CN113832251B - SNP locus combination for detecting tomato mosaic virus resistance and application thereof - Google Patents
SNP locus combination for detecting tomato mosaic 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 SNP locus combination for detecting resistance of tomato mosaic virus 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 tomato mosaic virus resistance gene Tm-2 a segment haplotype, 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 carry out tomato mosaic virus resistance identification in a tomato seedling stage, reduces the workload of artificial 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 SNP locus combination for detecting resistance of tomato mosaic virus 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. Tomato mosaic virus (Tomato Mosaic Virus, toMV) is a widely damaging RNA virus that has a serious impact on tomato yield and quality. The virus can survive over one year on the surface of seeds or in soil through field operation or mechanical damage such as aphid piercing and sucking, and can maintain infectivity even at high temperature. TMV mainly infects tobacco, tomatoes and other solanaceae plants, and often causes infected leaves to become twisted, shrunken, yellow and even necrotic in the strip spots of flowers and leaves, the growth of top leaves is stagnated, the plants are obviously dwarfed, and simultaneously phenomena such as flower falling and bud falling are accompanied, and the like, and the internal necrosis of fruits can be caused, so that the great loss of the yield and quality of the fruits is caused. At present, the cultivation and popularization of new varieties resistant to tomato mosaic virus are still considered as the most effective and environment-friendly methods. Therefore, the development of an effective molecular marker for detecting the resistance of the tomato mosaic virus is very important and urgent for efficient auxiliary breeding of new varieties resistant to the mosaic virus.
A plurality of major disease-resistant genes, which are named Tm-1 (located on chr.02), tm-2 and Tm-2 a (located on chr.09 long arm) and the like in sequence, are found in research on tomato mosaic virus resistance at home and abroad, and are cloned at present. Wherein Tm-2 a is considered to be an allele of Tm-2 (with four amino acid differences) and can respond to more diverse mosaic virus strains, is also the most durable and most widely used source of resistance, and few viral isolates can evade their resistance, even though those escaping their resistance are generally less toxic. The studies also find that Tm-2 gene is closely linked with morphological marker recessive gene (nv) for controlling plant type, and can cause plant dwarf and yellowing. Therefore, the use of more and more efficient molecular markers for carrying out the accurate detection and auxiliary breeding of the Tm-2 a gene has important significance.
In terms of molecular marker development for detection of the Tm-2 a gene, there are currently some stably mature tightly linked CAPS (CLEAVED AMPLIFIED Polymorphism Sequences) markers such as ToMV, SCN13 and NCTm-019, and some specific PCR markers InSW.about.3 and Tmfk-g\TmR801\ Tmfg-c\TmR2. In addition, there are ARMS (Amplification Refractory Mutation System) markers TMV-2262F/2678R/SNP2494F/2493R and HRM (High resolution melting, high resolution dissolution profile) markers Tm-2-SNP1, tm-2-SNP2 developed based on the anti-sense parent Tm-2 2 gene interior SNP (Single Nucleotide Polymorphism). Because the Tm-2 a gene is linked to many bad forms, it is very important to develop functional molecular markers directly by utilizing genomic variation of the target gene region. And CAPS marks are required to have complicated enzyme digestion procedures on PCR products, ARMS marks have extremely high and unstable requirements on amplification conditions, HRM marks have high detection cost and low flux and automation level, and SCAR marks are required to be manually operated and are easy to make mistakes, so that the method is not suitable for high-flux automatic detection.
Meanwhile, competitive allele specific PCR (KASP, kompetitive ALLELE SPECIFIC PCR) markers are taken as the mainstream genotyping method in the current world, have the advantages of high specificity, high accuracy, rapidness, high efficiency, strong flexibility, low cost of single data points, easy realization of automatic high-throughput operation and the like, are very suitable for high-throughput automatic detection of a small number of marker loci of a large number of samples, and are widely used in commercial breeding of animals and plants and basic research related to genotyping.
At present, a plurality of KASP mark detection technical schemes are provided for tomato mosaic virus resistance gene Tm-2 a, but most of target SNP selection in the schemes is based on single or few anti-sense parent sequence differences, or functional molecular marks directly related to target genes are not available, so that the effectiveness, universality and universality of tomato resources with different genetic backgrounds and commercial breeding cannot be ensured, the selection failure is possibly caused, or linkage encumbrance brings other unfavorable gene segments, and the tomato mosaic virus resistance detection efficiency and the breeding efficiency of new antiviral varieties are limited.
Therefore, the development of a high-efficiency, representative and general SNP locus combination and high-throughput detection application technical scheme which can rapidly and effectively detect the resistance of tomato mosaic virus 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 resistance of tomato mosaic virus and application thereof. A plurality of SNP loci capable of rapidly and effectively detecting tomato mosaic virus resistance are obtained through analysis and identification of a large number of mutation group data in the upstream and downstream adjacent areas of known Tm-2 a genes and in the genes, and accurate identification and selection of target gene regions can be realized, so that the problems that the 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, the target genes are not selected, or linkage redundancy brings other non-target adverse gene segments and the like are solved.
To achieve the above object, the first aspect of the present invention provides a SNP site combination for detecting tomato mosaic virus resistance, the SNP site combination comprising one or more of the following SNP sites located inside and on both sides of tomato mosaic virus resistance gene Tm-2 a:
SNP01 locus, which is located at 13624915 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, and its base is C or T;
SNP02 locus, located at 13623713 nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, the base of which is C or T;
SNP03 locus, locate at tomato Heinz 1706 reference genome SL2.50 version 09 on chromosome 13623321, its base is C or T;
SNP04 locus, which is located at 13622207 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, and its base is T or C;
SNP05 locus, which is located at 13621498 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, and the base is T or C; and
SNP06 locus, located at the 13621305 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, whose base is T or C.
The genomic information corresponding to the SNP locus combination according to the first aspect of the invention is derived from a database https:// solgenomics.
Based on the SNP locus combination of the first aspect of the invention, high-throughput SNP typing detection of tomato mosaic 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 target gene regions can be realized.
In one embodiment of the present invention, the SNP01 site-SNP 06 site and their respective flanking sequences are respectively shown in SEQ ID nos: 1-6, wherein the SNP01 locus to the SNP06 locus are respectively positioned in the SEQ ID No:1 to 6, and position 102 in the sequence.
In one embodiment of the invention, the SNP site combination includes one or more of the SNP02 site, the SNP03 site, the SNP04 site, the SNP05 site, and the SNP06 site.
The second aspect of the present invention provides a primer combination for amplifying the above SNP site combination, comprising one or more of the following primer sets 01 to 05:
primer set 01: SEQ ID No: 7-9 sequences, primers for amplifying the SNP02 locus;
primer set 02: SEQ ID No: 10-12 sequences, primers for amplifying the SNP03 locus;
primer set 03: SEQ ID No: 13-15 sequences, primers for amplifying the SNP04 sites;
Primer set 04: SEQ ID No: 16-18 sequences, primers for amplifying the SNP05 locus;
primer set 05: SEQ ID No: 19-21, a primer for amplifying the SNP06 locus.
In a third aspect the invention provides a kit for detecting tomato mosaic virus resistance comprising one or more of the primer sets of the 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:22, 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:23, 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:24, 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:25, 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) Use in the detection or assisted detection of tomato mosaic virus resistance;
(2) The application in preparing products for detecting or assisting in detecting tomato mosaic virus resistance;
(3) Application in tomato mosaic virus 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 mosaic virus 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.
According to the need, 1 or several or all of the 6 SNP sites of the SNP site combination according to the first aspect of the invention can be selected for SNP site polymorphism or genotype detection. In some embodiments, the presence of the Tm-2 a gene and/or the haplotype of the Tm-2 a gene segment in the tomato variety to be tested (homozygous anti-mosaic tomato Tm-2 a/Tm-2a, heterozygous anti-mosaic tomato Tm-2 a/tm-2a, or homozygous mosaic-infected tomato Tm-2 a/tm-2a) is identified by detecting 1 SNP locus therein. In other embodiments, the detection of 2 or more or all of the SNP sites is used to identify whether the tomato variety to be tested contains the Tm-2 a gene and/or to identify the Tm-2 a gene segment haplotype in the tomato variety to be tested. Preferably, whether the tomato variety to be detected contains the Tm-2 a gene or not and/or the segment haplotype of the Tm-2 a gene in the tomato variety to be detected is identified by detecting 1 or more of the SNP02 site, the SNP03 site, the SNP04 site, the SNP05 site, and the SNP06 site in the SNP site combination.
The fifth aspect of the invention provides a method for detecting resistance to tomato mosaic virus, wherein SNP typing detection is carried out on tomato varieties to be detected, and the method comprises 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, in the method according to the fifth aspect of the present invention, the detection of SNP typing of tomato variety to be detected adopts a KASP detection method 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 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, 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:22, 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:23, 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:24, 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:25, 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 label 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 state of the target SNP locus 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 the Tm-2 a gene segment haplotype (Tm-2 a/Tm-2a,Tm-2a/tm-2a or Tm-2 a/tm-2a) in the detected tomato material is judged, and rapid and accurate transfer application of the tomato mosaic virus resistant gene is further assisted.
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 variable group data in the upstream and downstream adjacent areas of known Tm-2 a genes and the inside of the genes, realizes the molecular marker-assisted effective selection of target tomato disease and insect resistance genes through the wide verification of different resource materials, and breaks the unfavorable linkage of the tomato disease and insect 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 substance/product (such as primer combination, kit and the like) developed based on the SNP locus combination can realize rapid, accurate and high-flux detection of the haplotype of the Tm-2 a section of the tomato mosaic virus resistance gene, has the advantages of simplicity in 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 tomato mosaic virus resistance in a tomato seedling stage, reduces the workload of artificial 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 a combination of the universal SNP locus and KASP primer for detecting the tomato mosaic virus resistance gene Tm-2 a;
FIG. 2 shows the variant group of 5 common SNP sites (SNP 02 to SNP 06) in the upstream and downstream and internal region of tomato mosaic virus resistance gene Tm-2 a (Solyc g 018220) and its positional information on tomato reference genome (SL 2.50 version); in the figure, KTm S02 is KASP mark and its position information disclosed in the prior art, TS-2, TS-7 and REFERENCE GENOME (Heinz 1706) are disease-resistant genotype contrast, and the rest TS-11-TS 315 are disease-resistant genotype contrast;
FIG. 3 shows typing of 5 KASP markers developed at the locus of the general SNPs of the tomato mosaic virus resistance gene Tm-2 a in a large population; in the figure, A is primer set 01 (Chr 09:13,623,713), B is primer set 02 (Chr 09:13,623,321), C is primer set 03 (Chr 09:13,622,207), D is primer set 04 (Chr 09:13,621,498), E is primer set 05 (Chr 09:13,621,305), the abscissa represents FAM fluorescence signal value (dot at I, disease resistance genotype), the ordinate represents HEX fluorescence signal value (dot at III, disease resistance genotype), the dot at middle II represents heterozygous disease resistance genotype, and the dot near the 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 of the development and application of the combination of the universal SNP locus and KASP primer for detecting the Tm-2 a gene of tomato mosaic virus disease. 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, chromatography, sequencing, gene chips, other PCR technologies and the like.
Example 1 screening of SNP site combinations
1. Experimental materials
Variant group data of 660 representative tomato germplasm resources of different source types in the world are selected for SNP locus screening in the embodiment, wherein the source of the variant group data of the tomato is mainly based on the earlier work (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.Achemical genetic roadmap to improved tomato flavor[J].Science,2017,355(6323):391). of the project group where the inventor is positioned, and part of genotype data of the tomato germplasm resources and resistance phenotype data of Tm-2 a genes are derived from the existing public database (https:// solgenemics.net /).
2. Screening of SNP site combinations
Universal SNP sites were screened within 2kb of the internal and upstream region by using the target gene Tm-2 a (Solyc g018220, database source https:// solgenomics.
Through the analysis of genome-wide variation patterns of different varieties such as big-fruit tomatoes, cherry tomatoes, fresh tomatoes, processing tomatoes and other wild disease-resistant donor germplasm resources, SNP loci with consistent differences in the Tm-2 a gene or on both sides of the resistance and sensation materials are obtained. Tomato germplasm resources TS-2 (Moneymaker), TS-7 (Micro-Tom), TS-253 (Heinz 1706, tomato reference genome) and the like are used as known disease resistance genotypes (Tm-2/Tm-2) for comparison, TS-11 (KR 2), TS-12 (yoku improvement), TS-151 (T-5), TS-211 (NC 84173), TS-52 (05-4126), TS-40 (CERISE VFNT) and the like are used as known disease resistance genotypes (Tm-2/Tm-2) for comparison, wherein TS-40 is cherry tomato, and the rest is large fruit cultivated tomato with different geographic sources and growing periods. And selecting according to different positions on the upstream and downstream of the gene and in the gene, and carrying out screening analysis on each position according to the typing result. Finally, 6 high-quality universal SNP loci (namely SNP locus combination of the invention: SNP01 locus to SNP06 locus) which show highly consistent and stable differences in the variation group of known resistance and inductance genotype control are successfully obtained from chromosome 9. Since these 6 SNP sites show highly consistent variation between different types and sources of resistant and susceptible varieties, a significant associative characterization effect on tomato mosaic virus resistance can be expected.
In consideration of diversity, success rate and fundamental principle universality of SNPs (single nucleotide polymorphisms) such as sequencing, PCR (polymerase chain reaction) amplification and gene chips and uncertainty of cloned gene functions and molecular regulation network mechanisms, the invention further provides 6 SNP loci of the Tm-2 a region of the anti-mosaic virus gene and respective flanking sequences thereof, and the application of the SNP loci in the method is used for detecting tomato mosaic virus resistance, preparing related detection or auxiliary detection products, and auxiliary breeding and germplasm resource protection and innovation of tomatoes.
SNP01 locus to SNP06 locus and their respective flanking sequences are shown in SEQ ID No:1 to 6.
Example 2 primer Synthesis and kit preparation
1. Primer design and screening
According to flanking sequences of SNP01 locus to SNP06 locus provided in example 1, two forward primers are designed on the upstream of SNP locus by Primer3.0 software and one reverse primer is designed on the downstream according to KASP mark design development principle (target product 80-150 bp, primer specifically matches target region on reference genome and locates in non SNP dense region, avoiding A\T or G\C high-level complex sequence region, etc.).
The tomato sample to be tested comprises 21 samples randomly selected from part of germplasm resources with known genotypes and mosaic virus resistance phenotypes and breeding materials as anti-infection or sense or heterozygous controls, and finally 3 ddH 2 O are added as NTC blank controls for 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; 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 of ddH 2 O are added, and in other embodiments, the same detection purpose can be achieved by using other reasonable primer mixture ratios). Wherein, the primer mixture consists of the primer sequences of the same primer group in the primer combination. The PCR premix includes fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, KLEARTAQ DNA polymerase, dNTPs and MgCl 2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:22, 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:23, 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:24, 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:25, 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; the fluorescent signal was read at 30℃for 1 min. And finally selecting primer combinations (comprising 5 primer groups, namely primer groups 01-05, wherein SNP information corresponding to each primer group is shown in figure 2, and the size and haplotype information of corresponding amplified target bands are shown in table 1) with obvious aggregation and typing trends of FAM signals, VIC signals and heterozygous fluorescent signals at the upstream and downstream or inside of a target gene, and carrying out subsequent large-population Tm-2 a region genotype or haplotype verification and breeding application.
Primer set 01: SEQ ID No: 7-9 sequences, primers for amplifying the SNP02 locus;
primer set 02: SEQ ID No: 10-12 sequences, primers for amplifying the SNP03 locus;
primer set 03: SEQ ID No: 13-15 sequences, primers for amplifying the SNP04 sites;
Primer set 04: SEQ ID No: 16-18 sequences, primers for amplifying the SNP05 locus;
primer set 05: SEQ ID No: 19-21, a primer for amplifying the SNP06 locus.
TABLE 1 primer combination for detecting tomato Tm-2 a gene and SNP locus, amplified fragment length and primer information corresponding thereto
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 includes fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, ROX internal reference dye, KLEARTAQ DNA polymerase, dNTPs and MgCl 2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:22, 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:23, 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:24, 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:25, 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 application of efficient KASP marker of tomato mosaic virus resistance gene Tm-2 a
The verification and breeding application work of the efficient KASP marker of the tomato mosaic virus resistance gene Tm-2 a are carried out by using the kit (comprising primer combination) provided in the example 2 based on the SNP locus provided in the example 1.
Representative tomato samples including main stream commercial varieties, core open germplasm of national resource library, breeding intermediate materials, new hybrid combinations and the like are selected 381 parts, and cultivated tomatoes of different types and different sources such as big-fruit tomatoes, cherry tomatoes, tandem tomatoes, fresh tomatoes, processing tomatoes, farms (local varieties) and the like are related, wherein 156 parts of Tm-2 a genotype data (comprising 25 parts of tomato germplasm resources and commodity varieties with known mosaic virus resistance phenotypes) are obtained by a third-party commercial establishment through the use of closely linked CAPS markers, and are shown in Table 2 in detail. Furthermore, the sites disclosed in the prior art (Kong Huili et al, application publication No. CN 108330201A) (i.e., KTm2S02, SL2.50ch09:13,623,466) serve as technical controls in this example.
Specifically, the embodiment provides a method for detecting resistance of tomato mosaic virus, 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 2 O are added as NTC blank control, and 384 parts are added.
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; the final concentration of each primer is 100pmol/L, the mixing ratio of the primers is 12 mu L of each specific typing 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 a IntelliQube platform operation manual, and executing an operation program {95 ℃ presegeneration 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 25 samples tested with known mosaic virus resistance phenotypes, the resistance phenotype, CAPS marker identification genotype and KASP marker (i.e., primer combination) identification gene data provided by the present invention were completely identical, i.e., identical p=100% (as shown in table 2). In addition, in 156 samples to be tested with reference to the results of the linked CAPS markers, the consistency P of the 5 primer sets provided in example 2 with the gene internal function markers KTm S02 { the consistency calculation formula was p= (number of effective test samples-number of difference samples)/100% of effective test samples } were 99.3%, 98.7%, 99.3% and 99.3%, respectively, and the consistency P with the commercial linked CAPS markers was 88.7%, 88.6%, 88.1% and 88.8%, respectively, whereas the consistency P between the 5 primer sets provided in example 2 was more than 99%. From this, the consistency between the detection results of the 5 primer sets provided in example 2 and the gene internal function marker KTm S02 is significantly higher than that of the third party detection of the gene-linked CAPS marker. Further analysis shows that contradiction of the detection results of samples of the same repeated materials (for example, numbers Ta135 and Ta137, and Ta141 and Ta142 are two sets of the same repeated materials), but the detection results of the 5 primer sets provided by the invention do not exist. In addition, for the detection of Ta128, the detection results of the gene internal functional markers KTm S02 are inconsistent with the detection results of the 5 primer sets provided by the invention, the detection results of the reference markers KTm S02 are homozygous genotypes (C: C), and the detection results of the 5 primer sets provided by the invention and the third-party CAPS markers are consistent, and the detection results are heterozygous genotypes. The results demonstrate that the detection accuracy and stability of the 5-group KASP markers provided by the invention are significantly higher than those of the existing commercial CAPS markers and the existing related KASP markers.
In addition, the embodiment also carries out Tm-2 a locus genotype detection on 225 other breeding intermediate materials, and the result shows that the genotype detection result of the sample to be detected is basically consistent with the genealogy relation, and the tomato mosaic virus resistance of offspring or father can be accurately predicted through the genotype detection result of the locus Tm-2 a 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 CAPS markers, and each group of KASP markers can be independently applied to molecular detection of tomato mosaic virus resistance. By analogy, the remaining 1 SNP site that also meets the aforementioned variant set features can be verified and obtained with 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 commercial breeding of tomato mosaic virus resistant molecules.
TABLE 2 mosaic Virus resistance phenotype and marker genotype of 156 tomato germplasm resources, variety and breeding intermediate
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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 tomato mosaic virus disease resistance phenotype, "S" represents disease susceptibility, "R" represents disease resistance, "HR" represents high resistance, and "IR" represents medium resistance; wherein the "CAPS" column represents detection results of third party commercial institutions by utilizing Tm-2 a gene linked CAPS markers, "SS" represents homozygous disease-causing genotype, "RR" represents homozygous disease-resistant genotype, and "H" represents heterozygous genotype; wherein "KTm S2" is a KASP marker preferred in the prior art scheme (Kong Huili et al, application publication No. CN 108330201A) as described above, and as a technical control in this example, "T: T" in the detection result represents a homozygous disease-resistant genotype, "C: C" represents a homozygous disease-resistant genotype, and "T: C" represents a heterozygous genotype; wherein in the detection result of the primer group 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 02, T represents homozygous disease-resistant genotype, C represents homozygous disease-resistant genotype, and T represents heterozygous genotype; wherein, in the detection results of the primer group 03, the primer group 04 and the primer group 05, G represents a homozygous disease resistance genotype, A represents a homozygous disease resistance genotype, and G represents a heterozygous genotype; "-" represents a data deletion; the ". Times. -mark represents the result of disagreement between the Tm-2 a gene-linked CAPS marker, the reference marker KTm S02 and the KASP marker provided by the present invention or between 5 primer sets within the 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 application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application 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> Institute of agricultural genome of China academy of agricultural sciences
Shenzhen agricultural genome institute of Chinese academy of agricultural sciences
<120> SNP locus combination for detecting resistance to tomato mosaic virus and use thereof
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Claims (12)
1. Use of a reagent for detecting a combination of SNP sites for detecting tomato mosaic virus resistance, characterized in that said combination of SNP sites comprises one or more of the following SNP sites located inside and on both sides of the tomato mosaic virus resistance gene Tm-2 a:
SNP02 locus, located at 13623713 nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, the base of which is C or T;
SNP03 locus, locate at tomato Heinz 1706 reference genome SL2.50 version 09 on chromosome 13623321, its base is C or T;
SNP04 locus, which is located at 13622207 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, and its base is T or C;
SNP05 locus, which is located at 13621498 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, and the base is T or C; and
SNP06 locus, located at the 13621305 th nucleotide locus on chromosome 09 of tomato Heinz 1706 reference genome SL2.50 version 09, whose base is T or C.
2. The use according to claim 1, wherein the SNP02 site-SNP 06 site and their respective flanking sequences are as set forth in SEQ ID nos: 2-6, wherein the SNP02 site-SNP 06 site are respectively located in the SEQ ID No: 2-6, and position 102 in the sequence.
3. A primer combination for amplifying the SNP site combination as set forth in claim 1, characterized in that the primer combination comprises one or more of the following primer sets 01 to 05:
primer set 01: SEQ ID No: 7-9 sequences, primers for amplifying the SNP02 locus;
primer set 02: SEQ ID No: 10-12 sequences, primers for amplifying the SNP03 locus;
primer set 03: SEQ ID No: 13-15 sequences, primers for amplifying the SNP04 sites;
Primer set 04: SEQ ID No: 16-18 sequences, primers for amplifying the SNP05 locus;
primer set 05: SEQ ID No: 19-21, a primer for amplifying the SNP06 locus.
4. A kit for detecting tomato mosaic virus resistance comprising one or more primer sets of the primer combination of claim 3 in powder or liquid form.
5. The kit of claim 4, further comprising a PCR pre-mix comprising a fluorescent probe, a quenching probe, a ROX reference dye, KLEARTAQ DNA polymerase, dntps, and MgCl 2.
6. The SNP site combination of claim 1 or 2, or the primer combination of claim 3, or the kit of any one of claims 4 or 5 for use as follows:
(1) Use in the detection or assisted detection of tomato mosaic virus resistance;
(2) The application in preparing products for detecting or assisting in detecting tomato mosaic virus resistance;
(3) Application in tomato mosaic virus resistance breeding;
(4) Application in identification and protection of tomato germplasm resources and new varieties;
(5) Application in tomato germplasm resource improvement and innovation.
7. The use according to claim 6, 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 claim 1 or 2, the detection method comprising one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
8. The use according to claim 6, characterized in that it is carried out by the following technical means:
Developing a PCR marker and/or a gene chip using sequence information of one or more SNP sites in the SNP site combination AS claimed in claim 1 or 2, the PCR marker comprising one or more of a PCR-RFLP marker, a TaqMan marker, a KASP marker, an AS-PCR marker and a HRM marker.
9. The use according to claim 6, characterized in that it is carried out by the following technical means:
molecular breeding improvement and germplasm resource innovation of tomato mosaic virus resistance are achieved by performing molecular manipulation, including genetic editing or genetic transformation, using one or more SNP sites in the SNP site combination of claim 1 or 2.
10. The method for detecting the resistance of the tomato mosaic virus is characterized by carrying out SNP genotyping 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 3, 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 3.
12. The method according to claim 10, 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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