CN112195264B

CN112195264B - SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of tomato hybrid and application

Info

Publication number: CN112195264B
Application number: CN202011133310.2A
Authority: CN
Inventors: 温常龙; 张建; 杨静静; 张晓飞; 罗江; 杨明珠
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-06-01
Anticipated expiration: 2040-10-21
Also published as: CN112195264A

Abstract

The invention discloses a method for identifying purity of tomato hybrid and an SNP primer combination used by the method. The SNP primer combination provided by the invention consists of 8 primer groups; each primer group consists of 3 primer sequences and is used for amplifying one SNP locus; the nucleotide sequence of each primer is shown as SEQ ID NO: 1 to SEQ ID NO: as shown at 24. The SNP primer combination can be used for early identification in the seed or seedling stage of tomato hybrid, ensures the purity of the hybrid, practically protects the rights and interests of producers and breeders, and provides technical support for the seed quality management of tomato varieties. The method provided by the invention has the advantages of high efficiency, accuracy, low cost and the like, and has a very wide application prospect.

Description

SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of tomato hybrid and application

Technical Field

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to an SNP (single nucleotide polymorphism) locus, a primer set, a kit, a detection method and application for identifying purity of tomato hybrid.

Background

Tomato (Solanum lycopersicum) belongs to the genus Solanum of the family Solanaceae, and plants are grown for one or more years, with origins in Central and south America. The product has unique taste, is rich in microelements required by human body, and has high nutritive value, thus being popular with consumers. Tomatoes have been widely distributed around the world for hundreds of years from cultivation to the present, and are the second largest vegetable crop in the world. The annual output of tomatoes in China exceeds 5000 ten thousand tons, and a considerable part is harvested by hybrid planting. The castration treatment is needed in the tomato hybrid seed production process, but in the actual production, the purity of the seeds is reduced due to improper operation of pollinators or other uncertain factors, and the quality of the tomato seeds and the subsequent production are seriously influenced. According to the specification of the crop seed quality standard (GB 16715.3-2010), the purity of the tomato hybrid is not less than 96%. The industrial seedling raising needs more than 99 percent of purity. Therefore, the tomato seeds need to be subjected to purity identification before being marketed, which is a requirement for controlling the production quality of the seeds and is also an important basis for trading.

The traditional seed purity identification mainly comprises seed form identification, field phenotype identification and the like, the period is long, labor and time are wasted, meanwhile, the influence of subjectivity and environmental factors is large, and the accuracy is difficult to guarantee.

In recent years, with the development of molecular biology, molecular marking methods have been widely applied to hybrid purity identification work of some crops. The molecular marker technology directly reveals the difference of parents from the DNA level, so the experimental result is accurate and reliable, and the identification can be carried out at the seedling stage and even at the seed level, thereby greatly shortening the experimental period. SSR markers and InDel markers are most commonly used in tomato production, but the SSR markers and the InDel markers are not widely used in seed purity identification work, such as purity identification of hybrids of Xinjiang main-cultivated tomato varieties TH8 and TH17 by using the InDel markers (Zhangxia et al, 2016); wangkai screened a pair of SSR primers SSR87 (Wangkai, 2015) capable of detecting the purity of tomato combination T8. In the domestic prior art, the application number is CN200710020044.0, and in a patent published on 6/3/2009, 1 pair of RAPD markers and 1 pair of SSR markers are disclosed, which can both identify the purity of tomato hybrid perilla powder No. 8. The application number is CN201810013749.8, a pair of EST-SSR markers are found in a patent published in 2018, 4, 13, the purity of tomato stock variety hobby hybrids can be identified, and 126/132bp difference bands can be amplified in an F1 generation. The purity of tomato hybrid 56115 was identified by a pair of Indel markers as disclosed in patent No. CN201811285955.0, published on 15.1.2019. The disclosed technology identifies the heterozygosis differential site of the hybrid from the DNA level, and has reliable result and higher application value. However, currently, there is no research on purity identification of many varieties by using molecular markers, and a primer combination capable of identifying many varieties cannot be found. Meanwhile, the amplification result of the currently used SSR primer has the problems of false positive and false negative, and the detection result is not stable enough; the InDel marker has the problems of relatively few sites and low detection accuracy although the stability is strong. Considering that the tomatoes have various varieties and abundant variation, and are screened accurately, efficiently and low-cost molecular marker combinations which are suitable for the purity detection requirements of the tomato seeds and are suitable for a large number of varieties, the molecular marker combination is a work to be urgently developed in the tomato breeding industry.

The third generation of molecular marker SNP is paid attention to its advantages such as large quantity, wide distribution, stable heredity, etc. With the development of high-throughput sequencing technology and the continuous reduction of sequencing cost, large-scale tomato re-sequencing becomes possible. Based on the analysis of tomato variation group information, more stable and efficient SNP sites can be mined. SNP typing does not need to contrast varieties, can detect differential sites rapidly and accurately with high throughput, is not influenced by environment, cultivation technology and artificial observation, has low cost and good repeatability, and is the most suitable selection for seed purity identification work. However, there is no report of identifying the purity of tomato hybrids using SNP markers. In view of this, there is an urgent need to develop a set of SNP sites, primer sets and detection methods suitable for identifying the purity of tomato hybrids in both scientific research and practice, and the methods are applied to identify the purity of tomato hybrids.

Reference documents:

wangkai, comparison test of varieties of small fruit type tomatoes in sunlight greenhouses and purity detection of hybrid seeds [ D ] [ Master thesis ]. Hebei academy of science and technology, 2015.

Zhangqingxia, Ganzhongxiang, Libeijin, Weiqiang, Yan Dellin, Zhang Yong, Chongqing Yong, Penggang utilizing InDel marker to identify and process tomato hybrid purity [ J ]. molecular plant breeding, 2016,14(06): 1533-.

Disclosure of Invention

The invention provides an SNP locus and a primer set for identifying the purity of a tomato hybrid, and a kit, a detection method and application based on the SNP locus and the primer set.

The invention is realized by the following technical scheme:

an SNP site for identifying purity of a tomato hybrid, selected from any 1 to 8 of the following first to eighth SNP sites: a first SNP locus, wherein the first SNP locus is located at 146411 th nucleotide of 9 th chromosome of a tomato reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C or T; a second SNP locus, wherein the second SNP locus is located at 348230 th nucleotide of 1 st chromosome of a tomato reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is A or G; a third SNP locus, wherein the third SNP locus is located at 45112865 th nucleotide of 6th chromosome of a tomato reference genome or a corresponding locus on homologous genome fragments among varieties thereof, and the nucleotide base of the locus is A or G; a fourth SNP locus, wherein the fourth SNP locus is located at 50349769 th nucleotide of 11 th chromosome of a tomato reference genome or a corresponding locus on an interspecific homologous genome fragment of the tomato reference genome, and the nucleotide base of the locus is C or T; a fifth SNP locus, wherein the fifth SNP locus is located at 2872268 th nucleotide of the 7 th chromosome of a tomato reference genome or a corresponding locus on an interspecific homologous genome fragment of the tomato reference genome, and the nucleotide base of the locus is A or T; a sixth SNP locus, wherein the sixth SNP locus is located at 21501063 th nucleotide of 2 nd chromosome of a tomato reference genome or a corresponding locus on homologous genome fragments among varieties thereof, and the nucleotide base of the locus is A or C; a seventh SNP locus, wherein the seventh SNP locus is located at 2072712 th nucleotide of the 4th chromosome of a tomato reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C or T; an eighth SNP locus, wherein the eighth SNP locus is located at 8449453 th nucleotide of 10 th chromosome of a tomato reference genome or a corresponding locus on an interspecific homologous genome fragment of the tomato reference genome, and the nucleotide base of the locus is C or T; wherein the tomato reference genome is tomato Heinz1706 reference genome.

In some embodiments, the sequence of the first SNP site and bases upstream and downstream thereof is SEQ ID NO: 25 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 25 has a homology of greater than or equal to 95%, 96%, 97%, 98% or 99%; the sequences of the second SNP locus and bases at the upstream and downstream of the second SNP locus are SEQ ID NO: 26 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 26 is greater than or equal to 95%, 96%, 97%, 98% or 99% homologous; the sequences of the third SNP locus and bases at the upstream and downstream are SEQ ID NO: 27 or an interspecies homologous genomic fragment thereof, more preferably a fragment that hybridizes to SEQ ID NO: 27 has a homology of greater than or equal to 95%, 96%, 97%, 98% or 99% to the nucleotide sequence; the fourth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 28 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 28 is greater than or equal to 95%, 96%, 97%, 98% or 99% homologous; the fifth SNP locus and the sequences of the upstream and downstream bases thereof are SEQ ID NO: 29 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 29 by greater than or equal to 95%, 96%, 97%, 98% or 99%; the sequences of the sixth SNP locus and the upstream and downstream bases thereof are SEQ ID NO: 30 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 30, or greater than 95%, 96%, 97%, 98%, or 99%; the seventh SNP locus and the sequences of bases on the seventh SNP locus and upstream and downstream thereof are SEQ ID NO: 31 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 31 by greater than or equal to 95%, 96%, 97%, 98% or 99%; the sequences of the eighth SNP locus and bases at the upstream and downstream are SEQ ID NO: 32 or an interspecies homologous genomic fragment thereof, more preferably a fragment identical to SEQ ID NO: 32 has a homology of 95%, 96%, 97%, 98% or 99% or more.

An SNP primer set for identifying the purity of tomato hybrids, which is used for amplifying the SNP sites respectively, and comprises: a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site.

In some embodiments, the first SNP primer set, the specific portion of the first forward primer, the specific portion of the second forward primer, and the downstream primer are each identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second SNP primer set are respectively matched with the sequence shown in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the third SNP primer set are respectively matched with the sequences shown in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fourth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the fifth SNP primer group, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, or more than 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the sixth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and in the eighth SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively matched with the sequences shown in SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM, HEX.

An SNP kit for identifying the purity of tomato hybrid is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises: the SNP primer sets preferably comprise a first upstream primer, a second upstream primer and a downstream primer of each primer set, wherein the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer in the SNP primer sets in the system is 2:2: 5.

A detection method for identifying the purity of tomato hybrids comprises the following steps: DNA extraction step: extracting the genome DNA of the N tomato hybrid seeds to be detected of a tomato variety to be detected; n is a natural number greater than 12, preferably greater than 95; screening a target primer group: respectively carrying out competitive allele specific PCR amplification reaction by using the primer group 4 by using the genomic DNA of more than or equal to 8 plants in the N tomato hybrid seeds to be detected as a template to obtain PCR reaction products; detecting the PCR reaction product to obtain the number of plants with the genotype of the SNP locus being a heterozygote, and obtaining a primer group with the largest number of the heterozygote plants as a target primer group; and (3) a target primer group PCR amplification step: taking the genome DNA of the N tomato hybrid seeds to be detected as a template, and respectively carrying out competitive allele specific PCR amplification reaction by using the target primer group to obtain a PCR reaction product of the target primer group; and (3) purity detection: detecting the PCR reaction product of the target primer group, and calculating the purity of the tomato hybrid to be detected according to the detection result; preferably, in the step of screening the target primer set and the step of detecting the purity, the method for detecting is selected from the group consisting of: fluorescence signal detection and direct sequencing.

In some embodiments, in the step of screening the target primer set and the step of detecting the purity, when the fluorescent signal detection is adopted, the number of strains showing the fluorescent signal indicating the color of the heterozygote in the primer set is counted; the primer group with the largest number of fluorescent strains showing the color indicating the heterozygote is the target primer group; when direct sequencing is adopted, counting the number of plants with the genotype of the SNP locus as a heterozygote; the primer group with the largest number of heterozygote strains is the target primer group.

In some embodiments, in the purity detecting step, the purity of the tomato hybrid to be detected is calculated by: when the fluorescent signal detection is adopted, counting the number of strains of which the colors of the heterozygotes are indicated by each target primer group and are fluorescent and the number of strains without fluorescence, respectively calculating the purity, and then calculating the average value, wherein the average value is the purity of the tomato hybrid to be detected; no fluorescence strain-N-strain showing color fluorescence indicative of heterozygotes-strain showing color fluorescence indicative of a first homozygote-strain showing color fluorescence indicative of a second homozygote; purity ═ 100% x [ number of strains indicating color fluorescence of heterozygote in target primer set/(N-number of strains with no fluorescence in target primer set) ]; when direct sequencing is used: counting the number of strains of each target primer group, which are heterozygous based on the genotype of the SNP locus, and the number of strains of which PCR amplification products are not obtained, respectively calculating the purities, and then calculating an average value, wherein the average value is the purity of the tomato hybrid to be detected; the purity was ═ 100% by number of strains whose target primer set was heterozygous based on the genotype of the SNP site/(N-number of strains from which PCR amplification products were not obtained with the target primer set).

In some embodiments, the tomato hybrid to be tested is Dou, L-402, Zhongza No. 8, Xueli, Anhui powder No. 3, Tianfu 501, 09FP53, black pearl, Zhongying No. 6, Peiteng, Shou-sheng-hong cherry, Beibei, Pingtao, Zhe powder 202, Jiangsu 152, Jiahong No. 4, Dongnong 715, Dongnong 706, European crown, Zhongza 101, Jinling Jiayu, Su 2, Su powder No. 15, Aolin 12, Rexing No. 5, Peiteng No. 5, camouflage No. 1, Su powder No. 16, Su powder 2331, Jingu No. 3, Shengyan No. 1, TP1303, Red ear, Beisi, Huamei 201, Dahong Wang No. 4, Zhonghaobao, Yan powder No. 1, Zibei, Gui Po, Pou 1478, Du, Dou, L-420, Red 968, Zhejiang 202, Shujiang No. 2, Shihong Shi No. 2, Shihong No. 4, Zhongbao, Zhonghuo No. 1, Shi No. 3, Shi Fangsu No. 1, Shi Fang, De Australian patent 302, golden shed No. 1, Xiaohong, Bofite 216, golden shed No. one, Juli, Nordun 0471, Baojiali No. two, golden shed No. one, Hongbao 304, HR57, Beijing white jade Tang, Qidali, 09FP47-1, SV0244TG, Beiying, Aijihongxiu, Petitng No. 1, De Australian 2030, Pink 2338, Shouyangzhen orange cherry, European Shanghai Red 968, Zhejiang Hetao 203, Naoxing No. 2, Zhongza 105, Jiaza No. 1, Russian MNP-1, golden shed No. 1, Osheng Sheng 126, HNX 80189, West nong 128, Ming Nu, Ke stock No. 2011 powder, Zhe powder 701, Qizali 203, Qida Liannau M208, Zhongza 206, Lifu red 16, Tian Liao, Tian Yi Su powder No. 14, Kaiyi Luo, SV 126, SV0384TG, Shi Mei Shu Yuan No. 216, Gui Mei Yi Yu Yi Fang No. 204, Zhen Yihao Yuan, Zhen Yu No. 2, Zhen Ying Yu Fang No. 204, Zhen Ying Yu No. 204, Zhen Ying Yu Ying Yu No., Red rice 35, red rice 10, jiahong No. 5, H9776, zhong za No. 9, zhe za 205, salon, melisa, hehang No. 1, zhong za No. 9, diffenib, niresta, wan za 15, new nobility, jinling sweet jade, tieti No. 1, zhe powder 702, su powder 11, HT12044, jinling beautiful jade, GBS103, 09FP253, 09FP29-4, SV4224TH, berculosis, small tom, shogao, millennium cherry tomato, SV7846TH, okouke, dongfeng No. one, zitao No. 9, zhong za No. 9, shenshui powder 998, sibe beide, zuhong No. 3, agyuhongyu yu, milandda, manan, jing T301, guan qun No. three, dong li, yan yao, yan fei, and fen 1.

The SNP locus, the SNP primer set, the SNP kit or the application of the detection method in detecting the purity of the tomato hybrid to be detected or preparing a reagent for detecting the purity of the tomato hybrid to be detected.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention screens a group of SNP marker combinations, which can be applied to the link of seed quality control, can quickly and accurately identify the purity of 163 parts of tomato varieties with high throughput, so that the links of seed production and quality control are more efficient and stable, and the speed of the varieties on the market is improved.

2. The 8 SNP loci and the primer combination thereof are confirmed to be suitable for identifying the purity of 163 tomato hybrid species, have stable and accurate detection effect and high detection speed, can be identified in the seed or seedling stage, are suitable for high-throughput detection equipment, are simple to operate, save time and labor, have wide application prospect, and can provide technical support for the seed quality management of tomato species.

Drawings

FIG. 1 is a graph showing the effect of SNP typing in a part of tomato hybrids tested in example 2 using 8 primer sets.

FIG. 2 is a graph showing the distribution of the number of heterozygous sites in a part of the tomato hybrids tested in example 2 for 8 primer sets.

FIG. 3 is a graph showing the effect of SNP typing in 96 Aijihongxiu hybrids of primer set 3 and primer set 7 in example 3.

Detailed Description

Defining:

tomato Heinz1706 reference genome: the genome of tomato variety Heinz 1706. A genome downloadable by the following address: ftp:// ftp. solgenomics. net/tomato _ genome/association/build-3.00/.

The hybrid of the invention: refers to the first generation of hybrid, and the hybrid used in production may have mixed parents.

Purity of hybrid seeds: a particular hybrid species is tested for its seeds or individuals using molecular markers that are polymorphic between parents (e.g., SNPs) and that are heterozygous for a particular molecular marker as a percentage of the total number of heterozygous and homozygous heterozygous individuals for that molecular marker.

Interspecies homologous genomic fragments: refers to a non-repetitive (single copy) homologous genomic fragment of the same segment of the same chromosome as a reference genomic fragment (e.g., the sequence shown in SEQ ID NO: 25 carrying the first SNP on the reference genome of tomato HEINZ 1706), in different varieties (or germplasm resources, such as, but not limited to, a tomato of 163 standard varieties of the invention) of a species (e.g., a genomic fragment in the tomato genome other than the reference genome, which is identical or homologous to the sequence shown in SEQ ID NO: 25 carrying the first SNP of the invention), and a genomic fragment extending or shortening several bases upstream and/or downstream of the genomic fragment. Shortening requires that the sequence specificity and the position recognition characteristics of the genomic fragment in different germplasms of the species (such as tomato) cannot be changed, shortening requires that when the reference genomic fragment contains molecular markers (such as polymorphic site markers like SNP sites), the molecular markers in the homologous genomic fragment cannot be spanned, shortening does not affect the degree of characterizing the molecular markers, and extending requires that the two ends of the genomic fragment cannot exceed the length that PCR can effectively amplify. Non-duplicative means that the genomic fragment is present only at one genomic position in one breed, and may be homozygous or heterozygous for polyploidy, and that the genomic fragment with high homology is not present at other positions in the genome of one breed, but highly homologous genomic fragments are ubiquitous in different breeds, for example, in the present invention, SEQ ID NO: 25 shows one of the interspecies homologous genomic fragments bearing the first SNP site of tomato. For example, SEQ ID NO: 25, the 11 th to 31 th sequences are genomic fragments formed by respectively extending 5 to 100 bases upwards and downwards on a reference genome of tomato HEINZ1706, or homologous sequences of the genomic fragments on other tomato germplasm genomes are regarded as homologous genomic fragments among varieties aiming at tomatoes.

Corresponding sites on the interspecies homologous genomic fragments: refers to a species (or germplasm resources, such as but not limited to one of the 163 standard varieties of tomato) in a species as a reference variety, which has a specific interspecies polymorphic genomic segment with molecular markers (e.g., SNP sites, position 21 of the sequence shown in SEQ ID NO: 25) that are highly polymorphic (e.g., predominantly C or T, and possibly other bases, at position 21 of the sequence shown in SEQ ID NO: 25) between varieties, and a sequence upstream and downstream (e.g., positions 1-20 and 22-41 of the sequence shown in SEQ ID NO: 25) that is highly conserved (this region may have small mutations that are random, not spread into the population, and that are not prevalent within a species, ignoring the mutation and looking at the SNP, the SNP has intra-breed commonality), then in an inter-breed homologous genomic fragment of a different breed of the species, the site of the molecular marker (e.g., highly polymorphic base) in the highly conserved genomic fragment is the corresponding site on the inter-breed homologous genomic fragment.

According to the invention, a group of tomato SNP primer combinations are provided through a large amount of screening preparation work, and the tomato SNP primer combinations are proved to be applicable to purity identification of 163 tomato varieties and can provide better technical support for the tomato breeding industry.

In a first aspect, the present invention provides a SNP site for identifying purity of a tomato hybrid, the SNP site being selected from any one of 1 to 8 of the following first to eighth SNP sites:

a first SNP site (LeSNP01) which is located at the 146411 th nucleotide of the 9 th chromosome of a tomato reference genome or a corresponding site on an interspecies homologous genome fragment thereof, and the nucleotide base of the site is C or T;

a second SNP site (LeSNP02) which is located at the 348230 th nucleotide of the 1 st chromosome of a tomato reference genome or a corresponding site on a homologous genomic fragment between varieties thereof, and the nucleotide base of the site is A or G;

a third SNP site (LeSNP03) which is positioned at the 45112865 th nucleotide of the 6th chromosome of a tomato reference genome or a corresponding site on a homologous genome fragment between varieties thereof, and the nucleotide base of the site is A or G;

a fourth SNP site (LeSNP04) which is positioned at the 50349769 th nucleotide of the 11 th chromosome of a tomato reference genome or a corresponding site on a homologous genome fragment between varieties thereof, and the nucleotide base of the site is C or T;

a fifth SNP locus (LeSNP05) which is positioned at the 2872268 th nucleotide of the 7 th chromosome of a tomato reference genome or a corresponding locus on a homologous genome fragment between varieties thereof, and the nucleotide base of the locus is A or T;

a sixth SNP locus (LeSNP06) which is positioned at the 21501063 th nucleotide of the 2 nd chromosome of a tomato reference genome or a corresponding locus on a homologous genomic fragment between varieties thereof, and the nucleotide base of the locus is A or C;

a seventh SNP locus (LeSNP07) which is positioned at the 2072712 th nucleotide of the 4th chromosome of a tomato reference genome or a corresponding locus on an interspecies homologous genome fragment thereof, and the nucleotide base of the locus is C or T;

an eighth SNP locus (LeSNP08) which is positioned at the 8449453 th nucleotide of the 10 th chromosome of a tomato reference genome or a corresponding locus on a homologous genomic fragment between varieties thereof, and the nucleotide base of the locus is C or T;

wherein the tomato reference genome is tomato Heinz1706 reference genome.

In a second aspect, the present invention provides an SNP primer set for identifying purity of tomato hybrid, comprising: a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site.

In some embodiments, the first SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2) of the first SNP primer set, the downstream primer (R) of the first SNP primer set, are identical to SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the second SNP primer set, which includes a specific portion of the first upstream primer (F1) of the second SNP primer set, a specific portion of the second upstream primer (F2) of the second SNP primer set, and a downstream primer (R) of the second SNP primer set, are identical to SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the third SNP primer set, which includes a specific portion of the first upstream primer (F1) of the third SNP primer set, a specific portion of the second upstream primer (F2) of the third SNP primer set, and a downstream primer (R) of the third SNP primer set, are identical to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fourth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fourth SNP primer set, are linked to SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the fifth SNP primer set, including the specific portion of the first upstream primer (F1), the specific portion of the second upstream primer (F2), and the downstream primer (R), of the fifth SNP primer set, are linked to SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, or more than 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the sixth SNP primer set, including the specific portion of the first upstream primer (F1) of the sixth SNP primer set, the specific portion of the second upstream primer (F2) of the sixth SNP primer set, and the downstream primer (R) of the sixth SNP primer set, are linked to SEQ ID NOs: 16. SEQ ID NO: 17. SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the seventh SNP primer set, including the specific portion of the first upstream primer (F1) of the seventh SNP primer set, the specific portion of the second upstream primer (F2) of the seventh SNP primer set, and the downstream primer (R) of the seventh SNP primer set, are linked to SEQ ID NOs: 19. SEQ ID NO: 20. SEQ ID NO: 21 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eighth SNP primer set, including the specific portion of the first upstream primer (F1) of the eighth SNP primer set, the specific portion of the second upstream primer (F2) of the eighth SNP primer set, and the downstream primer (R) of the eighth SNP primer set, are linked to SEQ ID NOs: 22. SEQ ID NO: 23. SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%.

In some embodiments, the above-described SNP primer combinations are selected from one or more of primer sets 01-08; the DNA sequence information of the primer group 01-08 is shown in a sequence table SEQ ID: 1-24, see table 2.

In the primer group, the 5' end of the upstream primer can be provided with a fluorescent tag sequence for fluorescent PCR detection, the first upstream primer and the second upstream primer in each group of primers are connected with different fluorescent molecules, more preferably, the fluorescent molecules are selected from FAM and HEX, and further preferably, the first upstream primer in each group of primers is connected with FAM, and the second upstream primer is connected with HEX; for example, the fluorescence signal of FAM fluorescent tag sequence is blue, and the fluorescence signal of HEX fluorescent tag sequence is red.

In any of the above primer sets, the molar ratio of the first forward primer (the primer named "F1"), the second forward primer (the primer named "F2") and the downstream primer (the primer named "R") may be specifically 2:2: 5.

In a third aspect, the present invention provides a SNP kit for identifying purity of tomato hybrids, the SNP reagent being formulated as a competitive allele-specific PCR reaction system, preferably comprising:

in the SNP primer sets, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer set in the system is 2:2: 5;

reagents, consumables and instruments in the reaction system were provided by LGC company, including reagent amounts, usage and the whole experimental procedure were performed according to the LGC company's operating manual KASP user guide and manual (www.lgcgenomics.com), KASPar reaction was performed in 384 well plates (Part No. KBS-0750-001) or 96 well plates (Part No. KBS-0751-001), and the reaction system was 3. mu.l or 10. mu.l, as shown in the following table.

Table: KASP reaction system of 384-well plate or 96-well plate

The preparation method of the kit also belongs to the protection scope of the invention, and the method comprises the step of separately packaging each primer in any primer group.

In a fourth aspect, the present invention provides an authenticity detection method for identifying a tomato variety, the method comprising the steps of:

s1, DNA extraction: extracting the genome DNA of the N tomato hybrid seeds to be detected of a tomato variety to be detected; n is a natural number greater than 95; the larger the numerical value of N is, the higher the accuracy of identifying the purity of the tomato hybrid to be detected is; if the numerical value of N is too small, the purity detection is not accurate.

S2, screening of target primer groups:

s2-1: using genomic DNA of more than or equal to 8, for example, 8-12 (such as 8-10, 10-12, 8, 10 or 12, the number of samples is convenient for arrangement in a PCR plate) of the tomato hybrid to be tested from the N tomato hybrids as a template, and respectively performing competitive allele specific PCR amplification (KASP) with the 8 primer groups to obtain PCR reaction products;

s2-2: detecting the PCR reaction product to obtain the number of plants which are heterozygous based on the genotype of the SNP locus, and obtaining a primer group with the largest number of heterozygous plants as a target primer group;

s3, PCR amplification of a target primer group: using the genomic DNA of the tomato hybrid to be tested of the N strains as templates, and respectively carrying out competitive allele specific PCR amplification reaction (KASP) by using the target primer group to obtain PCR reaction products of the target primer group;

s4, purity detection: detecting the PCR reaction product of the target primer group, and calculating the purity of the tomato hybrid to be detected according to the detection result;

in the step S2 of screening the target primer set and the step S4 of detecting the purity, the detection method is fluorescence signal detection or direct sequencing. (1) When the fluorescent signal detection is adopted, counting the number of strains of green (namely indicating the color of heterozygote) fluorescent signals displayed by the primer group (containing a fluorescent label sequence); the primer group with the largest number of green fluorescent strains is the target primer group; (2) when direct sequencing is adopted, counting the number of plants with the genotype of the SNP locus as a heterozygote; the primer group with the largest number of heterozygote strains is the target primer group.

In the primer set screening step S2 and the purity detecting step S4, the reaction program of KASP may specifically be: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. If the fluorescence signal is weak after the PCR amplification is finished and the data analysis is influenced, the cycle (denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min and 5 cycles) can be added until the result is satisfactory.

In the purity detecting step S4, the method for calculating the purity of the tomato hybrid to be tested includes:

(1) when the fluorescence signal is adopted for detection:

when the fluorescent signal detection is adopted, counting the number of strains of which the colors of the target primer groups indicate heterozygote fluorescence (green) and the number of strains without fluorescence, respectively calculating the purity, and then calculating the average value, wherein the average value is the purity of the tomato hybrid to be detected;

no fluorescence strain-N-strain showing color fluorescence indicative of heterozygotes-strain showing color fluorescence indicative of a first homozygote-strain showing color fluorescence indicative of a second homozygote;

purity ═ 100% x [ number of strains indicating color fluorescence of heterozygote in target primer set/(N-number of strains with no fluorescence in target primer set) ];

the color fluorescence for the above indicated heterozygotes is green, the color fluorescence for the first homozygote is red, and the color fluorescence for the second homozygote is blue.

(2) When direct sequencing is used:

counting the number of strains of each target primer group which are heterozygous based on the genotype of the SNP locus and the number of strains of which PCR amplification products are not obtained, respectively calculating the purity, and then calculating the average value, wherein the average value is the purity of the tomato hybrid to be detected;

the purity was ═ 100% by number of strains whose target primer set was heterozygous based on the genotype of the SNP site/(N-number of strains from which PCR amplification products were not obtained with the target primer set).

The tomato hybrid to be detected comprises the following components:

duoli, L-402, Zhonghao No. 8, Xueli, Anhui powder No. 3, Tianfu 501, 09FP53, black pearl, Zhongyu No. 6, Petitng, shou-yan-kusheng-hong cherry, Beibei, Pingtao No. 1, Zhe powder 202, Jiangsu No. 152, Jiahong No. 4, Dongnong 715, Dongnong 706, European crown, Zhonghao 101, Jinling Jiayu, Su No. 2, Su powder No. 15, Aolin 12, Rexing No. 5, Petitng No. 5, camouflage No. 1, Su powder No. 16, Pink 2331, Jinglu No. 3, Shengyan No. 1, TP1303, Red, Beisi, Huamei 201, Dawang, Jinhong No. 4, Zhongbao, Pink Fengbao No. 1, Zibei Bei, Hu Pai No. 8, Multi-420, Puhong 968, Zhe powder 202, Jiang No. 2, Shihong No. 14, L-402, Zhonghao No. 9, Weishitang No. 1, Shi No. 12, Shi Fu No. 1, Shi Fu No. 12, Shi Fu No. 216, cornel, nudun 0471, baojiali No. two, jinggang No. one, Hongbao 304, HR57, Beijing white jade Tang, zidarli, 09FP47-1, SV0244TG, Bei Yijihongxiu, Peiteng No. 1, De Aote 2030, Pink 2338, shou-an orange cherry, European Puhong 968, Zhe za 203, navigation Yi 2, Zhongza 105, Zhongza No. 1, Russian MNP-1, Jinchang No. 1, Odun 126, HNX12880189, Wen nong 2011, Ming-an, Ke 1, Sheng powder 701, Zhe za 203, Qidalli, Nu mu 208, Zhongza 206, Zhongfu red 16, Tianfu 363, Su powder No. 14, Kaiyuo, Fp126, SV03 0384TG, Zhuo powder 225, Pinus-a 138, Mei-Mei No. 2, east Mei-Li 2, Ying-Hai No. 2, Zhongfu red No. 205, Zhen-Hai-a Yihao No. 5, Zhen-a Zhen-Hai No. 5, Zhen-a, Zhen-Hai-a Yingza 976, Zhen-a, Zhen-Hai No. 1, Zhen-Hai-a Zhen-a, Zhe, Salon, Meilisa, Hehang No. 1, Zhongza No. 9, DiFenni, Niruisa, Anhui miscellaneous 15, New noble, Jinling sweet jade, Pingyi No. 1, Zhejiang powder 702, Su powder No. 11, HT12044, Jinling Xiuyu jade, GBS103, 09FP253, 09FP29-4, SV4224TH, Burjue, Xiaotomu, Shouying yellow cherry, millennium cherry tomato, SV7846TH, Ouchao, Dongfeng I, Zitao No. 9, Zhongza No. 9, Zhengfen, Shenfen 998, Sibeide, Anhui Red No. 3, Aijihongyu, Miranda, Rieman, Jing T301, Guanqu No. three, Dongnong nong No. 713, Difany Ni, Pingyan No. 1.

In a fifth aspect, the present invention provides the above-mentioned SNP sites, SNP primer sets, SNP kits, kits based on the SNP sites and primer sets, and applications of the detection methods: detecting the purity of the tomato hybrid to be detected, or preparing a reagent for detecting the purity of the tomato hybrid to be detected.

It should be noted that the present invention is only applicable to the purity identification of hybrid generation with only parental confounding, and is not applicable to the identification of several varieties confounding due to mechanical confounding.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1

SNP locus for identifying purity of tomato hybrid and acquisition of primer combination thereof

Determination of one, 8 SNP sites

Based on the inventor's laboratory 96 parts tomato resequencing data and tomato reference genome data, according to the screening conditions: MAF is greater than 0.1, deletion rate is less than 0.1, heterozygosity is less than 0.1, chromosomes are uniformly distributed, the Pearson correlation coefficient of the genetic distance with the whole genome SNP is greater than 0.9, PCA clustering effect is good, discrimination is high, the two wings of 50bp sequences are conserved (no InDel, no SSR and no other SNP), and 32 high-quality SNPs are selected.

163 parts of tomato hybrid variety genotypes are obtained by using the 32 pairs of SNP-KASP primers, then the optimal combination with good SNP typing effect and at least 1 heterozygous locus in 163 parts of hybrid is screened, and finally 8 SNP loci suitable for identifying the purity of tomato hybrids are determined. The basic information of the 8 SNP sites is detailed in Table 1. Wherein the position of the SNP locus on the chromosome is determined based on the alignment of tomato Heinz1706 reference genome sequences, and the tomato Heinz1706 reference genome download address is: ftp:// ftp. solgenomics. net/tomato _ genome/association/build _3.00 >

Table 1: basic information of 8 SNP sites

II, obtaining of SNP primer combination for identifying purity of tomato hybrid

According to the 8 SNP sites discovered in the first step, the inventor develops the SNP primer combination with higher polymorphism for identifying the purity of tomato hybrids.

The SNP primer set consists of 8 primer sets, and the name of each primer set is shown in the 2 nd column in Table 2. Each primer group consists of 3 primer sequences, comprises a first upstream primer, a second upstream primer and a downstream primer, and is used for amplifying one SNP site. The nucleotide sequences of the primers in the 8 primer sets are shown in Table 2, column 4, and the FAM fluorescent tag sequence is underlinedGAAGGTGACCAAGTTCATGCTDouble underlined HEX fluorescent tag sequence

The sequence of the specific part is not underlined.

Table 2: SNP primer nucleic acid sequence information table of 8 SNP sites

Example 2

This example is a validation test of the SNP primer combination developed in example 1. 163 parts of the tomato hybrid to be tested were all common excellent hybrids or foreign introduced hybrids. The details are shown in Table 3 below:

table 3: basic information of 163 test tomato hybrids

1. Acquisition of genomic DNA of tomato hybrids tested:

and (3) respectively extracting the genomic DNA of 163 leaves (30 seeds of each hybrid grow out of true leaves, and equal amount of leaves are picked and mixed) of the tomato hybrid to be tested by a Cetyl Trimethyl Ammonium Bromide (CTAB) method to obtain the genomic DNA of the tomato hybrid to be tested.

The CTAB method is specifically operated as follows: quickly grinding the mixed blades in liquid nitrogen into powder, and putting the powder into a centrifugal tube of 1.5 ml; adding 800 μ l CTAB buffer solution preheated to 65 deg.C for extraction, and extracting in 65 deg.C water bath for 30 min; adding a chloroform isoamyl alcohol mixed solution with the same volume, wherein the volume ratio of chloroform to isoamyl alcohol is 24:1, uniformly mixing, and rotating at the rotating speed of 8000r/min for 10 min; transferring the supernatant into a new centrifuge tube, adding isopropanol with the volume of 2/3 of the supernatant, and slightly and uniformly mixing the supernatant and the isopropanol in an upside-down manner; centrifuging at 10000r/min for 10 min; pouring out supernatant, washing precipitate with 75% ethanol, draining, standing at room temperature for 3min, and adding 100 μ l ddH₂O (containing 0.1% RNase) dissolves the precipitate and the resulting tomato genomic DNA is stored at 4 ℃ until use.

The quality and concentration of the stored genome DNA both need to meet the PCR requirement, and the standard of the standard is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; detecting that the ratio of A260 to A280 is about 1.8 and the ratio of A260 to A230 is more than 1.8 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of DNA ranged from 10-30 ng/. mu.L.

2. Obtaining a PCR amplification product: using 163 parts of genomic DNA of tomato hybrid to be tested as a template, and respectively adopting 8 primer groups to carry out competitive allele-specific PCR amplification. In each PCR reaction system, the concentration ratio of the first forward primer (named as "F1"), the second forward primer (named as "F2"), and the downstream primer (named as "R") was 2:2: 5.

Reagents, consumables and instruments in the reaction system were provided by LGC company, including reagent amounts, usage and the whole experimental procedure were performed according to the LGC company's operating manual KASP user guide and manual (www.lgcgenomics.com), KASPar reaction was performed in 384 well plates (Part No. KBS-0750-001) or 96 well plates (Part No. KBS-0751-001), and the reaction system was 3. mu.l or 10. mu.l, as shown in Table 4 below.

Table 4: KASP reaction system of 384-well plate or 96-well plate

Kits supplied by LGC company or otherwise having AS-PCR detection capability

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. The resulting amplification product was stored at 4 ℃ before electrophoresis.

3. And (3) fluorescent signal detection: after the step 2 is completed, when the temperature of the PCR amplification product is reduced to below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading value is observed when the FAM fluorescent label sequence is at 485nm of exciting light and 520nm of emitting light, reading value is observed when the HEX fluorescent label sequence is at 528nm of exciting light and 560nm of emitting light), and judging the genotype of 163 tomato hybrids to be tested based on each SNP site according to the color of a fluorescence signal.

The specific genotype judgment principle is as follows:

if a certain test tomato hybrid shows a blue fluorescent signal based on a certain SNP locus, the genotype of the test tomato hybrid based on the SNP locus is homozygote of the complementary base of the 1 st base at the 3' end of the first upstream primer for amplifying the SNP locus;

if a certain test tomato hybrid shows a red fluorescent signal based on a certain SNP locus, the genotype of the test tomato hybrid based on the SNP locus is homozygote of ' complementary base of 1 st base at the 3 ' end of a second upstream primer for amplifying the SNP locus ';

if a test tomato hybrid shows a green fluorescent signal based on a certain SNP site, the genotype of the test tomato hybrid based on the SNP site is a hybrid type, one base is a complementary base of the 1 st base at the 3 'end of the first upstream primer for amplifying the SNP site, and the other base is a complementary base of the 1 st base at the 3' end of the second upstream primer for amplifying the SNP site.

If the fluorescence signal is weak after the PCR amplification, which affects the data analysis, cycles (denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min) can be added until the result is satisfactory.

As shown in fig. 1, the fluorescence signal of PCR amplification product of 163 test tomato hybrids at each SNP site clearly appears in 3 forms: 1) the aggregate appears blue in the sample near the X-axis, the genotype is the allele that joins the HEX fluorescent tag sequence; 2) the aggregate appears red in the sample near the Y-axis, and the genotype is the allele that joins the FAM fluorescent tag sequence; 3) samples on the X and Y axes are shown in green and the genotype is a heterozygote of the two alleles. There were also few samples with no fluorescence signal or no discrimination, showing pink color, and amplification products were not clearly typed, possibly due to poor DNA quality or too low a concentration. Therefore, each primer group can obtain good typing effect in the tomato hybrid to be tested.

4. Heterozygous site number distribution and efficiency assessment

(1) And (3) counting the number of heterozygous loci of each tomato hybrid to be tested according to the 8 SNP locus-based genotypes of 163 tomato hybrids to be tested.

The results of the number distribution of heterozygous sites for 163 tomato hybrids tested, established on 8 primer sets, are shown in FIG. 2. The results showed that 8 primer sets enabled at least one heterozygous site per tomato hybrid tested.

(2) The purity identification of the hybrid seeds can reduce the workload by adopting a sequential analysis mode.

The result shows that the coverage rate of the heterozygous sites of the 8 primer groups in 163 test tomato hybrids reaches 100%.

Therefore, the SNP primer combination developed in example 1 can be applied to purity identification of tomato hybrids.

Example 3

In this example, the SNP primer combination developed in example 1 was used to detect the tomato hybrid to be tested: purity of Eleoxix hybrid. The detection method of the embodiment comprises the following steps:

1. acquisition of genomic DNA of Eleophilus elegans hybrid

(1) 200 commercially available Aijihongxiu hybrid seeds are planted in a conventional manner to obtain Aijihongxiu hybrid seedlings.

(2) Randomly taking leaf or root samples of 96 Aijihongxiu hybrid seedlings, replacing the tomato hybrid to be tested with Aijihongxiu according to the method of the step 1 in the example 2, and extracting genome DNA by adopting a CTAB method without changing other steps to sequentially obtain the genome DNA of 96 Aijihongxiu hybrids.

2. Screening of target primer set

(1) Randomly selecting 8 genomic DNAs from 96 Agelex hybrids, respectively using the 8 genomic DNAs of the Agelex hybrids as templates, replacing 163 genomic DNAs of tomato hybrids to be tested with 8 genomic DNAs of the Agelex hybrids according to the method of step 2 in example 2, and performing competitive allele-specific PCR reaction to obtain PCR products of 8 Agelex hybrids without changing other steps.

The PCR reaction program is: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. See table 4 of example 2 for the PCR reaction system.

(2) And (2) after the step (1) is completed, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm exciting light and 520nm emitting light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm exciting light and 560nm emitting light), and obtaining the color of a fluorescence signal. Comparing the strains of the 8 primer groups which show green fluorescent signals, wherein the primer group which shows the most green fluorescent strains is the screened primer group.

The results showed that the number of strains showing green fluorescence signals in the primer set 3 and the primer set 7 was the largest and 8 strains were both present. Therefore, the primer group 3 and the primer group 7 are the screened target primer groups, and subsequent experiments are carried out.

3. Obtaining the purity of the Aijihongxiu hybrid

(1) And (3) performing PCR amplification by using the genome DNA of 96 Agelex hybrids as a template and respectively adopting a primer group 3 and a primer group 7 to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the first upstream primer named "F1", the second upstream primer named "F2" and the downstream primer named "R" was 2:2: 5.

the reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. See table 4 of example 2 for the PCR reaction system.

(2) And (2) after the step (1) is completed, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm exciting light and 520nm emitting light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm exciting light and 560nm emitting light), and obtaining the color of a fluorescence signal.

The SNP typing results are shown in FIG. 3, in which the left panel is primer set 3 and the right panel is primer set 7.

(3) After completion of step (2), the number of strains showing green fluorescence and the number of strains showing no fluorescence (96-number of strains showing green fluorescence-number of strains showing red fluorescence-number of strains showing blue fluorescence) were counted for each of the primer set 3 and the primer set 7; calculating the purity of the Aijihongxiu hybrid according to the following formula; the average was further calculated to obtain the average purity.

Purity ═ 100% by number of strains exhibiting green fluorescence in the primer set/(96-strains exhibiting no fluorescence in the primer set).

The results showed that the number of strains showing green fluorescence of the primer set 3 was 96 strains, the number of strains showing no fluorescence was 0 strains, and the purity was 96/(96-0) ═ 100%; the number of strains showing green fluorescence of the primer group 7 is 95, the number of strains showing no fluorescence is 1, and the purity is 95/(96-1) ═ 100%; the average purity of the Eleophilus indica hybrid was (100% + 100%)/2 ═ 100%. In the same batch of seeds, 200 Aijihongxiu hybrid seeds are additionally selected for accelerating germination and raising seedlings, 100 single plants are randomly selected and transplanted to a field, phenotype investigation is carried out from a flowering period to a fruiting period, and no heterotypic plant is found in 100 single plants. The purity of the product is 100% by calculation, which proves that the field investigation result is basically consistent with the molecular identification result of the embodiment.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

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Claims

1. An SNP primer combination for identifying purity of tomato hybrids, which is used for amplifying the following SNP locus sets consisting of a first SNP locus to an eighth SNP locus respectively:

a first SNP locus, wherein the first SNP locus is located at 146411 th nucleotide of 9 th chromosome of a tomato reference genome, and the nucleotide base of the locus is C or T;

a second SNP site, wherein the second SNP site is located at 348230 th nucleotide of 1 st chromosome of a tomato reference genome, and the nucleotide base of the site is A or G;

a third SNP site, wherein the third SNP site is located at 45112865 th nucleotide of 6th chromosome of a tomato reference genome, and the nucleotide base of the third SNP site is A or G;

a fourth SNP site, wherein the fourth SNP site is located at 50349769 th nucleotide of 11 th chromosome of a tomato reference genome, and the nucleotide base of the fourth SNP site is C or T;

a fifth SNP locus, wherein the fifth SNP locus is located at 2872268 th nucleotide of the 7 th chromosome of a tomato reference genome, and the nucleotide base of the locus is A or T;

a sixth SNP locus, wherein the sixth SNP locus is located at 21501063 th nucleotide of 2 nd chromosome of a tomato reference genome, and the nucleotide base of the locus is A or C;

a seventh SNP locus, wherein the seventh SNP locus is located at 2072712 th nucleotide of the 4th chromosome of a tomato reference genome, and the nucleotide base of the locus is C or T;

an eighth SNP locus, wherein the eighth SNP locus is located at 8449453 th nucleotide of 10 th chromosome of a tomato reference genome, and the nucleotide base of the locus is C or T;

wherein the tomato reference genome is tomato Heinz1706 reference genome;

the SNP primer combination comprises:

a first SNP primer set for amplifying the first SNP site; a second SNP primer set for amplifying the second SNP site; a third SNP primer set for amplifying the third SNP site; a fourth SNP primer set for amplifying the fourth SNP site; a fifth SNP primer set for amplifying the fifth SNP site; a sixth SNP primer set for amplifying the sixth SNP site; a seventh SNP primer set for amplifying the seventh SNP site; an eighth SNP primer set for amplifying the eighth SNP site.

2. The SNP primer set according to claim 1, wherein:

the sequences of the first SNP locus and upstream and downstream bases thereof are shown as SEQ ID NO: 25 is shown;

the sequences of the second SNP locus and bases on the second SNP locus and the upstream and downstream thereof are shown as SEQ ID NO: 26 is shown;

the sequences of the third SNP locus and the upstream and downstream bases thereof are shown as SEQ ID NO: 27 is shown;

the fourth SNP locus and the sequences of the upstream and downstream bases thereof are shown as SEQ ID NO: 28 is shown;

the sequences of the fifth SNP locus and bases on the fifth SNP locus are shown as SEQ ID NO: 29 is shown;

the sequences of the sixth SNP locus and the upstream and downstream bases thereof are shown as SEQ ID NO: 30 is shown in the figure;

the seventh SNP locus and the sequences of the upstream and downstream bases thereof are shown as SEQ ID NO: 31, shown in the figure;

the sequences of the eighth SNP locus and bases at the upstream and downstream are shown as SEQ ID NO: shown at 32.

3. The SNP primer set according to claim 1 or 2, wherein:

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the first SNP primer set are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3 is shown in the specification;

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the second SNP primer set are respectively shown in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6 is shown in the specification;

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the third SNP primer set are respectively shown in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 is shown in the figure;

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the fourth SNP primer set are respectively shown in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 is shown in the specification;

and the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the fifth SNP primer set are respectively shown as SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 is shown in the figure;

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the sixth SNP primer set are respectively shown in SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18 is shown in the figure;

and in the seventh SNP primer set, the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer are respectively shown as SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21 is shown in the figure;

the specific part of the first upstream primer, the specific part of the second upstream primer and the downstream primer of the eighth SNP primer set are respectively shown in SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: shown at 24;

the first upstream primer and the second upstream primer in each set of primers are linked to different fluorescent molecules.

4. The SNP primer set according to claim 3, wherein:

the fluorescent molecule is selected from FAM and HEX.

5. An SNP kit for identifying the purity of tomato hybrids is characterized in that: the SNP kit is prepared into a competitive allele specificity PCR reaction system; the reaction system comprises:

the SNP primer set according to any one of claims 1 to 4.

6. The SNP kit according to claim 5, wherein:

in the SNP primer combination, the concentration ratio of the first upstream primer, the second upstream primer and the downstream primer of each primer group in the system is 2:2: 5.

7. A detection method for identifying the purity of tomato hybrid is characterized in that: the detection method comprises the following steps:

DNA extraction step: extracting the genome DNA of the N tomato hybrid seeds to be detected of a tomato variety to be detected; n is a natural number greater than 12;

screening a target primer group: respectively carrying out competitive allele specific PCR amplification reaction by using the genomic DNA of more than or equal to 8 strains in the N tomato hybrid to be detected as a template and using a primer group in the primer combination according to any one of claims 1-4 to obtain a PCR reaction product; detecting the PCR reaction product to obtain the number of plants which are heterozygous based on the genotypes of the eight SNP sites, and obtaining a primer group with the largest number of heterozygous plants as a target primer group;

and (3) a target primer group PCR amplification step: taking the genome DNA of the N tomato hybrid seeds to be detected as a template, and respectively carrying out competitive allele specific PCR amplification reaction by using the target primer group to obtain a PCR reaction product of the target primer group;

and (3) purity detection: and detecting the PCR reaction product of the target primer group, and calculating the purity of the tomato hybrid to be detected according to the detection result.

8. The detection method according to claim 7, characterized in that: n is a natural number greater than 95.

9. The detection method according to claim 7, characterized in that: in the step of screening the target primer group and the step of detecting the purity, the detection method is selected from the following steps: fluorescence signal detection and direct sequencing.

10. The detection method according to claim 7, characterized in that:

in the step of screening the target primer group and the step of detecting the purity,

counting strains of the fluorescent signal indicating the color of heterozygote, which is displayed by the primer combination according to any one of claims 1 to 4, when the fluorescent signal detection is used; the primer group with the largest number of fluorescent strains showing the color indicating the heterozygote is the target primer group;

when direct sequencing is adopted, counting the number of plants of which the genotypes of the eight SNP sites are heterozygous; the primer group with the largest number of heterozygote strains is the target primer group.

11. The detection method according to claim 9, characterized in that:

in the purity detection step, the calculation method of the purity of the tomato hybrid to be detected comprises the following steps:

when the fluorescent signal detection is adopted, counting the number of strains of which the colors of the heterozygotes are indicated by each target primer group and are fluorescent and the number of strains without fluorescence, respectively calculating the purity, and then calculating the average value, wherein the average value is the purity of the tomato hybrid to be detected;

non-fluorescent strain = N-strain showing color fluorescence indicative of heterozygotes-strain showing color fluorescence indicative of first homozygotes-strain showing color fluorescence indicative of second homozygotes;

purity = [ number of strains showing color fluorescence indicating heterozygote of target primer set/(N-number of strains showing no fluorescence of target primer set) ] × 100%;

when direct sequencing is used:

counting the number of strains of each target primer group, which are heterozygous based on the genotype of the SNP locus, and the number of strains of which PCR amplification products are not obtained, respectively calculating the purities, and then calculating an average value, wherein the average value is the purity of the tomato hybrid to be detected;

purity = [ number of strains whose target primer set is heterozygous based on genotype of SNP site/(N-number of strains whose PCR amplification product is not obtained by target primer set) ] × 100%.

12. The detection method according to any one of claims 7 to 11, characterized in that:

the tomato hybrid to be detected is Douli, L-402, Zhongza No. 8, Xueli, Anhui powder No. 3, Tianfu 501, 09FP53, black pearl, Zhongying No. 6, Huiten, Shouyangsheng, Shisheng, Japanese cherry, Beibei, Pingtao, Zhe powder 202, Jiangsu 152, Jiahong No. 4, Dongnong 715, Dongnong 706, European crown, Zhonghao 101, Jinling Jiayu, Su sweet No. 2, Su powder No. 15, Aolin 12, Rexing No. 5, Huiteng No. 5, camouflage No. 1, Su powder No. 16, 2331, Jingu No. 3, Sheng 1, TP, red spike, Beisi, Huamei 201, Dawang, Jinhong No. 4, Zhongbao, Ping No. 1, Zibei, Shanbei, Huli 1478, Duoli, L-420, Red 968, Zhejiang No. 2, Shihong No. 14, Shiluo No. 9, Shiluo No. 3, Shifang No. 1, Shifang, Shi No. 1, Shi No. 1, Shi, Small red, Bofeldt 216, golden shed I, July, nudun 0471, Baojiali No. two, golden shed I, Hongbao 304, HR57, Beijing white jade hall, Qidali, 09FP47-1, SV0244TG, Beiying, Aijihongxiu, Petitng No. 1, De Aoyao D2030, Pink 2338, shou Jiang orange cherry, European Shanpu Red 968, Zhejiang Hetao 203, Zhongza No. 2, Zhongza 105, Jiza No. 1, Russian MNP-1, golden shed No. 1, European Shield 126, HNX12880189, West nong Sheng 2011, Neuman, Keyu anvil No. 1, Zhejiang powder 701, Zhejiang Hetao 203, Qida, Neuman M208, Zhongza 206, Tianfu red 363, Su powder No. 14, Kailou, FP 09, SV0384, Zhu Mei powder 225, Gui Mei Shu No. 2, Gui Mei Shu Hua Li No. 2, Zhen Mei Hua Yu No. 3, Zhen Mei Hua Yu No. 204, Zhen Yihao No. 20, Zhen Yiwei Cheng Hua Yihua Hua Yisu 26, Zhen Yisu No. 10, Zhen Yimu No. 20, Zhen Yisu No. 3, Zhen Yimu Yisu Hua Yi, Jiahong No. 5, H9776, zhongza No. 9, zhejiang No. 205, salon, meilisha, ananghang No. 1, zhongza No. 9, diffenib, nerisia, anhui No. 15, new nobility, jinling sweet jade, buyan No. 1, zhe powder 702, su powder No. 11, HT12044, jinling beautiful jade, GBS103, 09FP253, 09FP29-4, SV4224TH, boja, little tom, shogao, millennium tomato, SV7846TH, ouke, dongfeng No. one, zitao No. 9, zhongza No. 9, zheng powder, shenqi 998, sobedde, beijing red No. 3, agjihong yu yulan dada, riman, rien T301, guan san, dong nong 713, fendne, and feast No. 1.

13. Use of the SNP primer combination according to any one of claims 1 to 4, the SNP kit according to claim 5 or 6, or the detection method according to any one of claims 7 to 12 for detecting the purity of a tomato hybrid to be detected or for preparing a reagent for detecting the purity of a tomato hybrid to be detected.