CN113736866B - SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof - Google Patents
SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant biology, in particular to an SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof. Based on the above, the invention develops a primer combination, a kit and a detection method which can rapidly, intuitively and effectively identify the genotype state of the target SNP locus. The method can realize the rapid, accurate and high-throughput detection of the tomato yellow leaf curl virus resistance gene Ty-1/Ty-3 and Ty-2 segment haplotype, has the advantages of simple operation, low cost, automation, high throughput efficiency, stable marking, safety, no toxicity, no harm and the like, can rapidly, accurately and high-throughput identify the tomato yellow leaf curl virus resistance in the tomato seedling stage, reduce the workload of artificial inoculation identification and field transplantation, improve the breeding efficiency, reduce the breeding cost and accelerate the breeding process, and is very suitable for modern commercial breeding application and large-scale genetic improvement research.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to an SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof.
Background
Tomatoes are important vegetable economic crops in the world and have important production application and basic research values. With the gradual expansion of the planting area of tomatoes all over the world, the influence of tomato plant diseases and insect pests is increasing day by day. Among them, Tomato Yellow Leaf Curl Virus (TYLCV) is a virus disease of solanaceous crops whose harmful regions are widely distributed in tropical and subtropical regions. The tomato whitefly is carried and spread mainly, after the tomato is infected, the plant performance is obviously dwarfed, the leaf margin is yellowed, the leaf becomes small and curly, the growth, the flowering and the fruit setting are seriously damaged, the fruit can not normally turn color, and even the destructive and dead yield is caused. Tomato yellow leaf curl virus disease resistance is mainly controlled by major genes of a Ty family, wherein Ty-1 and Ty-3 are one of the most widely researched and utilized genes (located on the long arm of Chr.06), and are alleles of each other, so that molecular markers such as TG97, P6-25 and the like which are closely linked with the Ty-1 or Ty-3 gene are successfully developed and commercially applied at present; ty-2 is also an important gene (located on the Chr.11 long arm) for resisting tomato yellow leaf curl virus, has been widely used in tomato breeding, and has been cloned at present (Solyc11g 069660).
The common types in the research of tomato molecular marker assisted breeding application at present mainly comprise first-generation molecular markers such AS microsatellite locus SSR markers and the like, second-generation molecular markers such AS target locus sequence specificity STS markers, insertion deletion InDel markers, restriction enzyme polymorphism CAPS markers and the like, and third-generation SNP molecular markers such AS allele specificity PCR (AS-PCR), high resolution melting curve (HRM) and competitive allele specificity PCR (KASP) and the like. The KASP (Kompetitive Allle Specific PCR) marker is taken as the mainstream SNP typing method in the world at present, has the advantages of high specificity, high accuracy, rapidness, high efficiency, strong flexibility, low cost of a single data point, easiness in realization of automatic high-throughput operation and the like, is very suitable for high-throughput automatic detection of a small number of marker sites of a large number of samples, and is widely mature and applied to animal and plant commercial breeding and gene typing related basic research.
At present, there are several KASP marker detection technical schemes for tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3, but most of the target SNP selection in the schemes is based on single anti-sense parental sequence difference, or is not a functional molecular marker directly related to a target gene, so that the effectiveness, the universality and the universality in tomato resources and commercial breeding of different genetic backgrounds cannot be guaranteed, selection failure may be caused, or other unfavorable gene fragments are brought by linkage drag, and the tomato yellow leaf curl virus resistance detection efficiency and the breeding efficiency of new disease-resistant varieties are limited. And at present, no commercial KASP marker detection technical scheme aiming at the Ty-2 gene exists.
Therefore, the efficient, representative and universal SNP locus combination and high-throughput detection application technical scheme capable of quickly and effectively detecting the tomato yellow leaf curl virus resistance is developed, and the practical and theoretical significance is very important for the commercial breeding application and the genetic research of tomatoes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof. A plurality of SNP loci capable of quickly and effectively detecting tomato yellow leaf curl virus resistance are obtained by analyzing and identifying a large amount of variation group data in upstream and downstream adjacent regions and genes of known Ty-1/Ty-3 and Ty-2 genes, and accurate identification and selection of a target gene region can be realized, so that the problems that the universality of the SNP loci is insufficient, or the target gene selection is invalid due to the fact that the SNP loci are not directly related to a target character/gene (only are linked markers), or other non-target adverse gene fragments are caused by linkage drag in the prior art are solved.
To achieve the above objects, the present invention provides, in a first aspect, a SNP site combination for detecting resistance to tomato yellow leaf curl virus, the SNP site combination including a first SNP site combination located inside and on both sides of a tomato yellow leaf curl virus resistance gene Ty-1 and a second SNP site combination located inside and on both sides of a tomato yellow leaf curl virus resistance gene Ty-2, the first SNP site combination including one or more of the following Ty1-SNP01 site to Ty1-SNP08 site, the second SNP site combination including one or more of the following Ty2-SNP01 site to Ty2-SNP08 site:
in the table, the gene sequences and SNP physical location information correspond to tomato (Heinz 1706) reference genome SL2.50 version.
The genome information of tomato yellow leaf curl virus resistance genes Ty-1/Ty-3(Solyc06g051170, Solyc06g051180 and Solyc06g051190) corresponding to the SNP locus combination in the first aspect of the invention is derived from databases https:// solgenomics, net/locus/26402,26403,26404/view, and the genome information of Ty-2(Solyc11g069660) is derived from databaseshttps://solgenomics.net/locus/40343/view。
Based on the SNP locus combination of the first aspect of the invention, the high-throughput SNP typing detection of tomato yellow leaf curl virus resistance can be realized, the result accuracy is high, the consistency is good, the universality is strong, and the accurate identification and selection of a target gene region can be realized.
In one embodiment of the present invention, the Ty1-SNP01 site-Ty 1-SNP08 site, Ty2-SNP01 site-Ty 2-SNP08 site and their respective flanking sequences are respectively shown in SEQ ID No: 1-16;
the Ty1-SNP01 site-Ty 1-SNP08 site and the Ty2-SNP01 site-Ty 2-SNP08 site are respectively positioned at the positions of SEQ ID No: bits 102 of 1-16.
In one embodiment of the present invention, the first SNP site combination includes one or more of the Ty1-SNP02 site, the Ty1-SNP04 site, the Ty1-SNP05 site, the Ty1-SNP06 site, and the Ty1-SNP08 site, and the second SNP site combination includes one or more of the Ty2-SNP04 site, the Ty2-SNP05 site, the Ty2-SNP07 site, and the Ty2-SNP08 site.
In a second aspect, the present invention provides a primer combination for amplifying the above-mentioned SNP site combinations, wherein the primer combination includes a first primer combination including one or more of the following primer sets 1-01 to 1-05, and a second primer combination including one or more of the following primer sets 2-01 to 2-04:
primer set 1-01: SEQ ID No: 17-19 sequences, and primers for amplifying the Ty1-SNP02 sites;
primer set 1-02: SEQ ID No: 20-22 sequences, and primers for amplifying the Ty1-SNP04 sites;
primer set 1-03: SEQ ID No: 23-25 sequences used for amplifying primers of the Ty1-SNP05 sites;
primer set 1-04: SEQ ID No: 26-28 sequences and primers for amplifying the Ty1-SNP06 sites;
primer set 1-05: SEQ ID No: 29-31 sequences, and primers for amplifying the Ty1-SNP08 sites;
primer set 2-01: SEQ ID No: 32-34 sequences, and primers for amplifying the Ty2-SNP04 sites;
primer set 2-02: SEQ ID No: 35-37 sequences, and primers for amplifying the Ty2-SNP05 sites;
primer set 2-03: SEQ ID No: 38-40 sequences, and primers for amplifying the Ty2-SNP07 sites;
primer set 2-04: SEQ ID No: 41-43 sequence and a primer for amplifying the Ty2-SNP08 site.
In a third aspect, the present invention provides a kit for detecting tomato yellow leaf curl virus disease resistance, which comprises one or more primer sets of the first primer combination and the second primer combination of the second aspect of the present invention in a powdered or liquid state.
In one embodiment of the present invention, the kit of the third aspect of the present invention further comprises a PCR premix, wherein the PCR premix comprises a fluorescent reagentLight probe, quenching probe, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2。
Preferably, the fluorescent probe comprises a fluorescent probe A and a fluorescent probe B, and the quenching probe comprises a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 44, the 5' end of the derivative is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 45, and a fluorescent group VIC or HEX is connected to the 3' end of the compound;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 46, and a quenching group BHQ is connected to the 3' end of the derivative;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 47, wherein the 3' end is connected with a quenching group BHQ.
In a fourth aspect, the present invention provides any one of the following applications of the SNP combination of the first aspect, the primer combination of the second aspect, or the kit of the third aspect:
(1) the application in detecting or assisting in detecting the tomato yellow leaf curl virus resistance;
(2) the application in preparing products for detecting or assisting in detecting tomato yellow leaf curl virus resistance;
(3) the application in tomato yellow leaf curl virus resistance breeding;
(4) the application in identifying and protecting tomato germplasm resources and new varieties;
(5) the application in improvement and innovation of tomato germplasm resources.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
detecting the polymorphism or genotype of one or more SNP sites in the SNP site combination provided by the first aspect of the invention, wherein the detection method comprises one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
the sequence information of one or more SNP sites in the SNP site combination provided by the first aspect of the invention is utilized to develop PCR markers and/or gene chips, wherein the PCR markers comprise one or more of PCR-RFLP markers, TaqMan markers, KASP markers, AS-PCR markers and HRM markers.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
molecular breeding improvement and germplasm resource innovation of tomato yellow leaf curl virus resistance are realized by carrying out molecular operation by using one or more SNP loci in the SNP locus combination provided by the first aspect of the invention, wherein the molecular operation comprises gene editing or genetic transformation.
The above applications can be optimized and adjusted or replaced according to different project requirements and purposes.
If necessary, 1 or several or all of the first SNP site combinations and the second SNP site combinations according to the first aspect of the invention may be selected for SNP site polymorphism or genotype detection. In some embodiments, 1 SNP site is detected to identify whether the variety of tomato to be tested contains Ty-1/Ty-3 and Ty-2 genes and/or identify the haplotype of the Ty-1/Ty-3 (which are alleles of each other) and Ty-2 gene segments (Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2; Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2; Ty-1/Ty-2/Ty-2) /Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2 or Ty-1/Ty-1/Ty-2/Ty-2). In other embodiments, the determination of whether a tomato variety to be tested contains a Ty-1/Ty-3 and Ty-2 gene and/or the determination of the haplotype of the Ty-1/Ty-3 and Ty-2 gene segments in a tomato variety to be tested is made by detecting 2 or more than 2 or all of the SNP sites therein. Preferably, 1 or more of Ty1-SNP02 locus, Ty1-SNP04 locus, Ty1-SNP05 locus, Ty1-SNP06 locus and Ty1-SNP08 locus in the first SNP locus combination are detected to identify whether the tomato variety to be detected contains the Ty-1/Ty-3 gene and/or identify the haplotype of the Ty-1/Ty-3 gene segment in the tomato variety to be detected; and (3) identifying whether the variety of the tomato to be detected contains the Ty-2 gene and/or identifying the haplotype of the Ty-2 gene segment in the variety of the tomato to be detected by detecting 1 or more of the Ty2-SNP04 locus, the Ty2-SNP05 locus, the Ty2-SNP07 locus and the Ty2-SNP08 locus in the second SNP locus combination.
The fifth aspect of the invention provides a method for detecting tomato yellow leaf curl virus resistance, which is characterized in that the method for carrying out SNP typing detection on a tomato variety to be detected comprises the following steps:
(1) extracting DNA of the tomato variety to be detected;
(2) performing PCR amplification on the DNAs by using the primer combination of the second aspect of the present invention;
(3) checking the amplification result, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
In a specific embodiment, the DNA of the tomato variety to be tested may be taken from any one of leaves, roots, stems, flowers, fruits and seeds of a tomato plant.
In one embodiment of the present invention, the SNP typing detection of the tomato variety to be detected employs a KASP detection method, which comprises:
(1) adding a primer mixed solution and a PCR premixed solution into the leaf DNA of the tomato variety to be detected, and carrying out KASP amplification;
(2) detecting the PCR product by adopting fluorescent quantitative equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group;
the primer mixture solution is composed of the primer sequences of the same primer group in the primer combination according to the second aspect of the invention.
Preferably, the PCR premix comprises a fluorescent probe, a quenching probe, a ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2。
Preferably, the fluorescent probe comprises a fluorescent probe A and a fluorescent probe B, and the quenching probe comprises a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 44, the 5' end of the derivative is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 45, and a fluorescent group VIC or HEX is connected to the 3' end of the compound;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 46, and a quenching group BHQ is connected to the 3' end of the derivative;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 47, wherein the 3' end is connected with a quenching group BHQ.
Preferably, the fluorescent quantitative equipment comprises various brands of fluorescent quantitative PCR instruments, enzyme labeling instruments, high-throughput genotyping systems (automatic workstations) such as IntelliQube, GeneMatrix and the like.
The invention successfully develops a primer combination capable of rapidly, intuitively and effectively identifying and distinguishing the genotype state of the target SNP locus based on the SNP locus combination provided by the first aspect of the invention, and carries out SNP typing detection by adopting a KASP detection method, thereby judging the haplotypes (Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2; Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2) of the gene segments in the detected tomato material, Ty-1/Ty-2; Ty-1/Ty-1/Ty-2/Ty-2, Ty-1/Ty-1/Ty-2/Ty-2 or Ty-1/Ty-1/Ty-2/Ty-2), thereby assisting in developing the application of rapid and accurate transfer of the tomato yellow leaf curl disease resistant gene.
In one embodiment of the present invention, according to the method of the fifth aspect of the present invention, the reaction system of the PCR is: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; 0.03 mu L of primer mixed solution, wherein the final concentration of each primer is 100 pmol/L;
the reaction conditions of the PCR are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 28-32 cycles.
The method provided by the fifth aspect of the invention is simple to operate, and only needs to add the primer mixed solution and the PCR premixed solution into the PCR micropore reaction plate containing the DNA sample for PCR amplification, and then adopts fluorescent quantitative equipment to detect and analyze the PCR product and perform data analysis.
Different from the prior art, the invention has the following beneficial effects:
(1) according to the invention, a large amount of variation group data in the upstream and downstream adjacent regions and the interior of the known Ty-1/Ty-3 and Ty-2 genes are directly analyzed and identified to obtain a high-efficiency, representative and universal SNP site combination, and the molecular marker-assisted effective selection of the target tomato yellow leaf curl virus resistance gene is realized through the wide verification of different resource materials, so that the adverse linkage of the tomato yellow leaf curl virus resistance gene is broken;
(2) the SNP locus combination (and respective flanking sequence information thereof) can provide powerful support help for other technical development or research such as targeted sequencing, gene chips, probes, PCR markers, gene cloning, functional research and the like;
(3) the detection substance/product (such as the primer combination, the kit and the like) developed based on the SNP locus combination can realize the rapid, accurate and high-flux detection of the haplotype of the TYLCV resistance genes Ty-1/Ty-3 and Ty-2, has the advantages of simple operation, low cost, automation, high flux efficiency, stable marking, safety, no toxicity, no harm and the like, can rapidly, accurately and high-flux identify the TYLCV resistance in the tomato seedling stage, reduce the workload of artificial inoculation identification and field transplantation, improve the breeding efficiency, reduce the breeding cost and accelerate the breeding process, and is very suitable for modern commercial breeding application and large-scale genetic improvement research.
Drawings
FIG. 1 is a flow chart of the universal SNP site discovery and KASP primer combination development and application of detecting tomato yellow leaf curl virus resistant genes Ty-1/Ty-3 and Ty-2 of the invention;
FIG. 2 shows variation groups of tomato yellow leaf curl virus resistant gene Ty-1/Ty-3(Solyc06g051170, Solyc06g051180, Solyc06g051190) upstream and downstream and internal region 5 universal SNP sites (Ty1-SNP02, Ty1-SNP04, Ty1-SNP05, Ty1-SNP06 and Ty1-SNP08) and position information thereof on tomato genome (SL2.50 version); in the figure, TS-2, TS-3, Reference genome (Heinz 1706) and the like are disease-susceptible genotype controls, and the rest of TS-307 to TS313 are disease-resistant genotype controls;
FIG. 3 shows the variation set of 4 universal SNP sites (Ty2-SNP04, Ty2-SNP05, Ty2-SNP07 and Ty2-SNP08) in the upstream and downstream and internal regions of tomato yellow leaf curl virus resistance gene Ty-2(Solyc11g069660) and the position information thereof on tomato genome (SL2.50 version); in the figure, TS-2, TS-3, Reference genome (Heinz 1706) and the like are taken as disease-sensitive genotype controls, and the rest TS-402 to TS408 are taken as disease-resistant genotype controls;
FIG. 4 shows the typing of 5 KASP primer combinations developed at the sites of the universal SNPs of the tomato yellow leaf curl virus resistance gene Ty-1/Ty-3(Solyc06g051170, Solyc06g051180, Solyc06g051190) in a large population; in the figure, A is a primer group 1-01(Chr 06: 34385952), B is a primer group 1-02(Chr 06: 34361543), C is a primer group 1-03(Chr 06: 34378550), D is a primer group 1-04(Chr 06: 34385942), E is a primer group 1-05(Chr 06: 34387918), the abscissa represents a FAM fluorescence signal value (dots at I, which represents a disease-resistant genotype), the ordinate represents a HEX fluorescence signal value (dots at III, which represents a disease-sensitive genotype), the middle dots at II represent a heterozygous disease-resistant genotype, and NTC close to the origin IV represents a negative control;
FIG. 5 shows the typing of 4 KASP primer combinations developed at the universal SNPs site of the tomato yellow leaf curl virus resistance gene Ty-2(Solyc11g069660) in a large population; in the figure, A is a primer group 2-01(Chr 11: 54290582), B is a primer group 2-02(Chr 11: 54288056), C is a primer group 2-03(Chr 11: 54288729), D is a primer group 2-04(Chr 11: 54289564), the abscissa represents the FAM fluorescence signal value (dot at I, representing the disease-resistant genotype), the ordinate represents the HEX fluorescence signal value (dot at III, representing the disease-sensitive genotype), the dot at the middle II represents the heterozygous disease-resistant genotype, and the dot near the origin IV represents the NTC negative control.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged. In the following examples, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA, unless otherwise specified. Some of the known resistant tomato detection materials used in the following examples, including TS series tomato germplasm resources, are published at home and abroad, and the social public can ask the agricultural genome institute of Chinese academy of agricultural sciences or other research units to repeat the following experiments, and other tomato commercial varieties can be obtained through regular commercial approaches according to the sources listed in Table 2.
In a specific embodiment, the tomato sample (DNA) to be tested can be taken from any one of leaves, roots, stems, flowers, fruits and seeds of a tomato plant. In the following examples, the leaves of tomato plants are used to extract DNA, but this is not intended to limit the scope of the invention. The PCR reagent, reaction system, platform device and amplification detection procedure used in the following examples are preferred embodiments of the present invention, and other similar reasonable reagents, equipment platforms, reaction systems and amplification procedures made in China or imported could also achieve the same detection purpose, and are not intended to limit the scope of the present invention.
FIG. 1 is a flow chart of the universal SNP site discovery and KASP primer combination development and application of detecting tomato yellow leaf curl virus resistant gene Ty-1/Ty-3 and Ty-2. As shown in FIG. 1, in the embodiment of the present invention, the KASP detection method is used for SNP typing detection, but it is not intended to limit the scope of the present invention. The SNP locus provided by the invention can be used by the technicians in the field to carry out SNP typing detection by means of mass spectrum, chromatographic sequencing, gene chips, other PCR technologies and the like.
Example 1 screening of SNP site combinations
1. Experimental materials
660 representative tomato germplasm resource mutation group data with different source types in the world are selected for SNP site screening in the embodiment, and the tomato mutation group data source is mainly based on the previous work of the project group of the inventor (Lin, T, Zhu, G, Zhang, J.et al.genomic analysis programs into the history of bottom breaking [ J ]. Nat Genet,2014,46: 1220-1226; Tieman D, Zhu G, resource M F R, et al.A chemical genetic code to improved bottom leaf [ J ]. Science,2017,355(6323): 391). Wherein, the genotype data of partial tomato germplasm resources and the resistance phenotype data of Ty-1/Ty-3 and Ty-2 genes are derived from the prior public database (https:// solgenomics.
2. Screening for SNP site combinations
Utilizing the target genes Ty-1/Ty-3(Solyc06g051170, Solyc06g051180, Solyc06g051190, database source https:// solgenomics. net/accum/26402,26403,26404/view) and Ty-2(Solyc11g069660, database sourcehttps://solgenomics.net/locus/40343/ view) Universal SNP sites were screened within 2kb of the internal and upstream regions.
Through the analysis of the whole genome variation maps of different types of tomato with big fruits, cherry tomato, fresh-eating tomato, processed tomato and other wild disease-resistant donor germplasm resources, SNP loci with consistent difference inside or at both sides of Ty-1/Ty-3 genes (Solyc06g051170, Solyc06g051180, Solyc06g051190) and Ty-2 genes (Solyc11g069660) between resistant materials and sensitive materials are obtained. TS-2(Moneymaker), TS-3(M-82), Reference genome (Heinz 1706) and the like are taken as known homozygous susceptible genotype (Ty-1/Ty-1, Ty-2/Ty-2) controls, TS-307-TS-313 and the like are taken as known homozygous disease-resistant genotype (Ty-1/Ty-1) controls, TS-402-TS-408 and the like are taken as known homozygous disease-resistant genotype (Ty-2/Ty-2) controls, and the controls comprise tomato variety resources of different types and geographical sources such as large-fruit tomatoes, cherry tomatoes, wild tomatoes and the like. And selecting according to different positions of upstream and downstream genes and inside the genes, and screening and analyzing each site according to a typing result. Finally, 16 high-quality universal SNP sites (namely the SNP site combination of the invention: Ty1-SNP01 site-Ty 1-SNP08 site and Ty2-SNP01 site-Ty 2-SNP08 site) which show highly uniform stable difference in the variation group of the known anti-and sensitive genotype control are successfully obtained. Since the 16 SNP loci show highly consistent variation among resistant and susceptible varieties of different types and sources, the remarkable association characterization effect on tomato yellow leaf curl virus resistance can be expected.
Considering the diversity, success rate and universality of basic principles of different detection methods of SNPs such as sequencing, PCR amplification, gene chips and the like, and the uncertainty of cloned gene functions and molecular regulation network mechanisms, the invention further provides 16 SNP sites and respective flanking sequences of the Ty-1/Ty-3 and Ty-2 regions of the tomato yellow leaf curl virus resistance-resistant genes, and the application of the 16 SNP sites and respective flanking sequences in the preparation of related detection or auxiliary detection products, tomato auxiliary breeding and germplasm resource protection and innovation.
Ty1-SNP01 site-Ty 1-SNP08 site, Ty2-SNP01 site-Ty 2-SNP08 site and respective flanking sequences thereof are respectively shown as SEQ ID No: 1 to 16.
Example 2 primer Synthesis and kit preparation
1. Primer design and screening
According to flanking sequences of Ty1-SNP01 site-Ty 1-SNP08 site and Ty2-SNP01 site-Ty 2-SNP08 site provided in example 1, aiming at each SNP site, two forward primers are designed on the upstream of the SNP site by using Primer3.0 software according to the KASP marker design development principle (a target product is 80-150 bp, the primers are specifically matched with a target region on a reference genome and are positioned in a non-SNP dense region, complex sequence regions with high A \ T or G \ C content and the like are avoided, and a reverse primer is designed on the downstream.
The tomato sample to be tested comprises part of germplasm resources with known genotype and yellowing leaf curl virus resistance phenotype and 21 randomly selected samples in breeding materials as resistance, infection or heterozygosis control, and finally 3 ddH are added2O as NTC blank, 24 parts in total.
DNA extraction adopts a conventional CTAB method or a domestic magnetic bead kit to extract genome DNA from leaves of a tomato sample, utilizes Nanodrop 1000 to measure the concentration of nucleic acid, dilutes and controls the concentration of a DNA template to be 10-20 ng/mu L.
The reaction system for PCR (10. mu.L) was: 10 to 20 ng/. mu.L5 mu L of DNA template; 5 mu L of PCR premix; the final concentration of each primer was 100pmol/L (preferably, the primer mixture ratio is 12. mu.L for each specific typing primer, 30. mu.L for the common primer, and 46. mu.L ddH for each common primer)2O; in other embodiments, the same detection objective can be achieved using other reasonable primer mix ratios). Wherein, the primer mixed solution consists of the primer sequences of the same primer group in the primer combination. The PCR premix solution comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 44, and a fluorescent group FAM is connected to the 5' end of the fluorescent substance FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 45, and a fluorescent group VIC or HEX is connected to the 3' end of the compound; the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 46, and a quenching group BHQ is connected to the 3' end of the derivative; the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 47, wherein the 3' end is connected with a quenching group BHQ.
Editing sample and primer arrangement template according to the operation manual of a fluorescent quantitative PCR instrument (Applied Biosystems Quant Studio 3, ABI-Q3) of Thermo Fisher company, and executing an operation program { reading a fluorescent signal at 30 ℃ for 1 min; denaturation at 94 deg.C for 15 min; denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, repeating the step for 10 cycles, wherein Touch-Down temperature is set to be reduced by 0.6 ℃ in each cycle, and the final annealing temperature is reduced to 55 ℃; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and repeating the step for 28-32 cycles; reading the fluorescence signal for 1min at 30 ℃, analyzing the data result, finally selecting 5 groups and 4 groups of FAM signals respectively, the primer combination of the invention is composed of primers which are positioned at the upstream and downstream or in the target gene and have remarkable VIC signal and heterozygous fluorescent signal aggregation typing tendency (comprising a first primer combination, namely a primer group 1-01, a primer group 1-02, a primer group 1-03, a primer group 1-04 and a primer group 1-05, and a second primer combination, namely a primer group 2-01, a primer group 1-02, a primer group 1-03 and a primer group 1-04, wherein SNP information corresponding to each primer group is shown in figures 2 and 3, and the size and haplotype information of a corresponding amplification target strip are detailed in table 1), and subsequent genotype or haplotype verification and breeding application in the large population Ty-1/Ty-3 and Ty-2 regions is developed.
Primer set 1-01: SEQ ID No: 17-19 sequences used for amplifying primers of the Ty1-SNP02 sites;
primer set 1-02: SEQ ID No: 20-22 sequences, and primers for amplifying the Ty1-SNP04 sites;
primer set 1-03: SEQ ID No: 23-25 sequences used for amplifying primers of the Ty1-SNP05 sites;
primer set 1-04: SEQ ID No: 26-28 sequences and primers for amplifying the Ty1-SNP06 sites;
primer set 1-05: SEQ ID No: 29-31 sequences, and primers for amplifying the Ty1-SNP08 sites;
primer set 2-01: SEQ ID No: 32-34 sequences, and primers for amplifying the Ty2-SNP04 sites;
primer set 2-02: SEQ ID No: 35-37 sequences, and primers for amplifying the Ty2-SNP05 sites;
primer set 2-03: SEQ ID No: 38-40 sequences, and primers for amplifying the Ty2-SNP07 sites;
primer set 2-04: SEQ ID No: 41-43 sequence and a primer for amplifying the Ty2-SNP08 site.
TABLE 1 primer combination for detecting tomato Ty-1/Ty-3 and Ty-2 genes and corresponding SNP sites, amplified fragment lengths and primer information thereof
2. Preparation of the kit
This example applies the primer combination described above to the preparation of kits. The kit comprises a PCR premix besides a primer combination. The primer mixture consists of the primer sequences of the same primer group in the primer combination of the invention. The PCR premix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, ROX internal reference dye and KlearTaq DNA polymerizationEnzyme, dNTP and MgCl2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 44, the 5' end of the derivative is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 45, and a fluorescent group VIC or HEX is connected to the 3' end of the compound; the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 46, and a quenching group BHQ is connected to the 3' end of the derivative; the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 47, wherein the 3' end is connected with a quenching group BHQ. Three primers (each primer group) of each SNP locus are independently subpackaged and packaged together; and (5) independently packaging the PCR premix.
Example 3 verification and Breeding application of tomato yellow leaf curl virus resistant genes Ty-1/Ty-3 and Ty-2 efficient KASP markers
This example was carried out to verify the markers of tomato yellow leaf curl virus resistance genes Ty-1/Ty-3 and Ty-2 and to apply them to breeding by using the kit (including primer combination) provided in example 2 based on the SNP sites provided in example 1.
381 parts of representative tomato samples containing mainstream commercial varieties, national resource bank core open varieties, breeding intermediate materials, new hybridization combinations and the like are selected, and various types of cultivated tomatoes including big-fruit tomatoes, cherry tomatoes, string harvest tomatoes, fresh-eating tomatoes, processed tomatoes, farmed families (local varieties) and the like are selected, wherein 154 parts of the tomato samples obtain Ty-1/Ty-3 and Ty-2 genotype data (comprising 41 parts of tomato germplasm resources with known yellowing leaf curl virus disease resistance phenotypes and commercial varieties, which are detailed in tables 2-1 and 2-2) by using closely-linked SCAR markers through a third-party commercial institution.
Specifically, the embodiment provides a method for detecting tomato yellow leaf curl virus resistance, which adopts a KASP detection method to perform SNP typing detection on a tomato variety to be detected, and comprises the following steps:
(1) extracting leaf DNA of a tomato variety to be detected;
(2) adding a primer mixture and a PCR premix into the leaf DNA of the tomato variety to be detected by using the kit provided in example 2 to perform KASP amplification (the leaf DNA is subjected to KASP amplification by using each primer group in the primer combination provided in example 2);
(3) and detecting the PCR product by adopting a fluorescent quantitative PCR instrument, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
The tomato variety to be detected is 381 parts of germplasm resources and breeding materials containing part of known genotypes and phenotypes, and finally 3 ddH are added2O as NTC blank control, 384 parts in total.
The extraction method and conditions of the leaf DNA of the tomato variety to be tested are the same as those in example 2.
The PCR amplification reaction system (1.6. mu.L) was: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; 0.03 mu L of primer mixture, wherein the final concentration of each primer is 100pmol/L, the primer mixture ratio is that the specific typing primers are 12 mu L each, the common primer is 30 mu L, and 46 mu L ddH is added2O。
Editing a sample and a primer arrangement template according to an IntelliQube platform operation manual, and executing an operation program { pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 28-32 cycles in total }, reading fluorescence data, increasing the number of PCR cycles as appropriate, analyzing and removing partially uncertain data points with too low or abnormal fluorescence values, finally typing the large population as shown in figures 4 and 5, and deriving the Excel result (as shown in tables 2-1 and 2-2).
As a result, the 9 sets of KASP markers provided in example 2 obtain highly consistent and well-defined population typing effects in 381 large populations of tomato breeding with different types and sources (as shown in FIG. 4 and FIG. 5), that is, experiments prove that the SNP site combinations provided in example 1 have good universality and stability in tomato variety resources with different genetic backgrounds. Further, in 41 test samples with known yellowing leaf curl virus resistance phenotypes, the resistance phenotype, the SCAR marker identification genotype and the KASP marker (i.e., the primer combination) identification gene data provided by the present invention were completely consistent, i.e., the consistency P was 100% (as shown in tables 2-1 and 2-2). In addition, in 154 samples to be tested with the reference of the results of the linked SCAR markers, the consistency P between the 5 primer sets for detecting Ty-1/Ty-3 and the genotype identification result of the Ty-1 linked SCAR marker is 78.7%, the consistency P between the genotype identification result of the Ty-3 linked SCAR marker is 55.0%, and the consistency P between the Ty-1 and Ty-3 linked SCAR markers is 58.4%; the agreement P between the genotype identification results of 4 primer sets used to detect Ty-2 and the SCAR marker closely linked to the Ty-2 gene was 96.5%. And the consistency P among the results of 5 primer sets for detecting Ty-1/Ty-3 was more than 95%, and the consistency P among the results of 4 primer sets for detecting Ty-2 was more than 98% { consistency calculation formula is P ═ number of effective detection samples-number of differential samples)/number of effective detection samples 100% }. Further analysis shows that the detection results of the same repeated material samples in the SCAR marker detection results are inconsistent (for example, numbers Ta246 and Ta247, Ta235 and Ta292 are two groups of the same repeated materials, and intentional test design is repeated), but the detection results of the primer group provided by the invention do not have the phenomenon. The results prove that the detection accuracy and stability of the KASP marker provided by the invention are obviously higher than those of the conventional commercial SCAR marker.
In addition, the embodiment also carries out Ty-1/Ty-3 and Ty-2 locus genotype detection on 227 breeding intermediate materials, and the result shows that the genotype detection result of the sample to be detected is basically identical to the pedigree relationship, and the resistance of tomato yellow leaf curl virus of filial generation or father generation can be accurately predicted through the genotype detection results of the Ty-1/Ty-3 and Ty-2 loci of the sample to be detected.
In summary, the 9 sets of KASP markers provided by this embodiment have good versatility and stability in tomato cultivar resources of different types, sources or genetic backgrounds, the detection results of the markers are substantially consistent with each other, the accuracy and stability of the detection of each set of markers are significantly better than those of the existing commercial scarr markers, and each set of markers can be independently applied to molecular detection of tomato yellow leaf curl virus resistance. By analogy, the remaining 7 SNP sites that also meet the characteristics of the aforementioned variation set can be verified by sequencing, gene chip or other PCR markers and achieve the same expected effect. Meanwhile, the KASP markers are combined together, so that the detection accuracy can be further improved, the resistance identification errors caused by experimental errors such as false positives and genetic variation factors such as incomplete selection or partial loss of a target gene region can be avoided, and a better detection and judgment effect is obtained. Therefore, the high-efficiency KASP marker provided by the invention can be directly used for the commercial application of tomato yellow leaf curl virus disease resistant molecular breeding.
TABLE 2-1154 tomato germplasm resources, varieties and breeding intermediate materials for resistance phenotype and marker genotype of yellow leaf curl virus disease
TABLE 2-2154 tomato germplasm resources, varieties and breeding intermediate materials yellow leaf curl virus resistance phenotype and marker genotype
Note: the sample numbers in tables 2-1 and 2-2 are actual detection sample arrangement sequence numbers, so that the results are compared, analyzed and visually judged conveniently, and the sequencing is adjusted according to the source types; wherein the column "resistance" represents the tomato yellow leaf curl virus disease resistance phenotype, "S" represents susceptible, "R" represents resistant, "IR" represents resistant; wherein the column of SCAR represents the detection result of the SCAR marker linked with Ty-1, Ty-2 and Ty-3 genes by a third-party commercial institution, SS represents homozygous susceptible genotype, RR represents homozygous disease-resistant genotype, and H represents heterozygous genotype; wherein in the detection results of the primer group 1-01, the primer group 1-04, the primer group 2-01 and the primer group 2-03, T: T represents homozygous susceptible genotype, C: C represents homozygous disease-resistant genotype, and T: C represents heterozygous genotype; wherein, in the detection result of the primer group 1-02, C: C represents homozygous susceptible genotype, T: T represents homozygous disease-resistant genotype, and C: T represents heterozygous genotype; wherein in the detection results of the primer group 1-03 and the primer group 2-04, A: A represents homozygous susceptible genotype, G: G represents homozygous resistant genotype, and A: G represents heterozygous genotype; in the detection result of the primer group 1-05, G: G represents homozygous susceptible genotype, A: A represents homozygous disease-resistant genotype, and G: A represents heterozygous genotype; wherein, in the detection result of the primer group 2-02, C: C represents homozygous susceptible genotype, G: G represents homozygous disease-resistant genotype, and C: G represents heterozygous genotype; "-" represents a data miss; the ". sup." notation represents the result of the inconsistency between the gene-linked SCAR markers and the KASP markers (i.e., primer sets) provided herein or between different markers within the KASP markers provided herein.
While the preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts and related principles. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention, and without undue experimentation, the invention may be practiced in a wide range of equivalent parameters, concentrations, and conditions. It is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> institute of agricultural genomics of Chinese academy of agricultural sciences
Shenzhen agricultural genome institute of Chinese academy of agricultural sciences
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<223> n = A or T
<400> 7
atttgtcaat gacaatctct gaaagttgat atgaaggaaa aactaaccat cttgtacttc 60
tttataactt cattgcaaga ggtgattctt agttcacaat cnaagttcat ggcccgtgtc 120
atatcctttt tgtactcttc atatcttcct ctccacaatg tcatgcatct ttgagggatt 180
tcaacttcaa agcaaggcag ttt 203
<210> 8
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = G or A
<400> 8
tctgagggct tgcacaggcc aataagccca tctcaagctt tgatttagaa gaactttcac 60
cttgtaaacg tttaccctct aagcatagaa agtacaaaca tntagtaaga aatatagtaa 120
gaagggggtt tggctaaagc ttaaaagctg gtcaaacaga ctttgatagc ttactacgcg 180
tttggtaaac acccaagcct tca 203
<210> 9
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = G or A
<400> 9
aacaccctct tgcttatatg atttagagag taatagtcat tggttgaaag caatgtcctc 60
agctgctctg attcagagag ttgtttcaat ttctcaaagt gnccatcttc tcctctggaa 120
taggacatgt gccgactttg ttccaacata tcagcagatc cttcattctc ttccaaccta 180
atacaatgtt ttgaagatgc agt 203
<210> 10
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = A or T
<400> 10
cagagagttg tttcaatttc tcaaagtggc catcttctcc tctggaatag gacatgtgcc 60
gactttgttc caacatatca gcagatcctt cattctcttc cnacctaata caatgttttg 120
aagatgcagt ttgggccaaa tcattgataa ggtcgtgcat tacgaatccc tccgatttcc 180
attctgattc tgggaccctt tca 203
<210> 11
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = C or T
<220>
<221> misc_feature
<222> (119)..(203)
<223> n (85) = A, G, C or T
<400> 11
ttgggatata tcgcacaaaa agcaaaacat cgcttcaaat gtggaggaag atcattgtag 60
ctcaacatta acgctggtaa tataccattc caacaacttt gntgatccca tatttcacnn 120
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnn 203
<210> 12
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = T or C
<220>
<221> misc_feature
<222> (152)..(203)
<223> n (52) = A, G, C or T
<400> 12
taaataactt ggtctttgaa gaattcatga tctttgggat atatcgcaca aaaagcaaaa 60
catcgcttca aatgtggagg aagatcattg tagctcaaca tnaacgctgg taatatacca 120
ttccaacaac tttgctgatc ccatatttca cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180
nnnnnnnnnn nnnnnnnnnn nnn 203
<210> 13
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = C or G
<400> 13
cacatgcatt caccattgat gtataagatg ggaatttgag caatattttg ccagtatttc 60
cctttgtcaa attccagtag tggttcgagc aatgggcatc tnctaatatc tagtttagag 120
agggaagagg gcatctcttt tactggaagg gattggagat tagggcagtc ggagatgttc 180
agctgagaga gggaggaggg cag 203
<210> 14
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = T or C
<400> 14
tgctgattca gagagtgatt ggagattagg gcaatcgaag atgtgtagat gtataagtga 60
agtgaggtgg gaaagatgta gtgaatggag ctcatcatgg tnatgtaaat gtagctctga 120
aagagaggag ggaagcccac cttgttccac aagtgacggt atattaccct caacatatag 180
atattgaaga gaggtgaggc ttt 203
<210> 15
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = T or C
<400> 15
aacaccactc ctttcggctg ttcactactt tcttgcaatt actgatcata aggagttgta 60
aattaaagag atgtatttct ggcagacact tgagcttctc angattccga atatacaaat 120
acgtcaccgc agagtcaact gccaccgaaa gtatttcaac attctcacaa tcctgaatag 180
tgagactttc agtggcagta gga 203
<210> 16
<211> 203
<212> DNA
<213> Artificial sequence (Artificial)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n = A or G
<400> 16
caatttcaca agcttaagaa acaaaggatc agcaagccaa tttgaaaatt ttgcccctct 60
atatcctgtg atttctaatc gttttatatt tgtgtatggg tntagctcat caagtatctc 120
tctttcagtt tgagaatcgt aagcagaact accgtcactc cactgcaaat acaactcctc 180
aacatgctcc ttttccttca tat 203
<210> 17
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 17
gaaggtgacc aagttcatgc tttagcacat tcaatgaccg 40
<210> 18
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 18
gaaggtcgga gtcaacggat tttagcacat tcaatgacca 40
<210> 19
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 19
ttaaagaata caaggagacg ca 22
<210> 20
<211> 44
<212> DNA
<213> Artificial sequence (Artificial)
<400> 20
gaaggtgacc aagttcatgc tgagtacaaa aaggatatga caca 44
<210> 21
<211> 44
<212> DNA
<213> Artificial sequence (Artificial)
<400> 21
gaaggtcgga gtcaacggat tgagtacaaa aaggatatga cacg 44
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 22
tccacagcac catatagcaa 20
<210> 23
<211> 41
<212> DNA
<213> Artificial sequence (Artificial)
<400> 23
gaaggtgacc aagttcatgc ttcagggaag ctctttacac g 41
<210> 24
<211> 42
<212> DNA
<213> Artificial sequence (Artificial)
<400> 24
gaaggtcgga gtcaacggat tctcagggaa gctctttaca ca 42
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence (Artificial)
<400> 25
tcatagaagt gaaaccctga aatt 24
<210> 26
<211> 41
<212> DNA
<213> Artificial sequence (Artificial)
<400> 26
gaaggtgacc aagttcatgc tccacacaga aatcctaatg c 41
<210> 27
<211> 41
<212> DNA
<213> Artificial sequence (Artificial)
<400> 27
gaaggtcgga gtcaacggat tccacacaga aatcctaatg t 41
<210> 28
<211> 24
<212> DNA
<213> Artificial sequence (Artificial)
<400> 28
acctttatct tatttgtttt cagt 24
<210> 29
<211> 48
<212> DNA
<213> Artificial sequence (Artificial)
<400> 29
gaaggtgacc aagttcatgc tcccccttct tactatattt cttactat 48
<210> 30
<211> 48
<212> DNA
<213> Artificial sequence (Artificial)
<400> 30
gaaggtcgga gtcaacggat tcccccttct tactatattt cttactac 48
<210> 31
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 31
agaactttca ccttgtaaac g 21
<210> 32
<211> 43
<212> DNA
<213> Artificial sequence (Artificial)
<400> 32
gaaggtgacc aagttcatgc tgaatggtat attaccagcg ttg 43
<210> 33
<211> 44
<212> DNA
<213> Artificial sequence (Artificial)
<400> 33
gaaggtcgga gtcaacggat tggaatggta tattaccagc gtta 44
<210> 34
<211> 24
<212> DNA
<213> Artificial sequence (Artificial)
<400> 34
attcatgatc tttgggatat atcg 24
<210> 35
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 35
gaaggtgacc aagttcatgc tttcgagcaa tgggcatctg 40
<210> 36
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 36
gaaggtcgga gtcaacggat tttcgagcaa tgggcatctc 40
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial)
<400> 37
ctgccctaat ctccaatccc 20
<210> 38
<211> 43
<212> DNA
<213> Artificial sequence (Artificial)
<400> 38
gaaggtgacc aagttcatgc tgtatttctt gcagcttctc aca 43
<210> 39
<211> 43
<212> DNA
<213> Artificial sequence (Artificial)
<400> 39
gaaggtcgga gtcaacggat tgcagacact tgsgcttctc atg 43
<210> 40
<211> 24
<212> DNA
<213> Artificial sequence (Artificial)
<400> 40
agaatgttga aatactttcg gtgg 24
<210> 41
<211> 46
<212> DNA
<213> Artificial sequence (Artificial)
<400> 41
gaaggtgacc aagttcatgc tatygtttta tatttgtgtr tgggtg 46
<210> 42
<211> 46
<212> DNA
<213> Artificial sequence (Artificial)
<400> 42
gaaggtcgga gtcaacggat tatcgtttta tatttgtgta tgggta 46
<210> 43
<211> 23
<212> DNA
<213> Artificial sequence (Artificial)
<400> 43
ggtagttctg cttacgattc tca 23
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 44
gaaggtgacc aagttcatgc t 21
<210> 45
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 45
gaaggtcgga gtcaacggat t 21
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 46
agcatgaact tggtcacctt c 21
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 47
aatccgttga ctccgacctt c 21
Claims (14)
1. The application of the SNP site combination in detecting or assisting in detecting the tomato yellow leaf curl virus resistance is characterized in that the SNP site combination comprises a first SNP site combination positioned inside and on two sides of a tomato yellow leaf curl virus resistance gene Ty-1 and a second SNP site combination positioned inside and on two sides of a tomato yellow leaf curl virus resistance gene Ty-2, the first SNP site combination comprises one or more of the following Ty1-SNP02 site, Ty1-SNP04 site, Ty1-SNP05 site, Ty1-SNP06 site and Ty1-SNP08 site, and the second SNP site combination comprises one or more of the following Ty2-SNP04 site, Ty2-SNP05 site, Ty2-SNP07 site and Ty 2-08 site:
In the table, the gene sequences and SNP physical location information correspond to tomato Heinz 1706 reference genome version SL 2.50.
2. The use of claim 1, wherein the first combination of SNP sites further comprises one or more of a Ty1-SNP01 site, a Ty1-SNP03 site, and a Ty1-SNP07 site, and the second combination of SNP sites further comprises one or more of a Ty2-SNP01 site, a Ty2-SNP02 site, a Ty2-SNP03 site, and a Ty2-SNP06 site:
in the table, the gene sequences and SNP physical location information correspond to the tomato Heinz 1706 reference genome version SL 2.50.
3. The use of claim 1, wherein the Ty1-SNP01 site-Ty 1-SNP08 site, Ty2-SNP01 site-Ty 2-SNP08 site and their respective flanking sequences are respectively as set forth in SEQ ID Nos: 1-16;
the Ty1-SNP01 site-Ty 1-SNP08 site and the Ty2-SNP01 site-Ty 2-SNP08 site are respectively positioned in the nucleotide sequences shown in SEQ ID No: bits 102 of 1-16.
4. The application according to claim 1, wherein the application comprises:
(1) the application in preparing products for detecting or assisting in detecting tomato yellow leaf curl virus resistance;
(2) the application in tomato yellow leaf curl virus disease resistance breeding;
(3) the application in identifying and protecting tomato germplasm resources and new varieties;
(4) the application in improvement and innovation of tomato germplasm resources.
5. The application according to claim 4, characterized in that the application is carried out by adopting the following technical means:
and detecting the polymorphism or the genotype of the SNP locus in the SNP locus combination, wherein the detection method comprises one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
6. The application according to claim 4, characterized in that the application is carried out by adopting the following technical means:
developing PCR markers and/or gene chips by using the sequence information of the SNP sites in the SNP site combination, wherein the PCR markers comprise one or more of PCR-RFLP markers, TaqMan markers, KASP markers, AS-PCR markers and HRM markers.
7. The application according to claim 4, characterized in that the application is carried out by adopting the following technical means:
and carrying out molecular operation by utilizing the SNP loci in the SNP locus combination to realize molecular breeding improvement and germplasm resource innovation of tomato yellow leaf curl virus resistance, wherein the molecular operation comprises gene editing or genetic transformation.
8. A primer combination for amplifying the SNP site combination of claim 1, wherein the primer combination comprises a first primer combination comprising one or more of the following primer sets 1-01 to 1-05 and a second primer combination comprising one or more of the following primer sets 2-01 to 2-04:
primer set 1-01: SEQ ID No: 17-19 sequences used for amplifying primers of the Ty1-SNP02 sites;
primer set 1-02: SEQ ID No: 20-22 sequences, and primers for amplifying the Ty1-SNP04 sites;
primer set 1-03: SEQ ID No: 23-25 sequences used for amplifying primers of the Ty1-SNP05 sites;
primer set 1-04: SEQ ID No: 26-28 sequences and primers for amplifying the Ty1-SNP06 sites;
primer set 1-05: SEQ ID No: 29-31 sequences, and primers for amplifying the Ty1-SNP08 sites;
primer set 2-01: SEQ ID No: 32-34 sequences, and primers for amplifying the Ty2-SNP04 sites;
primer set 2-02: SEQ ID No: 35-37 sequences, and primers for amplifying the Ty2-SNP05 sites;
primer set 2-03: SEQ ID No: 38-40 sequences, and primers for amplifying the Ty2-SNP07 sites;
primer set 2-04: SEQ ID No: 41-43 sequence and a primer for amplifying the Ty2-SNP08 site.
9. A kit for detecting tomato yellow leaf curl virus disease resistance, comprising the primer combination of claim 8 in a powdered or liquid state.
10. The kit of claim 9, further comprising a PCR premix comprising a fluorescent probe, a quenching probe, a ROX internal reference dye, KlearTaq DNA polymerase, dntps, and MgCl2。
11. The primer combination of claim 8, or the kit of claim 9 or 10, for any one of the following applications:
(1) the application in detecting or assisting in detecting the tomato yellow leaf curl virus resistance;
(2) the application in preparing products for detecting or assisting in detecting tomato yellow leaf curl virus resistance;
(3) the application in tomato yellow leaf curl virus disease resistance breeding;
(4) the application in identifying and protecting tomato germplasm resources and new varieties;
(5) the application in improvement and innovation of tomato germplasm resources.
12. A method for detecting tomato yellow leaf curl virus disease resistance is characterized in that SNP typing detection is carried out on a tomato variety to be detected, and the method comprises the following steps:
(1) extracting DNA of the tomato variety to be detected;
(2) performing PCR amplification on the DNAs with the primer combination according to claim 8;
(3) checking the amplification result, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
13. The method of claim 12, wherein the SNP typing assay of the tomato variety to be tested employs a KASP assay comprising:
(1) adding a primer mixed solution and a PCR premixed solution into the leaf DNA of the tomato variety to be detected, and carrying out KASP amplification;
(2) detecting the PCR product by adopting fluorescent quantitative equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group;
the primer mixture is composed of the primer set according to claim 8.
14. The method of claim 12, wherein:
the reaction system of the PCR is as follows: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; the primer mixture is 0.03 mu L, wherein the final concentration of each primer is 100 pmol/L;
the reaction conditions of the PCR are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 28-32 cycles.
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