CN113736906B - SNP locus combination for detecting verticillium wilt resistance of tomatoes and application thereof - Google Patents
SNP locus combination for detecting verticillium wilt resistance of tomatoes and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant biology, in particular to a SNP locus combination for detecting verticillium wilt resistance of tomatoes and application thereof. Based on the above, the invention develops a primer combination, a kit and a detection method capable of rapidly, intuitively and effectively identifying the genotype status of the target SNP locus. The invention can realize rapid, accurate and high-flux detection of the haplotypes of the segments Ve1 and Ve2 of the verticillium wilt resistance genes of the tomatoes, 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 verticillium wilt resistance of the tomatoes in the seedling stage of the tomatoes, reduces the workload of manual inoculation identification and field transplanting, improves the breeding efficiency, reduces the breeding cost, accelerates 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 a SNP locus combination for detecting verticillium wilt resistance of tomatoes and application thereof.
Background
Tomatoes are important vegetable cash crops in the world and have important production application and basic research values. With the gradual expansion of tomato planting areas worldwide, the influence of tomato diseases and insect pests is increasing. Wherein tomato verticillium wilt (Verticillium Wilt) is a worldwide fungal disease caused by verticillium dahliae (Verticillium dahliae) and verticillium dahliae (Verticillium alboatrum Reinke and Berth) of the genus verticillium of the sub-phylum of the soil. It has now been found that two pathogenic physiological races, corresponding to two disease-resistant genes Ve1 and Ve2, respectively, are fully dominant genes derived from Peru tomato. Ve1 and Ve2 are located on chr.09s and have both been cloned (Kawchuk et al 2001), corresponding to Solyc09g005090 and Solyc09g005080, respectively. The study reports that the two nucleotide sequences are highly similar, but that Ve2 gene alone may not develop resistance itself. More than 4 groups of CAPS, SCAR, ARMS or HRM markers applied to tomato verticillium wilt resistant molecular marker assisted breeding have been successfully developed based on the nucleotide differences of the gene regions among the susceptible parents.
The existing molecular marker assisted breeding application research of tomato verticillium wilt resistance mainly utilizes functional molecular markers developed by target gene sequence differences, and mainly comprises second generation molecular markers such AS specific sequence amplification SCAR markers, allele specific amplification AS-PCR markers, restriction enzyme cleavage polymorphism CAPS markers and the like, and third generation SNP molecular markers such AS allele specific PCR (AS-PCR) and high resolution dissolution curve (HRM). The competitive allele specific KASP (Kompetitive Allele Specific PCR) marker is used as a main SNP typing method in the world today, has the advantages of high specificity, high accuracy, high speed, high efficiency, high flexibility, low cost of single data point, easy realization of automatic high-throughput operation and the like, is very suitable for high-throughput automatic detection of a small number of marker loci of a large number of samples, and has been widely used in commercial breeding of animals and plants and related basic research of genotyping.
At present, several KASP mark detection technical schemes are provided for tomato verticillium wilt resistance genes Ve1 and Ve2, but most of target SNP selection in the schemes is based on single resistance parent sequence difference, or functional molecular marks directly related to the target genes are not available, so that the effectiveness, universality and universality of the tomato can not be ensured in different genetic background tomato resources and commercial breeding, the selection failure can be caused, or linkage encumbrance brings other unfavorable gene segments, and the detection efficiency of tomato verticillium wilt resistance and the breeding efficiency of new disease-resistant varieties are limited. In addition, the existing CAPS, AS-PCR, tetraprimer ARMS, HRM and other molecular markers or experimental operation flows are complicated, or the problems of relatively poor PCR stability, higher requirements on a DNA template and a PCR system, too high dependence on manual operation and the like exist, and the method is not suitable for commercial breeding detection of high-throughput automation.
Therefore, the development of a high-efficiency, representative and general SNP locus combination and high-flux detection application technical scheme which can rapidly and effectively detect the verticillium wilt resistance of tomatoes has very important practical and theoretical significance for commercial breeding application and 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 verticillium wilt resistance of tomatoes and application thereof. A plurality of SNP loci capable of rapidly and effectively detecting verticillium wilt resistance of tomatoes are obtained through analysis and identification of a large number of mutation group data in the upstream and downstream adjacent areas of known Ve1 and Ve2 genes and in the genes based on mutation information of tomato genomes, and accurate identification and selection of target gene areas can be achieved, so that the problems that SNP loci in the prior art are insufficient in universality, or functional molecular markers (only linkage markers) directly related to target characters/genes are not needed, target gene selection is invalid, or linkage is cumbersome and causes other non-target adverse gene fragments and the like are solved.
To achieve the above object, the first aspect of the present invention provides an SNP site combination for detecting resistance to verticillium wilt of tomato, comprising a first SNP site combination located inside and on both sides of a verticillium resistance gene Ve1 of tomato and a second SNP site combination located inside and on both sides of a verticillium resistance gene Ve2 of tomato, wherein the first SNP site combination comprises one or more of the following Ve1-SNP01 site to Ve1-SNP04 site, and the second SNP site combination comprises one or more of the following Ve2-SNP01 site to Ve2-SNP05 site:
in the table, the gene sequence and SNP physical location information correspond to tomato (Heinz 1706) reference genome version SL 2.50.
The genomic information of the tomato verticillium resistance gene Ve1 (Solyc 09g 005090) corresponding to the SNP locus combination in the first aspect of the invention is derived from the database https:// solgenemics.
Based on the SNP locus combination in the first aspect of the invention, high-throughput SNP typing detection of tomato verticillium wilt resistance can be realized, the accuracy of the result is high, the consistency is good, the universality is strong, and the accurate identification and selection of target gene regions can be realized.
In one embodiment of the invention, the Ve1-SNP01 site-Ve 1-SNP04 site, ve2-SNP01 site-Ve 2-SNP05 site and their respective flanking sequences are shown in SEQ ID No:1 to 9;
the Ve1-SNP01 site-Ve 1-SNP04 site and the Ve2-SNP01 site-Ve 2-SNP05 site are respectively positioned in the SEQ ID No: 1-4 and SEQ ID No:5 to 9, 102 th position.
In one embodiment of the invention, the first SNP site combination comprises one or more of the Ve1-SNP02 site, the Ve1-SNP03 site, and the Ve1-SNP04 site, and the second SNP site combination comprises one or more of the Ve2-SNP03 site and the Ve2-SNP04 site.
The second aspect of the present invention provides a primer combination for amplifying the above SNP site combination, comprising a first primer combination comprising one or more of the following primer sets 1 to 01 to 1 to 03 and a second primer combination comprising one or more of the following primer sets 2 to 01 and 2 to 02:
primer set 1-01: SEQ ID No: 10-12 sequences, primers for amplifying the Ve1-SNP02 locus;
primer set 1-02: SEQ ID No: 13-15 sequences, primers for amplifying the Ve1-SNP03 locus;
primer set 1-03: SEQ ID No: 16-18 sequences, primers for amplifying the Ve1-SNP04 site;
primer set 2-01: SEQ ID No: 19-21, a primer for amplifying the Ve2-SNP03 site;
primer set 2-02: SEQ ID No: 22-24 sequences for amplifying the primers of the Ve2-SNP04 site.
In a third aspect the invention provides a kit for detecting verticillium wilt resistance of tomato comprising one or more of the first primer combination and the second primer combination of the second aspect of the invention in powder or liquid form.
In one embodiment of the invention, the kit according to the third aspect of the invention further comprises a PCR premix comprising a fluorescent probe, a quenching probe, a ROX reference dye, klearTaq DNA polymerase, dNTPs and MgCl 2 。
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:25, the 5' end of which is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:26, the 3' end of which is connected with a fluorescent group VIC or HEX;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No:27, the 3' end of which is connected with a quenching group BHQ;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No:28, the 3' end of which is linked to a quenching group BHQ.
The fourth aspect of the present invention provides the SNP site combination according to the first aspect of the present invention, or the primer combination according to the second aspect of the present invention, or any one of the following applications of the kit according to the third aspect of the present invention:
(1) The application in detecting or assisting in detecting verticillium resistance of tomatoes;
(2) The application in preparing products for detecting or assisting in detecting verticillium resistance of tomatoes;
(3) Application in tomato verticillium resistance breeding;
(4) Application in identification and protection of tomato germplasm resources and new varieties;
(5) Application in tomato germplasm resource improvement and innovation.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
detecting a polymorphism or genotype of one or more SNP sites in the SNP site combination provided by the first aspect of the invention, the detection method comprising 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 loci in the SNP locus combination provided in the first aspect of the invention is utilized to develop a PCR marker and/or a gene chip, wherein the PCR marker comprises one or more of a PCR-RFLP marker, a TaqMan marker, a KASP marker, an AS-PCR marker and an HRM marker.
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 verticillium resistance are realized by utilizing one or more SNP loci in the SNP locus combination provided in the first aspect of the invention to carry out molecular operation, wherein the molecular operation comprises gene editing or genetic transformation.
The application can be optimized and adjusted or replaced according to different project requirements and purposes.
1 or several or all of the first SNP site combination and the second SNP site combination according to the first aspect of the invention may be selected for SNP site polymorphism or genotype detection, as desired. In some embodiments, the presence of the Ve1 and Ve2 genes in the tomato variety to be tested and/or the haplotypes of the Ve1 and Ve2 gene segments in the tomato variety to be tested are identified by detecting 1SNP locus therein (Ve 1/Ve2, ve1/Ve2, ve1/Ve 2; ve1/Ve1/Ve2/Ve2, ve1/Ve1/Ve2/Ve2 or Ve1/Ve1/Ve2/Ve 2). In other embodiments, the detection of 2 or more or all of the SNP loci is used to identify whether the Ve1 and Ve2 genes are contained in the tomato variety to be tested and/or to identify the Ve1 and Ve2 gene segment haplotypes in the tomato variety to be tested. Preferably, whether the Ve1 gene is contained in the tomato variety to be tested and/or the Ve1 gene segment haplotype in the tomato variety to be tested is identified by detecting 1 or more of the Ve1-SNP02 locus, the Ve1-SNP03 locus and the Ve1-SNP04 locus in the first SNP locus combination; identifying whether the tomato variety to be tested contains the Ve2 gene and/or identifying the Ve2 gene segment haplotype in the tomato variety to be tested by detecting 1 or all of the Ve2-SNP03 locus and the Ve2-SNP04 locus in the second SNP locus combination.
The fifth aspect of the invention provides a method for detecting verticillium wilt resistance of tomatoes, which is characterized by carrying out SNP typing detection on tomato varieties to be detected, and comprising the following steps:
(1) Extracting DNA of the tomato variety to be detected;
(2) Respectively carrying out PCR amplification on the DNA by using the primer combination according to the second aspect of the invention;
(3) And (5) checking an amplification result, and determining the genotype of the tomato variety to be detected at the SNP locus corresponding to each primer set.
In a specific embodiment, the DNA of the tomato variety to be tested may be taken from any one of the leaves, roots, stems, flowers, fruits and seeds of the tomato plant.
In one embodiment of the present invention, the detection of SNP typing of tomato variety to be detected adopts a KASP detection method, and the KASP detection method comprises:
(1) Adding a primer mixed solution and a PCR premix solution into the leaf DNA of the tomato variety to be detected, and performing KASP amplification;
(2) Detecting PCR products by adopting fluorescent quantitative PCR equipment, and determining genotypes of SNP loci corresponding to each primer set of the tomato variety to be detected;
the primer mixture consists of primer sequences of the same primer set in the primer combination according to the second aspect of the present invention.
Preferably, the PCR premix comprises a fluorescent probe, a quenching probe, a ROX reference dye, kleataq DNA polymerase, dNTPs and MgCl 2 。
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:25, the 5' end of which is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:26, the 3' end of which is connected with a fluorescent group VIC or HEX;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No:27, the 3' end of which is connected with a quenching group BHQ;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No:28, the 3' end of which is linked to a quenching group BHQ.
Preferably, the fluorescence quantitative device comprises a high-throughput genotyping system (an automatic workstation) such as a fluorescence quantitative PCR instrument, an enzyme-labeled instrument, intelliQube, geneMatrix and the like of each brand.
According to the invention, the primer combination capable of rapidly and intuitively identifying and distinguishing the genotype states of target SNP loci is successfully developed based on the SNP locus combination provided by the first aspect of the invention, and SNP typing detection is carried out by adopting a KASP detection method, so that haplotypes (Ve 1/Ve2 ) of gene segments in detected tomato materials are judged; ve1/Ve1/Ve2/Ve2, ve1/Ve1/Ve2/Ve2 or Ve1/Ve1/Ve2/Ve 2) to assist in developing rapid and accurate transfer application of tomato verticillium wilt resistant genes.
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: 10-20 ng/. Mu.L template DNA 0.8. 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 reaction conditions of the PCR are as follows: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 20s, annealing at 61 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles, and final annealing temperature is reduced to 55 ℃; denaturation at 94℃for 20s and annealing at 55℃for 60s, for 28-32 cycles.
The method provided by the fifth aspect of the invention is simple to operate, and only the primer mixture and the PCR premix are added into a PCR micropore reaction plate containing a DNA sample to carry out PCR amplification, and then fluorescent quantitative PCR equipment is adopted to detect and analyze PCR products and carry out data analysis.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention obtains high-efficiency, representative and general SNP locus combination through analyzing and identifying a large number of variation groups in the upstream and downstream adjacent areas of the known Ve1 and Ve2 genes and the inside of the genes, realizes the molecular marker-assisted effective selection of the target tomato verticillium wilt resistance genes through the wide verification of different resource materials, and breaks the unfavorable linkage of the tomato verticillium wilt resistance genes;
(2) The SNP locus combination (and the respective flanking sequence information thereof) can provide powerful support help for other technical expansion or research such as targeted sequencing, gene chips, probes, PCR markers, gene cloning and function research;
(3) The detection substances/products (such as primer combination, kit and the like) developed based on the SNP locus combination can realize rapid, accurate and high-throughput detection of the haplotypes of the segments Ve1 and Ve2 of the verticillium wilt resistance genes of tomatoes, has the advantages of simplicity in operation, low cost, automation, high throughput efficiency, stable marking, safety, no toxicity, innocuity and the like, can rapidly, accurately and high-throughput identify the verticillium wilt resistance of tomatoes in the seedling stage of tomatoes, reduces the workload of manual inoculation identification and field transplanting, improves the breeding efficiency, reduces the breeding cost, accelerates 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 development and application of the combination of the universal SNP locus discovery and KASP primers for detecting the verticillium wilt-resistant genes Ve1 and Ve2 of tomatoes;
FIG. 2 shows the variant sets of tomato verticillium wilt resistance genes Ve1 (Solyc 09g 005090) and Ve2 (Solyc 09g 005080) upstream and downstream and 5 KASP tagged universal SNP sites in the internal region (Ve 1-SNP02 site, ve1-SNP03 site, ve1-SNP04 site, ve2-SNP03 site and Ve2-SNP04 site) and their positional information on the tomato genome (SL 2.50 version); in the figure, ts-2, ts-9 and the like are the genotype control of the disease, the rest of Ts-1, ts-3, ts-133, ts-147 and Ts-253 (Heinz 1706,Reference genome) are the genotype control of the disease, ve1-SNP05 is derived from C base deletion mutation existing in the anti-disease parent Ve1 gene sequence of the prior art Su Xiaomei and the like (2014) (the mutation is not successfully identified in 660 tomato variant group data and only serves as a control), and the mutation is not successfully developed into KASP markers with ideal typing effect;
FIG. 3 shows typing of 5 KASP primer combinations developed at the sites of the tomato verticillium wilt resistance genes Ve1 (Solyc 09g 005090) and Ve2 (Solyc 09g 005080) universal SNPs in a large population; in the figure, A is primer set 1-01 (Chr 09:56,183), B is primer set 1-02 (Chr 09:55,723), C is primer set 1-03 (Chr 09:54,848), D is primer set 2-01 (Chr 09:49,172), E is primer set 2-02 (Chr 09:48,822), the abscissa represents FAM fluorescence signal value (dot at I, disease resistant genotype), the ordinate represents HEX fluorescence signal value (dot at III, disease sensitive genotype), the dot at middle II represents heterozygous disease resistant genotype, and the dot near origin IV represents NTC negative control.
Detailed Description
In order to describe the technical content, constructional features, achieved objects and effects of the technical solution in detail, the following description is made in connection with the specific embodiments in conjunction with the accompanying drawings. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are provided, but the protection scope of the present invention is not limited to the following embodiments.
The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, 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. Some tomato detection materials with known resistance used in the following examples, including TS series tomato germplasm resources, are public resources at home and abroad, the public may ask the national academy of agricultural sciences for agricultural genome institute or other scientific research units to repeat the following experiments, and the rest of tomato commercial varieties may be obtained through regular commercial approaches according to the sources listed in Table 2.
In particular embodiments, the tomato sample (DNA) to be tested may be taken from any one of the leaves, roots, stems, flowers, fruits and seeds of a tomato plant. In the following examples, the leaves of tomato plants are used for DNA extraction, but this is not intended to limit the scope of the invention. The PCR reagents, reaction systems, platform devices and amplification detection procedures used in the following examples are preferred embodiments of the present invention, and other similar and reasonable domestic or imported reagents, device platforms, reaction systems and amplification procedures can achieve the same detection purpose, and are not intended to limit the scope of the present invention.
FIG. 1 is a flow chart showing the development and application of the combination of the universal SNP locus discovery and KASP primers for detecting the verticillium wilt-resistant genes Ve1 and Ve2 of tomatoes. As shown in FIG. 1, in the embodiment of the present invention, the KASP detection method is used for SNP typing detection, but the scope of the present invention is not limited thereto. Based on the SNP locus provided by the invention, a person skilled in the art can carry out SNP typing detection by means of mass spectrometry, chromatographic sequencing, gene chips, other PCR technologies and the like.
Example 1 screening of SNP site combinations
1. Experimental materials
Variant data of 660 representative tomato germplasm resources of different source types worldwide were selected for SNP locus screening in this example, the tomato variant data sources were mainly based on the earlier work of the project group where the inventors were located (Lin, T., zhu, G., zhang, J.et al. Genomic analyses provide insights into the history of tomato breeding [ J ]. Nat Genet,2014, 46:1220-1226;Tieman D,Zhu G,Resende M F R,et al.A chemical genetic roadmap to improved tomato flavor[J ]. Science,2017,355 (6323):391). Wherein, the genotype data of part of tomato germplasm resources and the resistance phenotype data of Ve1 and Ve2 genes are derived from the existing public database (https:// solgenomics.
2. Screening of SNP site combinations
Universal SNP sites were screened inside and within 2kb of the upstream and downstream region using the target genes Ve1 (Solyc 09g005090, database sources: https:// solgenomics. Net/locus/1531/view) and Ve2 (Solyc 09g005080, database sources: https:// solgenomics. Net/locus/1532/view).
SNP loci with consistent differences in the Ve1 gene (Solyc 09g 005090) and the Ve2 gene (Solyc 09g 005080) or on both sides of the resistant and susceptible materials are obtained through genome-wide variation spectrum analysis of different varieties such as large-fruit tomatoes, cherry tomatoes, fresh tomatoes, processing tomatoes and other wild disease-resistant donor germplasm resources. As a control of the known homozygous disease-resistant genotype (Ve 1/Ve1Ve2/Ve 2), ts-2 (Moneymaker), ts-9 (Ailsa Craig) and the like, as a control of the known homozygous disease-resistant genotype (Ve 1/Ve 2), ts-3 (M-82), ts-133, ts-147, ts-253 and the like, the selection was made at different positions upstream and downstream of the gene and within the gene, and the screening analysis was performed for each site based on the typing result. Finally, 9 high-quality universal SNP sites (namely, the SNP site combination of the invention: a Ve1-SNP01 site-Ve 1-SNP04 site and a Ve2-SNP01 site-Ve 2-SNP05 site) which show highly consistent and stable differences in the variant group of the known resistance and genotype-sensitive controls are successfully obtained. In FIG. 2, ve1-SNP05 was derived from a C base deletion mutation in Ve1 gene sequence SL2.50ch09:56,698 using the disease-resistant parent '9706' and the disease-resistant parent 'Moneymaker' by Su Xiaomei et al (2014), and the mutation was not successfully identified in 660 tomato variant group data, and was used as a control only. Since these 9 SNP loci show highly consistent variation between resistant and susceptible varieties of different types and sources, a significant associative characterization effect on tomato verticillium resistance can be expected.
In consideration of diversity, success rate and basic principle universality of SNPs (single nucleotide polymorphisms) such as sequencing, PCR (polymerase chain reaction) amplification, gene chips and the like and uncertainty of cloned gene functions and molecular regulation network mechanisms, the invention further provides 9 SNP loci of the tomato verticillium wilt-resistant gene Ve1 and Ve2 regions and respective flanking sequences thereof, and the application of the SNP loci in detection of tomato verticillium wilt resistance, preparation of related detection or auxiliary detection products, auxiliary breeding of tomatoes, germplasm resource protection and innovation.
The sequences of the Ve1-SNP01 site-Ve 1-SNP04 site, the Ve2-SNP01 site-Ve 2-SNP05 site and the respective flanking sequences thereof are respectively shown in SEQ ID No:1 to 9.
Example 2 primer Synthesis and kit preparation
1. Primer design and screening
According to the flanking sequences of the Ve1-SNP01 locus to the Ve1-SNP04 locus and the Ve2-SNP01 locus to the Ve2-SNP05 locus provided in example 1, two forward primers are designed on the upstream of the SNP locus by using Primer3.0 software according to the KASP marker design development principle (target products 80-150 bp, primers are specifically matched with target regions on a reference genome and are positioned in non-SNP dense regions, and the like), and one reverse primer is designed on the downstream of the SNP locus.
The tomato sample to be tested comprises 21 samples randomly selected from part of germplasm resources with known genotypes and verticillium wilt resistance phenotypes and breeding materials as anti-sense or heterozygous controls, and finally 3 ddH are added 2 O was used as NTC blank for a total of 24 parts.
The DNA extraction adopts a conventional CTAB method or a domestic magnetic bead kit to extract genome DNA from the leaves of a tomato sample, and uses Nanodrop 1000 to measure the concentration of nucleic acid, dilute and control the concentration of DNA template to 10-20 ng/. Mu.L.
The PCR reaction system (10. Mu.L) was: 10-20 ng/. Mu.L of DNA template; 5 mu L of PCR premix; 0.14. Mu.L of primer mixture, wherein the final concentration of each primer is 100pmol/L (preferably, the primer mixture ratio is 12. Mu.L for each specific typing primer, 30. Mu.L for common primer, and 46. Mu.L for ddH 2 O; in other embodiments, the same detection can be achieved using other reasonable primer mix ratiosMeasurement purpose). Wherein, the primer mixture consists of the primer sequences of the same primer group in the primer combination. The PCR premix comprises fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 . Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:25, the 5' end of which is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:26, the 3' end of which is connected with a fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown as SEQ ID No:27, the 3' end of which is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is shown as SEQ ID No:28, the 3' end of which is linked to a quenching group BHQ.
According to the operation manual of a fluorescence quantitative PCR instrument (Applied Biosystems Quant Studio, ABI-Q3) of Thermo Fisher company, editing a sample and a primer arrangement template, and executing an operation program {30 ℃ to read a fluorescence signal for 1min; denaturation at 94℃for 15min; denaturation at 94℃for 20s and annealing at 61℃for 60s, repeating this step for 10 cycles, each cycle setting Touch-Down to cool to 0.6℃and the final annealing temperature to 55 ℃; denaturation at 94℃for 20s and annealing at 55℃for 60s, and repeating this step for 28 to 32 cycles; and reading fluorescent signals for 1min at 30 ℃, analyzing data results, and finally selecting 3 groups and 2 groups of FAM signals, VIC signals and primer combinations with obvious aggregation and typing trends of heterozygous fluorescent signals, wherein the primer combinations are positioned at the upstream and downstream or inside of a target gene, and are used for verifying genotypes or haplotypes of areas of subsequent large groups Ve1 and Ve2 and breeding, wherein each primer combination comprises a first primer combination, a primer group 1-01, a primer group 1-02 and a primer group 1-03, and a second primer combination, a primer group 2-01 and a primer group 1-02, SNP information corresponding to each primer group is shown in figure 2, and the corresponding amplified target band sizes and haplotype information are shown in a table 1.
Primer set 1-01: SEQ ID No: 10-12 sequences, primers for amplifying the Ve1-SNP02 locus;
primer set 1-02: SEQ ID No: 13-15 sequences, primers for amplifying the Ve1-SNP03 locus;
primer set 1-03: SEQ ID No: 16-18 sequences, primers for amplifying the Ve1-SNP04 site;
primer set 2-01: SEQ ID No: 19-21, a primer for amplifying the Ve2-SNP03 site;
primer set 2-02: SEQ ID No: 22-24 sequences for amplifying the primers of the Ve2-SNP04 site.
TABLE 1 primer combinations for detecting tomato Ve1 and Ve2 genes and corresponding SNP sites, amplified fragment lengths and primer information
2. Preparation of the kit
The present example uses the above primer combination to prepare a kit. The kit comprises a primer combination and a PCR premix. The primer mixture consists of the primer sequences of the same primer group in the primer combination. The PCR premix comprises fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 . Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:25, the 5' end of which is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:26, the 3' end of which is connected with a fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown as SEQ ID No:27, the 3' end of which is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is shown as SEQ ID No:28, the 3' end of which is linked to a quenching group BHQ. Three primers (each primer group) of each SNP locus are independently packaged together; and (5) independently packaging the PCR premix.
Example 3 verification and Breeding applications of efficient KASP markers of tomato verticillium wilt resistance genes Ve1 and Ve2
This example was based on the SNP locus provided in example 1, and the kit (including primer combination) provided in example 2 was used to perform the verification and breeding application work of efficient KASP markers of tomato verticillium wilt resistance genes Ve1 and Ve 2.
Representative tomato samples containing main stream commercial varieties, core open germplasm of a national resource library, breeding intermediate materials, new hybrid combinations and the like are selected 381, and various different types of cultivated tomatoes such as big-fruit tomatoes, cherry tomatoes, tandem tomatoes, fresh tomatoes, processing tomatoes, farmyard varieties (local varieties) and the like are related. Of these 177, ve1 and Ve2 genotype data (including 15 tomato germplasm resources and commercial species with known verticillium resistance phenotypes) have been obtained by third party commercial establishments using closely linked SCARs markers, see table 2 for details.
Specifically, the embodiment provides a method for detecting resistance to verticillium wilt of tomatoes, which adopts a KASP detection method to carry out SNP typing detection on tomato varieties 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 to leaf DNA of a tomato variety to be detected by using the kit provided in example 2, and performing KASP amplification (KASP amplification is performed on leaf DNA with each primer set in the primer combination provided in example 2;
(3) Detecting PCR products by adopting a fluorescent quantitative PCR instrument, and determining the genotype of the tomato variety to be detected at the SNP locus corresponding to each primer group.
The tomato variety to be tested is 381 parts of germplasm resources and breeding materials containing part of known genotypes and phenotypes, and finally 3 ddH are added 2 O was used as NTC blank for 384 total parts.
The extraction method and conditions of 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: 10-20 ng/. Mu.L template DNA 0.8. 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 mixture ratio of the primers is 12 mu L of each specific parting primer, 30 mu L of common primer and 46 mu L of ddH 2 O。
Editing a sample and a primer arrangement template according to an IntelliQube platform operation manual, and executing an operation program {95 ℃ for 10min; denaturation at 95 ℃ for 20s, annealing at 61 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles, and final annealing temperature is reduced to 55 ℃; denaturation at 94℃for 20s, annealing at 55℃for 60s, total 28-32 cycles }, reading fluorescence data and increasing the number of PCR cycles as appropriate, analyzing and removing data points with partial uncertainty, too low fluorescence values or anomalies, and finally large population typing as shown in FIG. 3, and deriving Excel results (as shown in Table 2).
As a result, it was found that the 5 sets of KASP markers provided in example 2 obtained highly consistent and well-defined typing effect (as shown in FIG. 3) in 381 large populations of tomato breeds of different types and sources, i.e., experiments confirmed that the SNP locus combinations provided in example 1 had good versatility and stability in tomato variety resources of different genetic backgrounds. Further, in 15 samples tested with known verticillium wilt resistance phenotype, the SCAR marker identification genotype and the KASP marker (i.e., primer combination) identification gene data provided by the present invention were completely identical, i.e., consistency p=100% (as shown in table 2). Wherein, ta023 resistance phenotype is disease resistance R, and gene function markers SCAR-Ve1 and SCAR-Ve2 identify genotype as SS, 4 pairs of markers in KASP markers (namely primer combination) provided by the invention identify genotype as disease resistance type (in addition, 1 pair of primer group 2-02 is deletion data). In addition, in 177 samples to be tested with the reference of the results of the interlocked SCAR markers, the consistency P between the detection results of the 5 primer sets (primer set 1-01 to primer set 1-03 and primer set 2-01 to primer set 2-02) for detecting the Ve1 or Ve2 gene was more than 97% { the consistency calculation formula was P= (number of effective detection samples-number of difference samples)/number of effective detection samples × 100% }, and the consistency P between the functional SCAR markers of the Ve1 or Ve2 gene and the genotyping results of the 5 primer sets provided in example 2 was 81.8%. Further analysis shows that the detection results of the 5 primer sets provided by the invention do not have contradiction between the detection results of the same repeated material sample (for example, the numbers Ta115 and Ta116, and the numbers Ta125 and Ta294 are the same repeated material, and the intentional test design is repeated). The result proves that the detection accuracy and stability of the KASP mark (namely the primer combination) provided by the invention are obviously higher than those of the existing commercial SCAR mark.
In addition, in the embodiment, ve1 and Ve2 locus genotype detection is performed on 204 breeding intermediate materials with unknown genotypes and phenotypes, and the result shows that the genotype detection result of the sample to be detected is basically consistent with the genealogy relationship, and tomato verticillium resistance of offspring or father can be accurately predicted through the genotype detection result of the Ve1 and Ve2 locus of the sample to be detected.
In summary, the 5 groups of KASP markers provided in this embodiment have good versatility and stability in cultivar resources of tomatoes of different types, sources or genetic backgrounds, the detection results of the markers are basically consistent with each other, the accuracy and stability of detection of each group of markers are obviously superior to those of the existing commercial SCAR markers, and each group of KASP markers can be independently applied to molecular detection of tomato verticillium wilt resistance. By analogy, the remaining 4 SNP sites that also fit the aforementioned variant set features can be verified and achieve the same expected effect by sequencing, gene chip or other PCR markers. Meanwhile, the KASP markers are combined together, so that the detection accuracy can be further improved, experimental errors such as false positive and the like and resistance identification errors caused by 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 can be obtained. Therefore, the high-efficiency KASP marker provided by the invention can be directly applied to the commercialization of molecular breeding of tomato verticillium wilt resistance.
TABLE 2 verticillium wilt resistance phenotype and marker genotype of 177 parts of tomato germplasm resources, varieties and breeding intermediate materials
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Note that: sample numbers in the table are actual detection sample arrangement sequence numbers, and sorting is adjusted according to source types for facilitating result comparison analysis and visual judgment; wherein the "resistance" column represents the tomato verticillium wilt resistance phenotype, "S" represents disease susceptibility, "R" represents disease resistance, "T" represents disease resistance; wherein the SCAR column represents detection results of third-party commercial institutions by utilizing the Ve1 and Ve2 gene function SCAR marks, the SS represents homozygous disease genotype, the RR represents homozygous disease-resistant genotype, and the H represents heterozygous genotype; wherein in the detection result of the primer group 1-01, A represents homozygous disease-resistant genotype, T represents homozygous disease-resistant genotype, and A represents heterozygous genotype; wherein, in the detection result of the primer group 1-02, C represents homozygous disease-resistant genotype, G represents homozygous disease-resistant genotype, and C represents heterozygous genotype; wherein in the detection results of the primer group 1-03 and the primer group 2-01, A represents homozygous disease-resistant genotype, G represents homozygous disease-resistant genotype, and A represents heterozygous genotype; wherein in the detection result of the primer group 2-02, A represents homozygous disease-resistant genotype, C represents homozygous disease-resistant genotype, and A represents heterozygous genotype; the "-" indicates data loss and the "x" label indicates the result of an inconsistency between a gene functional SCAR marker and a KASP marker provided by the present invention (i.e., primer combination) or between different markers within a KASP marker provided by the present invention.
While 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 the associated principles. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
Various modifications and alterations of this invention will be apparent to those skilled in the art without departing from the spirit and scope of this invention, and it is intended that the invention be practiced in a broad range of equivalent parameters, concentrations and conditions without undue experimentation. The present invention is intended to include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
While the invention has been described with respect to specific embodiments, 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> Shenzhen agricultural genome research institute of China academy of agricultural sciences
Agricultural genome institute of Chinese academy of agricultural sciences
<120> SNP site combination for detecting resistance to verticillium wilt of tomato and application thereof
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<400> 21
cttggtgctg gtttcaactc t 21
<210> 22
<211> 43
<212> DNA
<213> Artificial sequence (Artifical)
<400> 22
gaaggtgacc aagttcatgc tcccaagaga tgcttctttt ccc 43
<210> 23
<211> 44
<212> DNA
<213> Artificial sequence (Artifical)
<400> 23
gaaggtcgga gtcaacggat tgcccaagag atgcttcttt tcca 44
<210> 24
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 24
ttcagttaca ccagatttga ccc 23
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 25
gaaggtgacc aagttcatgc t 21
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 26
gaaggtcgga gtcaacggat t 21
<210> 27
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 27
agcatgaact tggtcacctt c 21
<210> 28
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 28
aatccgttga ctccgacctt c 21
Claims (12)
1. Use of a reagent for detecting a SNP site combination comprising a first SNP site combination within and flanking a tomato verticillium wilt resistance gene Ve1 and a second SNP site combination within and flanking a tomato verticillium wilt resistance gene Ve2, the first SNP site combination comprising one or more of the Ve1-SNP02 site, the Ve1-SNP03 site and the Ve1-SNP04 site, the second SNP site combination comprising one or more of the Ve2-SNP03 site and the Ve2-SNP04 site, for detecting verticillium wilt resistance of tomatoes:
In the table, the gene sequence 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 SNP site combination further comprises a Ve1-SNP01 site and the second SNP site combination further comprises one or more of a Ve2-SNP01 site, a Ve2-SNP02 site, and a Ve2-SNP05 site:
In the table, the gene sequence and SNP physical location information correspond to tomato Heinz 1706 reference genome version SL 2.50.
3. The use according to claim 1 or 2, wherein the Ve1-SNP01 site-Ve 1-SNP04 site, ve2-SNP01 site-Ve 2-SNP05 site and their respective flanking sequences are as shown in SEQ ID nos: 1 to 9;
the Ve1-SNP01 site-Ve 1-SNP04 site and the Ve2-SNP01 site-Ve 2-SNP05 site are respectively positioned in the SEQ ID No: 1-4 and SEQ ID No:5 to 9, 102 th position.
4. A primer combination for amplifying the SNP site combination of claim 1, characterized in that the primer combination comprises a first primer combination comprising one or more of the following primer sets 1-01 to 1-03 and a second primer combination comprising one or more of the following primer sets 2-01 and 2-02:
primer set 1-01: SEQ ID No: 10-12 sequences, primers for amplifying the Ve1-SNP02 locus;
primer set 1-02: SEQ ID No: 13-15 sequences, primers for amplifying the Ve1-SNP03 locus;
primer set 1-03: SEQ ID No: 16-18 sequences, primers for amplifying the Ve1-SNP04 site;
primer set 2-01: SEQ ID No: 19-21, a primer for amplifying the Ve2-SNP03 site;
primer set 2-02: SEQ ID No: 22-24 sequences for amplifying the primers of the Ve2-SNP04 site.
5. A kit for detecting verticillium resistance in tomato comprising one or more of the first primer set and the second primer set of the primer set of claim 4 in powder or liquid form.
6. The kit of claim 5, further comprising a PCR pre-mix comprising a fluorescent probe, a quenching probe, a ROX reference dye, klearTaq DNA polymerase, dntps, and MgCl 2 。
7. The SNP site combination as set forth in claim 1 or 2, or the primer combination as set forth in claim 4, or the kit as set forth in claim 5 or 6, for any one of the following applications:
(1) The application in detecting or assisting in detecting verticillium resistance of tomatoes;
(2) The application in preparing products for detecting or assisting in detecting verticillium resistance of tomatoes;
(3) Application in tomato verticillium resistance breeding;
(4) The application in the identification and protection of tomato germplasm resources and new varieties.
8. The use according to claim 7, characterized in that it is carried out by the following technical means:
detecting a polymorphism or genotype of one or more SNP sites in the SNP site combination as defined in any one of claims 1-3, the detection method comprising one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
9. The use according to claim 7, characterized in that it is carried out by the following technical means:
a PCR marker and/or gene chip is developed using the sequence information of one or more SNP sites in the SNP site combination AS set forth in any one of claims 1-3, the PCR marker comprising one or more of a PCR-RFLP marker, a TaqMan marker, a KASP marker, an AS-PCR marker and an HRM marker.
10. The method for detecting the verticillium wilt resistance of the tomatoes is characterized by carrying out SNP typing detection on the tomato variety to be detected, and comprises the following steps:
(1) Extracting DNA of the tomato variety to be detected;
(2) Performing PCR amplification of the DNA with the primer combination of claim 4, respectively;
(3) And (5) checking an amplification result, and determining the genotype of the tomato variety to be detected at the SNP locus corresponding to each primer set.
11. The method according to claim 10, wherein said SNP typing of tomato variety to be tested is performed using a KASP assay comprising:
(1) Adding a primer mixed solution and a PCR premix solution into the leaf DNA of the tomato variety to be detected, and performing KASP amplification;
(2) Detecting PCR products by adopting fluorescent quantitative PCR equipment, and determining genotypes of SNP loci corresponding to each primer set of the tomato variety to be detected;
the primer mixture consists of the primer sequences of the same primer group in the primer combination of claim 4.
12. The method according to claim 11, wherein:
the PCR reaction system is as follows: 10-20 ng/. Mu.L template DNA 0.8. 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 reaction conditions of the PCR are as follows: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 20s, annealing at 61 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles, and final annealing temperature is reduced to 55 ℃; denaturation at 94℃for 20s and annealing at 55℃for 60s, for 28-32 cycles.
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| CN107090497A (en) * | 2017-04-01 | 2017-08-25 | 中国农业科学院棉花研究所 | With the SNP marker of upland cotton resisting verticillium major gene resistance close linkage |
| CN111916151A (en) * | 2020-07-21 | 2020-11-10 | 深圳海关动植物检验检疫技术中心 | Tracing detection method and application of verticillium wilt of alfalfa |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090497A (en) * | 2017-04-01 | 2017-08-25 | 中国农业科学院棉花研究所 | With the SNP marker of upland cotton resisting verticillium major gene resistance close linkage |
| CN111916151A (en) * | 2020-07-21 | 2020-11-10 | 深圳海关动植物检验检疫技术中心 | Tracing detection method and application of verticillium wilt of alfalfa |
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