Molecular markers for identifying or assisting in the identification of resistance to fusarium crown and root rot in tomato and uses thereof

Abstract

The application discloses a molecular marker for identifying or assisting in identifying tomato neck and root rot resistance, a specific primer pair thereof and application of the molecular marker in tomato breeding. Experiments prove that the SNP-Fr160 site typing result of the molecular marker and the primer pair of the application identified or assisted in identifying by using the molecular marker is completely consistent with the result of phenotype resistance determination, the accuracy is 100%, and the accuracy is significantly higher than that of the existing C2-25 molecular marker detection. It is indicated that the molecular marker and the corresponding primer pair of the application can effectively detect the single nucleotide type of the SNP-Fr160 site, can be used for identifying or assisting in identifying the tomato neck and root rot resistance, can be used for screening of the tomato neck and root rot resistance variety, can be used for tomato molecular marker assisted breeding, and can be used for breeding and cultivation of the tomato neck and root rot resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecular marker for identifying or assisting in identifying tomato Fusarium crown and root rot resistance and its application in the field of biotechnology. BACKGROUND

[0002] Tomato Fusarium crown and root rot is a devastating soil-borne disease caused by the necrotrophic pathogen Fusarium oxysporum f. sp. radicis-lycopersici (Forl), which can cause more than 65% reduction in tomato yield or even total loss. The disease was first discovered in Japan in the 1970s, and then spread to many countries. It has become one of the most serious diseases threatening tomato production in winter and spring.

[0003] Frl is the only known high-resistance gene to Fusarium crown and root rot, which is derived from wild Solanum peruvianum. Tomato materials containing Frl gene show a high level of immunity to Forl infection. In the early stage, when Frl gene was not cloned, people developed UBC#194, 116, 655 and C2-25, etc. molecular markers closely linked to Frl for its assisted selection (Staniaszek, M., Szczechura, W., and Marczewski, W. 2014. Identification of a new molecular marker C2-25 linked to the Fusarium oxysporum f. sp. Radicis-lycopersici resistance Frl gene in tomato. Czech J. Genet. Plant Breed. 50, 285-287). Since the above molecular markers are Frl linked molecular markers, they have a certain genetic distance from Frl gene, so there is a problem of low accuracy in using these molecular markers for assisted selection of Frl gene. Taking C2-25, which is used most in breeding, as an example, the accuracy of this molecular marker assisted selection is 95.83% (Liu L, Wang H, Li W L, Wang F 2018. Markers linked to tomato Fusarium crown and root rot resistance gene Frl and their application. Jiangsu Agricultural Sciences 46, 91-93). SUMMARY

[0004] The technical problem to be solved by the present application is how to accurately identify tomato Fusarium crown and root rot resistance.

[0005] In order to solve the above technical problems, the present application provides the use of a substance for detecting the polymorphism or genotype of SNP in the genome of Solanum lycopersicum, wherein the SNP is SNP-Frl60, located at the 60th position of Frl gene, the 229th nucleotide of SEQ ID No. 1, and the nucleotide type is A or G, in any one of A1-A3.

[0006] A1. Use in identifying or assisting in identifying the resistance of tomato to Verticillium wilt;

[0007] A2. Use in preparing a product for identifying or assisting in identifying the resistance of tomato to Verticillium wilt;

[0008] A3. Use in tomato breeding or use in preparing a product for tomato breeding.

[0009] The genotype of the SNP in the genome of Solanum lycopersicum can be Frl / Frl, frl / frl or Frl / frl. The Frl / Frl is a homozygous type of the SNP G in the genome of Solanum lycopersicum, the frl / frl is a homozygous type of the SNP A in the genome of Solanum lycopersicum, and the Frl / frl is a heterozygous type of the SNP A and G in the genome of Solanum lycopersicum. The resistance of the tomato to be tested to Verticillium wilt with the genotype Frl / Frl or Frl / frl is higher or is a candidate for being higher than that of the tomato to be tested with the genotype frl / frl.

[0010] In the above use, the substance is any one of the following:

[0011] D1) The substance contains PCR primers for amplifying a DNA fragment of Solanum lycopersicum genome containing the SNP;

[0012] D2) The substance is a PCR reagent containing the PCR primers of D1);

[0013] D3) The substance is a kit containing the PCR primers of D1) or the PCR reagent of D2);

[0014] D4) The substance contains the PCR primers of D1) and the restriction endonuclease SacI;

[0015] D5) The substance is a reagent containing the PCR reagent of D2) and the restriction endonuclease SacI.

[0016] In the above use, the PCR primers are a primer pair consisting of P1 and P2; the P1 is a single-stranded DNA specifically binding to the upstream of the 229th position of the double-stranded DNA shown in SEQ ID No. 1; and the P2 is a single-stranded DNA specifically binding to the downstream of the 229th position of the double-stranded DNA shown in SEQ ID No. 1.

[0017] In the above-mentioned application, the P1 is a single-stranded DNA as shown in SEQ ID No. 3, and the P2 is a single-stranded DNA as shown in SEQ ID No. 4.

[0018] In the above, the substance for detecting the polymorphism or genotype (i.e. allele) of the SNP in the genome of tomato can be a reagent and / or instrument required for determining the polymorphism or genotype of the SNP by at least one of the following methods: DNA sequencing, restriction enzyme digestion fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography, and SNP chip. Among them, the SNP chip includes chip based on nucleic acid hybridization reaction, chip based on single base extension reaction, chip based on allele-specific primer extension reaction, chip based on "one-step" reaction, chip based on primer ligation reaction, chip based on restriction enzyme reaction, chip based on protein DNA binding reaction, and chip based on fluorescence molecule DNA binding reaction.

[0019] The present application also provides a product containing the substance for detecting the polymorphism or genotype of the SNP in the genome of tomato, which is any one of the products of C1) to C3):

[0020] C1) a product for detecting the single nucleotide polymorphism or genotype associated with tomato resistance to collar and root rot disease;

[0021] C2) a product for identifying or assisting in identifying the resistance of tomato to collar and root rot disease;

[0022] C3) a product for tomato breeding.

[0023] The present application also provides a method for detecting the polymorphism or genotype of the SNP in the genome of tomato.

[0024] The method for detecting the genotype of the SNP in the genome of tomato provided by the present application is as follows I or II:

[0025] I, comprising the following K1) and K2):

[0026] K1) using the genomic DNA of the tomato to be tested as a template, performing PCR amplification with the above-mentioned primer pair to obtain a PCR product;

[0027] K2) detecting the PCR product obtained in step K1), and determining the genotype of the SNP of the tomato to be tested according to the PCR product:

[0028] the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being only G is Frl / Frl; the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being only A is frl / frl; and the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being G and A is Frl / frl.

[0029] II. comprising the following L1) and L2):

[0030] L1) using the to-be-tested tomato genomic DNA as a template, performing PCR amplification by using a primer pair composed of P1 and P2 to obtain a PCR product;

[0031] L2) the following L21) or L22):

[0032] L21) performing enzyme digestion on the PCR product obtained in step L1) by using Sac I, detecting the size of the enzyme digestion product, and determining the genotype of the SNP of the to-be-tested tomato according to the size of the enzyme digestion product:

[0033] the genotype of the to-be-tested tomato corresponding to the enzyme digestion product being only a 479 bp DNA fragment is Frl / Frl; the genotype of the to-be-tested tomato corresponding to the enzyme digestion product being a 228 bp DNA fragment and a 247 bp DNA fragment is frl / frl; and the genotype of the to-be-tested tomato corresponding to the enzyme digestion product being a 479 bp DNA, a 228 bp DNA fragment and a 247 bp DNA fragment is Frl / frl;

[0034] L22) detecting the sequence of the PCR product obtained in step L1), and determining the genotype of the SNP of the to-be-tested tomato according to the sequence:

[0035] the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being G is Frl / Frl; the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being A is frl / frl; and the genotype of the to-be-tested tomato corresponding to the nucleotide of SEQ ID No. 1 at position 229 being G and A is Frl / frl.

[0036] The application also provides a method for identifying or assisting in identifying the resistance of tomato to neck and foot rot, comprising detecting the genotype of a to-be-tested tomato by the method for detecting the genotype of the SNP in the genome of the tomato described above, and identifying or assisting in identifying the resistance of the tomato to neck and foot rot according to the genotype of the to-be-tested tomato; the to-be-tested tomato with the genotype of Frl / Frl or Frl / frl has higher or is a candidate for higher resistance to neck and foot rot than the to-be-tested tomato with the genotype of frl / frl.

[0037] Another technical problem to be solved by the application is how to breed tomatoes.

[0038] To solve the above technical problems, the application provides a method for breeding tomatoes, comprising detecting the genotype of a to-be-tested tomato by the method for detecting the genotype of the SNP in the genome of the tomato described above, and selecting a tomato with the genotype of Frl / Frl or Frl / frl as a parent for breeding.

[0039] The application also provides the following A1-A3 of the primer pair:

[0040] A1, application in identifying or assisting in identifying the resistance of tomato to neck and foot rot;

[0041] A2, application in preparing a product for identifying or assisting in identifying the resistance of tomato to neck and foot rot;

[0042] A3, application in breeding tomatoes or application in preparing a product for breeding tomatoes.

[0043] In the above application, the breeding of tomatoes is breeding or selecting tomatoes resistant to neck and foot rot.

[0044] In the above, the tomato can be the offspring of the self-fertilization of Kevin.

[0045] In one embodiment of the present application, the primer is used to amplify the genomic DNA of tomato including SNP-Fr160 site, the reaction product is subjected to enzyme digestion with Sac I, and the enzyme digestion product is subjected to gel electrophoresis to determine the nucleotide type of SNP-Fr160 site. The experiment proves that, for the to-be-tested population of 5 commercial tomato varieties and 24 F2 generation offspring of one of the commercial varieties, or for the population of 1000 to-be-tested tomatoes, the genotyping result of SNP-Fr160 site using the primer of the present application is completely consistent with the result of phenotypic resistance determination, and the accuracy is 100%, which is significantly higher than the accuracy of the existing C2-25 molecular marker detection. It is proved that the primer pair of the present application can effectively detect the single nucleotide type of SNP-Fr160 site. The resistance of the to-be-tested tomato variety with homozygous SNP-Fr160 site G (genotype Fr1 / Fr1) and the to-be-tested tomato variety with heterozygous SNP-Fr160 site G and A (genotype Fr1 / fr1) to neck and foot rot is significantly higher than or candidate higher than that of the to-be-tested tomato variety with homozygous SNP-Fr160 site A (genotype fr1 / fr1), which proves that SNP-Fr160 is an SNP molecular marker related to the resistance of tomato to neck and foot rot, and can be used for identifying or assisting in identifying the resistance of tomato to neck and foot rot, screening tomato varieties resistant to neck and foot rot, tomato molecular marker assisted breeding, and breeding tomato resistant to neck and foot rot. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Figure 2 shows the electrophoresis results of each to-be-tested tomato after Sac I enzyme digestion in Example 2 of the present application. Figure 1 M is DL2KPUS DNA marker (Transgene BM101-01), lane 1 is Kevin, lane 2 is Caesar, lane 3 is Okko, lane 4 is Obe, lane 5 is Julie No. 2, and lanes 6-29 are Kevin F2 generation. DETAILED DESCRIPTION

[0047] The present application will be further described in detail below in conjunction with specific embodiments. The examples provided below serve only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0048] In the following examples, the experimental methods are conventional methods unless otherwise specified.

[0049] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0050] The tomato commercial varieties Kevin, Kaiser, and Julie 2 used in the following examples were purchased from Shouguang Syngenta Seed Co., Ltd., while Ouke and Obe were purchased from Beijing Zenong Weiye Agricultural Technology Co., Ltd. Among them, the commercial tomato varieties Kevin, Kaiser, Julie 2, and Obe contain the disease-resistant allele Frl, exhibiting high resistance to tomato neck and root rot; the commercial varieties Kevin, Kaiser, Julie 2, and Obe are F1 hybrids, also exhibiting high resistance to tomato neck and root rot. The commercial variety Ouke does not contain the disease-resistant allele Frl and is highly susceptible to neck and root rot.

[0051] Example 1: Obtaining Molecular Markers

[0052] I. Obtaining molecular marker-related fragments

[0053] Unlike linkage markers, functional markers are designed based on sequence variations that determine the presence (or strength) of a target gene's function, and can accurately distinguish between different allelic forms. Using this type of marker for assisted selection of target genes has 100% accuracy.

[0054] Recently, the inventors successfully cloned the Frl gene, which exists in two allele forms: the disease-resistant allele Frl and the disease-susceptible allele frl. Further research revealed that the difference at position 60 of the two alleles is crucial in determining their resistance to neck and root rot diseases, and this SNP site was named SNP-Frl60. The disease-resistant allele Frl has a G (guanine) base at position 60, encoding a protein with 217 amino acid residues and normal disease resistance; while the disease-susceptible allele frl has an A (adenine) base at position 60, causing premature translation termination and encoding a protein with only 19 amino acid residues and no disease resistance function.

[0055] SNP-Frl60 is a diallelic polymorphic SNP site in the tomato Frl gene. The SNP-Frl60 site is located at position 60 of the Frl gene, which is position 229 of SEQ ID No. 1 (corresponding to position 225 of SEQ ID No. 2), and the nucleotide type is G or A (represented by the letter R).

[0056] The inventors also discovered that the susceptibility allele *frl* has a restriction endonuclease cleavage site (GAGCTC) at positions 59-64 (positions 224-229 of SEQ ID No. 2) at positions 59-64, which can be cleaved by *Sac I*; while the resistance allele *Frl* has a cleavage site (GGGCTC) at positions 59-64 (positions 228-233 of SEQ ID No. 1), which cannot be cleaved by *Sac I*. Therefore, whether a tomato variety is resistant to tomato neck and root rot can be determined by identifying whether the 60th base of the *Frl* gene in the tested tomato variety is A or G through *Sac I* restriction enzyme digestion.

[0057] II. Design of Molecular Marker-Related Primers

[0058] Based on the flanking sequences of the 60th base of the Frl gene, CAPS-based molecular markers were developed, and PCR amplification primers were located on both sides of the 60th base of the Frl gene.

[0059] The primer sequences for the molecular markers are as follows:

[0060] Primer 1: 5'-GAGTCCTGCATAGCATAATC-3' (as shown in SEQ ID No. 3);

[0061] Primer 2: 5'-CCTTATCATCAAGGAACTTAG-3' (as shown in SEQ ID No. 4).

[0062] 2. Establishment of a method for identifying whether a tomato plant is resistant to tomato neck rot and root rot.

[0063] 1) Extract genomic DNA from the tomatoes to be tested.

[0064] 2) PCR amplification

[0065] Using the extracted genomic DNA as a template, PCR amplification was performed using primers 1 and 2, which are PCR-based molecular markers, to obtain the PCR amplification products.

[0066] The PCR amplification system consisted of: 10 μL of Premix Taq DNA polymerase Mix (Nanjing Novizan Biotechnology Co., Ltd. P222-01), 0.8 μL each of primer 1 and primer 2 (both with a final concentration of 10 μM in the system), 1.5 μL of genomic DNA, and double-distilled water to a final volume of 20 μL.

[0067] The PCR amplification reaction conditions were: denaturation at 94℃ for 20 seconds, annealing at 56℃ for 20 seconds, extension at 72℃ for 20 seconds, for a total of 35 cycles.

[0068] The obtained PCR product was digested with the restriction endonuclease SacI to obtain the digested product.

[0069] The enzyme digestion reaction system was as follows: 1 μL of restriction endonuclease SacI (ThermoFisher FastDigest SacIFD1133), 2 μL of 10x FastDigest buffer, 10 μL of the above PCR product, and double-distilled water to a final volume of 20 μL.

[0070] Electrophoresis was performed on a 1% agarose gel to determine the size of the enzyme digestion products.

[0071] If the enzyme digestion product is only a band of 226-249 bp, then the 60th base of the Frl gene in the genome of the tomato to be tested is A, and the genotype is frl / frl. The tomato to be tested is not resistant or is a candidate for not resistant to tomato neck rot and root rot.

[0072] If the enzyme digestion product is only a single band of 479bp, then the 60th base of the Frl gene on both homologous chromosomes of the tomato genome being tested is G, then the genotype of the tomato being tested is Frl / Frl, and the tomato being tested is resistant or a candidate for resistance to tomato neck rot and root rot.

[0073] If the enzyme digestion product contains two bands, 479bp and 226-249bp, then the 60th base of the Frl gene on one homologous chromosome of the tomato genome being tested is A, and the 60th base of the Frl gene on the other homologous chromosome is G. The genotype of the tomato being tested is Frl / frl, and the tomato being tested is resistant or a candidate for resistance to tomato neck rot and root rot.

[0074] Example 2: Application of molecular marker-related fragments or primers in identifying whether a tomato sample is resistant to neck rot and root rot.

[0075] I. Testing whether the tomatoes to be tested are resistant to tomato neck rot and root rot.

[0076] 1. Extract genomic DNA from the tomatoes to be tested.

[0077] Genomic DNA was extracted from the leaves of the commercial tomato varieties shown in Table 1 and the F2 progeny of the commercial variety Kevin, thus obtaining the genomic DNA of each tomato variety.

[0078] Table 1 shows the resistance of commercial tomato varieties and their F2 progeny (obtained from Kevin's first generation of self-pollination), as indicated in column 4 of Table 1. The resistance of the commercial varieties is known, while the resistance of the F2 progeny of Kevin has been determined in a laboratory disease nursery using the following methods:

[0079] Tomato seeds were sown in seedling trays and cultured in a greenhouse for one month. The seedlings were then transplanted to a laboratory nursery for tomato neck and root rot, allowing them to continue growing. Plant survival was observed two weeks later. If the plants grew normally, they were considered resistant to neck and root rot; if the above-ground parts wilted, the stem base constricted, and the roots rotted and turned brown, the tomato plants were not resistant to neck and root rot. The results are shown in column 4 of Table 1.

[0080] 2. PCR amplification and enzyme digestion

[0081] Using the genomic DNA of each tomato variety as a template, PCR amplification was performed using primers 1 and 2, which are CAPS-based molecular markers designed in Example 1, to obtain the PCR amplification products of each tomato variety.

[0082] The amplification system consisted of: 10 μL of Premix Taq DNA polymerase Mix (Nanjing Novizan Biotechnology Co., Ltd. P222-01), 0.8 μL each of primer 1 and primer 2 (final concentration in the system was 10 μL), 1.5 μL of genomic DNA, and double-distilled water to a final volume of 20 μL.

[0083] The reaction conditions were: denaturation at 94℃ for 20 seconds, annealing at 56℃ for 20 seconds, extension at 72℃ for 20 seconds, for a total of 35 cycles.

[0084] The PCR product was digested with restriction endonuclease SacI (ThermoFisher FastDigest SacIFD1133) to obtain the digested product.

[0085] The enzyme digestion reaction system was as follows: 1 μL of restriction endonuclease SacI (ThermoFisher FastDigest SacIFD1133), 2 μL of 10x FastDigest buffer, 10 μL of the above PCR product, and double-distilled water to a final volume of 20 μL.

[0086] 3. Testing

[0087] The DNA of each tomato variety was subjected to PCR reaction and enzyme digestion, and the products were electrophoresed on a 1% agarose gel to detect the enzyme digestion products.

[0088] If the enzyme digestion product is only a band of 226-249 bp, then the 60th base of the Frl gene in the genome of the tomato to be tested is A, and the genotype is frl / frl. The tomato to be tested is not resistant or is a candidate for not resistant to tomato neck rot and root rot.

[0089] If the enzyme digestion product is only a single band of 479bp, then the 60th base of the Frl gene on both homologous chromosomes of the tomato genome being tested is G, then the genotype of the tomato being tested is Frl / Frl, and the tomato being tested is resistant or a candidate for resistance to tomato neck rot and root rot.

[0090] If the enzyme digestion product contains two bands, 479bp and 226-249bp, then the 60th base of the Frl gene on one homologous chromosome of the tomato genome being tested is A, and the 60th base of the Frl gene on the other homologous chromosome is G. The genotype of the tomato being tested is Frl / frl, and the tomato being tested is resistant or a candidate for resistance to tomato neck rot and root rot.

[0091] The results are as follows Figure 1As shown in Table 1, among the commercial varieties, the tomato genome of Ouke, after primer amplification, yielded a single band of 200-500 bp. This band was recovered and sequenced, revealing that it consisted of two DNA fragments: one 228 bp fragment (sequence 1-228 of SEQ ID No. 2) and another 247 bp fragment (sequence 229-475 of SEQ ID No. 2). Its genotype is frl / frl, indicating a lack of resistance to tomato neck and root rot. The commercial varieties Kevin, Caesar, Obe, and Julie II also yielded two bands of 200-500 bp after primer digestion. These two bands were recovered and sequenced, revealing that they consisted of three DNA fragments: one 228 bp fragment (sequence 1-228 of SEQ ID No. 2) and another 247 bp fragment (sequence 229-475 of SEQ ID No. 2). The third DNA fragment, located at positions 229-475 of No. 2, is 479 bp in size and has the sequence SEQ ID No. 1. Its genotype is Frl / frl, and it is resistant to tomato neck rot and root rot.

[0092] Table 1 shows the PCR-based molecular marker detection results of the tomatoes to be tested.

[0093]

[0094]

[0095] In Table 1, the first column is the lane number, the second column is the variety origin, the third column is the variety name, the fourth column is the known resistance of the variety to tomato neck rot and root rot, the fifth column is the size of the enzyme digestion product band, and the sixth column is the genotype.

[0096] The above results indicate that the CAPS-based molecular markers highly correlated with the resistance of tomatoes to neck and root rot, with an accuracy rate of 100%. They can be used for the identification of resistant and susceptible materials.

[0097] II. Comparison of detection results between CAPS-based molecular markers and control C2-25 molecular markers

[0098] Phenotypic detection, CAPS-based molecular marker detection (primers 1 and 2 were used, and the method was the same as in step one) and C2-25 molecular marker detection were performed on 1000 Kevin F2 progeny plants.

[0099] For the C2-25 molecular marker method, please refer to (Liu Lei, Wang Hui, Li Wenli, Wang Fu, 2018. Marker and application linked to the tomato neck and root rot resistance gene Frl. Jiangsu Agricultural Sciences, 46, 91-93).

[0100] The primers used for the C2-25 molecular marker are primer 3 and primer 4:

[0101] Primer 3: 5'-ATGGGCGCTGCATGTTTCGTG-3';

[0102] Primer 4: 5'-ACACCTTTGTTGAAAGCCATCCC-3'.

[0103] Table 2

[0104]

[0105] The detection results of the three methods are shown in Table 2. It can be seen that, compared with the phenotypic detection results, the accuracy of the CAPS-based molecular marker of the present invention is 100%, while the accuracy of the C2-25 molecular marker detection is only 98.12%. The results show that the accuracy of the resistance identification of the CAPS-based molecular marker of the present invention is higher than that of the C2-25 molecular marker.

[0106] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Xi'an Jinpeng Seedling Co., Ltd. <120> Molecular markers for identifying or assisting in the identification of resistance to tomato neck rot and root rot and their applications <130> GNCSY213270 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 479 <212> DNA <213> Artificial Sequence <400> 1 gagtcctgca tagcataatc caaatttaat cagtgctata gactccgaac acgagagaga 60 aaaaaacctc tataaaacaa gactaaacct caagggagaa aaaccacaca acaattccct 120 cgaatattat tgtaaagatt agaaaaaaat agttaaccaa aattgagaga tggcagatgt 180 taagttgctt ggtctatggt atagcccttt tagtcataga gttgagtggg ctctaaagat 240 caagggtgtt gaatatgaat acatagaaga tgatctacat aataagagtt ctctacttct 300 tgaatccaat ccaattcaca aaaaagttcc agtactaatt cacaatggca agccactttg 360 tgagtcaatg gtaattgttg aatacatcga tgagacattt gaaggtcctt caatcttacc 420 taaagaccct tatgatcgtg ctatagctcg tttctgggct aagttccttg atgataagg 479 <210> 2 <211> 475 <212> DNA <213> Artificial Sequence <400> 2 gagtcctgca tagcataatc caaatttaat caatggtata gacaccgaac acgagagaga 60 aaaaaacctc aataaaacaa gactaaacct caagggagaa aaaccacaca acaattccct 120 cgaatattat tgtaaagatt agaaaaagtt aaccaaagtt gagagatggc agatgttaag 180 ttgcttggtc tatggtatag cccttttagt cacagagttg agtgagctct aaagatcaag 240 ggtgttgaat atgaatacat agaagatgat ctatataata agagttctct acttctcgaa 300 tccaatccaa ttcacaaaaa tattccagta ctaattcaca atgggaagcc aatttgtgag 360 tcaatggtaa ttgttgaata cattgataag acatttgaag gcccttccat catacctaaa 420 gatccttatg attgtgctat tgctcgtttt tgggctaagt tccttgatga taagg 475 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gagtcctgca tagcataatc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 ccttatcatc aaggaactta g 21

Claims

1. The application of a reagent for detecting single nucleotide polymorphisms in the solanum genome in any of the A1-A3 ranges, characterized in that, The single nucleotide polymorphism is SNP-Frl60, which is the nucleotide at position 229 of SEQ ID No. 1, and its nucleotide type is A or G; A1. Application in identifying or assisting in the identification of resistance to tomato neck rot and root rot; A2. Application in the preparation of products for identifying or assisting in the identification of tomato neck rot and root rot resistance; A3. Application in tomato breeding or in the preparation of tomato breeding products; wherein the tomato breeding is for the cultivation of tomatoes resistant to neck rot and root rot or the selection of tomatoes resistant to neck rot and root rot.

2. The application according to claim 1, characterized in that: The reagent is any one of the following: D1) The reagent contains PCR primers for amplifying tomato genomic DNA fragments including the SNP-Frl60; D2) The reagent described in D2) is a PCR reagent containing the PCR primers described in D1); D3) The reagent is a kit containing the PCR primers described in D1) or the PCR reagents described in D2); D4) The reagent contains the PCR primers and restriction endonuclease described in D1). Sac I; The reagent described in D5) contains the PCR reagent described in D2) and the restriction endonuclease. Sac I reagent.

3. The application according to claim 2, characterized in that: The PCR primers are a primer pair consisting of P1 and P2; P1 is a single-stranded DNA that specifically binds to the upstream of position 229 of the double-stranded DNA shown in SEQ ID No. 1; and P2 is a single-stranded DNA that specifically binds to the downstream of position 229 of the double-stranded DNA shown in SEQ ID No.

1.

4. The application according to claim 3, characterized in that: P1 is the single-stranded DNA shown in SEQ ID No. 3, and P2 is the single-stranded DNA shown in SEQ ID No.

4.

5. A method for detecting the genotype of SNP-Frl60 as described in claim 1 in the tomato genome, characterized in that: It is either I or II as follows: I. Including K1) and K2): K1) Using the genomic DNA of the tomato to be tested as a template, PCR amplification was performed using the primer pair described in claim 3 to obtain the PCR product; K2) Detect the PCR product obtained in step K1), and determine the genotype of SNP-Frl60 in the tomato to be tested based on the PCR product: The genotype of the tomato being tested, where the nucleotide at position 229 of SEQ ID No. 1 corresponding to the PCR product is only G, is: Frl / Frl The genotype of the tomato being tested, where the nucleotide at position 229 of SEQ ID No. 1 corresponding to the PCR product is only A, is: frl / frl The genotype of the tomato being tested, whose PCR product corresponds to nucleotides G and A at position 229 of SEQ ID No. 1, is: Frl / frl ; II. Including L1) and L2): L1) Using the tomato genomic DNA to be tested as a template, PCR amplification was performed using the primer pair composed of P1 and P2 as described in claim 4 to obtain the PCR product; L2) (L21) or L22 below): L21) Sac The PCR product obtained from enzyme digestion step L1) is analyzed, and the size of the digested product is determined. Based on the size of the digested product, the genotype of the SNP-Frl60 in the tomato sample is determined. The enzyme digestion product is only a 479bp DNA fragment. The genotype of the tomato being tested is... Frl / Frl The enzyme digestion products consist only of a 228 bp DNA fragment and a 247 bp DNA fragment. The genotype of the tomato being tested is... frl / frl The enzyme digestion products are 479 bp DNA, 228 bp DNA fragments, and 247 bp DNA fragments. The genotype of the tomato being tested is... Frl / frl ; L22) Detect the sequence of the PCR product obtained in step L1), and determine the genotype of the SNP-Frl60 in the tomato to be tested based on the sequence: The genotype of the tomato being tested, where the PCR product corresponds to nucleotide G at position 229 of SEQ ID No. 1, is [not specified]. Frl / Frl ; The PCR product corresponds to nucleotide A at position 229 of SEQ ID No. 1, indicating that the genotype of the tomato being tested is [not specified]. frl / frl The genotype of the tomato being tested, whose PCR product corresponds to nucleotides G and A at position 229 of SEQ ID No. 1, is: Frl / frl .

6. A method for identifying, distinguishing, or assisting in the identification of resistance to tomato neck rot and root rot, characterized in that, The genotype of the tomato to be tested is detected using the method of claim 5, and the resistance to neck rot and root rot of the tomato is identified or assisted in the identification based on the genotype of the tomato; the genotype is... Frl / Frl or Frl / frl The tested tomato varieties showed higher or higher resistance to neck rot and root rot than candidate genotypes. frl / frl The resistance of the tomato to neck rot and root rot was tested.

7. A method for tomato breeding, characterized by: The tomato genotype was detected using the method of claim 5, and the genotype was selected as... Frl / Frl or Frl / frl Tomatoes were used as parent plants for breeding.

8. The application of the primer pair as described in claim 3 or 4 in any of A1-A3: A1. Application in identifying or assisting in the identification of resistance to tomato neck rot and root rot; A2. Application in the preparation of products for identifying or assisting in the identification of tomato neck rot and root rot resistance; A3. Applications in tomato breeding or in the preparation of tomato breeding products.

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