His Trp Gln Leu Pro Ser Ser Ile

1125 1130 1135

Thr Arg Leu Thr Ile Lys Asn Leu Lys Thr Leu Ser Ser Gln Val Leu

1140 1145 1150

Lys Ser Leu Thr Ser Leu Gln Tyr Leu His Val Glu Gly Asn Ile Pro

1155 1160 1165

Ser Ser Leu Val Glu Gln Gly Gly Leu Pro Ser Ser Leu Ser Glu Leu

1170 1175 1180

Tyr Leu Gly Tyr Asn Asp Glu Leu His Ser Leu His Leu Ser His Leu

1185 1190 1195 1200

Thr Ser Leu Ile Cys Leu His Ile Ser His Cys His Asn Leu Gln Ser

1205 1210 1215

Leu Ser Glu Ser Ala Leu Pro Ser Ser Leu Ser Glu Leu Thr Ile Phe

1220 1225 1230

Tyr Cys Pro Lys Leu Gln Ser Leu Ser Glu Ser Ala Leu Pro Phe Ser

1235 1240 1245

Leu Ser Gln Leu Thr Ile Ser Tyr Cys Asp Asn Leu Gln Ser Leu Ser

1250 1255 1260

Glu Ser Ala Leu Pro Ser Ser Leu Ser Gln Leu Thr Ile Ile Asn Cys

1265 1270 1275 1280

Arg Asn Leu Gln Ser Leu Ser Glu Ser Ala Leu Pro Ser Ser Leu Ser

1285 1290 1295

Gln Leu Thr Ile Ile Asn Cys Arg Asn Leu Gln Ser Leu Ser Glu Ser

1300 1305 1310

Ala Leu Pro Ser Ser Leu Ser Gln Leu Thr Ile Ile Asp Cys Arg Asn

1315 1320 1325

Leu Gln Ser Leu Pro Ala Lys Glu Met Pro Ser Ser Leu Ser Ile Leu

1330 1335 1340

His Ile Ser Arg Cys Pro Leu Leu Lys Pro Leu Leu Glu Phe Asp Lys

1345 1350 1355 1360

Gly Lys Tyr Trp Gln Asp Ile Ala Gln Ile Pro Ile Leu Tyr Ile Asn

1365 1370 1375

Asp Glu Trp Leu

1380

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agaaggttaa cgcgctaaat ta 22

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aagccaagaa gtttgaaaac ac 22

Claims (2)

1. The primer pair specific to the co-dominant SCAR molecular marker AW910 of the tomato yellow leaf curl virus Ty-2 resistance gene is characterized by comprising the following primers:

AW910 F:5’-AGAAGGTTAACGCGCTAAATTA-3’

AW910 R:5’-AAGCCAAGAAGTTTGAAAACAC-3’。

2. use of a pair of specific primers according to claim 1 for the detection of resistance to tomato yellow leaf curl virus.

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