CN113736907B - SNP locus combination for detecting tomato gray leaf spot resistance and application thereof - Google Patents
SNP locus combination for detecting tomato gray leaf spot resistance and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant biology, in particular to an SNP locus combination for detecting resistance of tomato gray leaf spot and application thereof. Based on the above, the invention develops a primer combination, a kit and a detection method which can rapidly, intuitively and effectively identify the genotype state of the target SNP locus. The method can realize rapid, accurate and high-flux detection of the haplotype of the resistance gene Sm segment of the tomato gray leaf spot, has the advantages of simple operation, low cost, automation, high flux efficiency, stable labeling, safety, no toxicity, no harm and the like, can rapidly, accurately and high-flux identify the resistance of the tomato gray leaf spot in the tomato seedling stage, reduce the workload of artificial inoculation identification and field transplantation, improve the breeding efficiency, reduce the breeding cost and accelerate the breeding process, and is very suitable for modern commercial breeding application and large-scale genetic improvement research.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to an SNP locus combination for detecting resistance of tomato gray leaf spot and application thereof.
Background
Tomatoes are important economic crops of vegetables in the world and have important production application and basic research value. With the gradual expansion of the planting area of tomatoes all over the world, the influence of tomato plant diseases and insect pests is increasing day by day. Among them, tomato Gray Leaf Spot Disease (GLSD) is a fungal disease which is newly developed and prevalent in recent years, and the incidence rate is increasing year by year. The pathogenic bacteria are fungi of the genus Tinospora of the class Deuteromycotina, and are mainly caused by infection of Stachybotrys solani (Stemphylium solani) and Stachybotrys stoloni (S.lycopersici). The disease occurs worldwide, and is particularly serious in warm and humid areas. In recent years, the method is commonly used in spring and summer in south and north of China, particularly in early spring protected land cultivation, the damage is serious, the quality of the tomatoes is reduced, the yield is sharply reduced by 20% -80%, and the production of the tomatoes is seriously influenced. Therefore, the development of effective molecular markers for detecting the resistance of the tomato gray leaf spot is very important and urgent for the efficient auxiliary breeding of new varieties of gray leaf spot resistant varieties.
Some tomato germplasm resources resisting the gray leaf spot have been excavated at present, and a tomato key gene Sm resisting the gray leaf spot, which is positioned on the short arm of Chr.11 and shows single-gene incomplete dominant inheritance, has been identified and utilized, and is widely applied to tomato commercial breeding at present. Scientists at home and abroad locate and lock the candidate region of the Sm gene by constructing different genetic groups and utilizing different types of molecular markers. Sm gene-linked RFLP markers CT55, C2_ At4g10050 and C2_ At2g14260, among others, have been reported, with CT55 later being transformed into a recessive CAPS marker, and a co-dominant CAPS marker T050. However, the restriction enzyme digestion process of PCR products is complicated due to the AFLP and CAPS markers, and the closely linked marker Sm-InDel disease-resistant band is very close to the disease-sensitive band and is not easy to distinguish. Moreover, most of the CAPS and InDel markers need manual operation, are easy to cause errors, and are not suitable for high-throughput automated detection.
Meanwhile, competitive Allele Specific PCR (KASP) markers are taken as the mainstream genotyping method in the world, have the advantages of high specificity, high accuracy, rapidness, high efficiency, strong flexibility, low cost of single data point, easiness in realizing automatic high-throughput operation and the like, are very suitable for high-throughput automatic detection of a few marker sites of a large number of samples, and are widely and mature applied to animal and plant commercial breeding and genotyping related basic research.
At present, there are several KASP marker detection technical schemes for the tomato gray leaf spot resistance gene Sm, but the selection of target SNPs or indels in these schemes is mostly based on single or few differences in sequences of resistant parents, or is not a functional molecular marker directly related to the target gene, so that the effectiveness, universality and universality in tomato resources and commercial breeding in different genetic backgrounds cannot be guaranteed, selection failure may be caused, or other unfavorable gene fragments are brought by linkage drag, and the detection efficiency of the tomato gray leaf spot resistance and the breeding efficiency of new disease-resistant varieties are limited.
Therefore, the development of an efficient, representative and universal SNP locus combination capable of quickly and effectively detecting the tomato gray leaf spot resistance and a high-throughput detection application technical scheme has very important practical and theoretical significance for the 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 the resistance of tomato gray leaf spot and application thereof. A plurality of SNP loci capable of rapidly and effectively detecting the resistance of tomato gray leaf spot are obtained by directly analyzing and identifying a large amount of variation group data in upstream and downstream adjacent regions and genes of the known Sm gene, and the accurate identification and selection of a target gene region can be realized, so that the problems that the universality of the SNP loci is insufficient, or the target gene selection is invalid due to the fact that the SNP loci are not directly related functional molecular markers (only linkage markers) of target characters/genes, or other non-target adverse gene fragments are caused by linkage drag and the like in the prior art are solved.
To achieve the above objects, the present invention provides, in a first aspect, a SNP site combination for detecting resistance to tomato gray leaf spot, the SNP site combination including one or more of the following SNP sites located inside and on both sides of the tomato gray leaf spot resistance gene Sm:
SNP01 site, located at 9318832 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is G or A;
SNP02 site, located at 9243756 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is C or T;
SNP03, located at 9241397 th nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706, wherein the base is T or C;
SNP04 site, located at 9246838 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is C or T;
SNP05 site, located at 9306833 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP06 site, located at 9314583 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP07 site, located at 9318930 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is C or T;
SNP08 site, located at 9322852 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is G or A;
SNP09 site, located at 9333469 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP10, located at 9334124 nucleotide site on chromosome SL2.50 version 11 of tomato Heinz1706, and base of SNP10 is A or G; and
SNP11, located at the 9335134 th nucleotide site on the SL2.50 version 11 chromosome of the tomato Heinz1706 reference genome, and the base of SNP11 is A or G.
The genome information corresponding to the SNP locus combination in the first aspect of the invention is derived from databases https:// solgenomics. net/locus/39057,39058,39059/view, and the sequences and SNP physical position information of corresponding tomato gray leaf spot resistance genes Sm (Solyc11g018710, Solyc11g018720 and Solyc11g018730) correspond to tomato (Heinz1706) reference genome SL2.50 version.
Based on the SNP locus combination of the first aspect of the invention, the high-throughput SNP typing detection of the tomato gray leaf spot resistance can be realized, the result accuracy is high, the consistency is good, the universality is strong, and the accurate identification and selection of the target gene region can be realized.
In one embodiment of the present invention, the SNP 01-SNP 11 and their respective flanking sequences are set forth in SEQ ID nos: 1-11, wherein the SNP01 site to the SNP11 site are respectively positioned in the nucleotide sequences shown in SEQ ID No: position 102 in the sequence 1-11.
In one embodiment of the present invention, the SNP combination site includes one or more of the SNP03 site, the SNP04 site, the SNP05 site, the SNP07 site, the SNP09 site, the SNP10 site, and the SNP11 site.
The second aspect of the invention provides a primer combination for amplifying the SNP locus combination, wherein the primer combination comprises one or more of the following primer groups 01-07:
primer set 01: SEQ ID No: 12-14 sequence, and primer for amplifying SNP03 site;
primer set 02: SEQ ID No: 15-17 sequence, and primer for amplifying the SNP04 locus;
primer set 03: SEQ ID No: 18-20 sequences and primers for amplifying the SNP05 locus;
a primer set 04: SEQ ID No: 21-23 sequence, and primer for amplifying the SNP07 locus;
primer set 05: SEQ ID No: 24-26 sequences and primers for amplifying the SNP09 locus;
primer set 06: SEQ ID No: 27-29 sequence, and primer for amplifying the SNP10 locus;
primer set 07: SEQ ID No: 30-32 sequence and primer for amplifying the SNP11 locus.
In a third aspect, the present invention provides a kit for detecting resistance to tomato gray leaf spot, comprising one or more primer sets of the second aspect of the present invention in a powdered or liquid state.
In one embodiment of the invention, the kit of the third aspect of the invention further comprises a PCR premix comprising a fluorescent probe, a quenching probe, a ROX internal reference dye, KlearTaq DNA polymerase, dntps and MgCl2。
Preferably, the fluorescent probe comprises a fluorescent probe A and a fluorescent probe B, and the quenching probe comprises a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 33, and the 5' end is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 34, and a fluorescent group VIC or HEX is connected to the 3' end of the derivative;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 35, and the 3' end of the derivative is connected with a quenching group BHQ;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 36, and a quenching group BHQ is connected to the 3' end of the derivative.
In a fourth aspect, the present invention provides any one of the following applications of the SNP combination of the first aspect, the primer combination of the second aspect, or the kit of the third aspect:
(1) the application in detecting or assisting in detecting the resistance of tomato gray leaf spot;
(2) the application in the preparation of products for detecting or assisting in detecting the resistance of tomato gray leaf spot;
(3) the application in tomato gray leaf spot resistance breeding;
(4) the application in identifying and protecting tomato germplasm resources and new varieties;
(5) the application in improvement and innovation of tomato germplasm resources.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
detecting the polymorphism or genotype of one or more SNP sites in the SNP site combination provided by the first aspect of the invention, wherein the detection method comprises one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
the sequence information of one or more SNP sites in the SNP site combination provided by the first aspect of the invention is utilized to develop PCR markers and/or gene chips, wherein the PCR markers comprise one or more of PCR-RFLP markers, TaqMan markers, KASP markers, AS-PCR markers and HRM markers.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
the molecular breeding improvement and germplasm resource innovation of tomato gray leaf spot resistance are realized by carrying out molecular operation by utilizing one or more SNP loci in the SNP locus combination provided by the first aspect of the invention, wherein the molecular operation comprises gene editing or genetic transformation.
The above applications can be optimized and adjusted or replaced according to different project requirements and purposes.
If necessary, 1 or several or all of the 11 SNP sites of the SNP site combination according to the first aspect of the invention may be selected for SNP site polymorphism or genotype detection. In some embodiments, the Sm gene is identified in the tomato variety to be tested by detecting 1SNP locus therein and/or the haplotype of the Sm gene segment in the tomato variety to be tested (homozygous Sm/Sm in tomato resistant to gray leaf spot, heterozygous Sm/Sm in tomato resistant to gray leaf spot, or homozygous Sm/Sm in tomato susceptible to gray leaf spot). In other embodiments, the Sm gene is identified in the tomato variety to be tested by detecting 2 or more than 2 or all of the SNP sites therein and/or the haplotype of the Sm gene segment in the tomato variety to be tested. Preferably, the Sm gene in the tomato variety to be tested is identified and/or the haplotype of the Sm gene segment in the tomato variety to be tested is identified by detecting 1 or more of SNP03 site, SNP04 site, SNP05 site, SNP07 site, SNP09 site, SNP10 site and SNP11 site in the SNP site combination.
The fifth aspect of the invention provides a method for detecting the resistance of tomato gray leaf spot, which carries out SNP typing detection on a tomato variety to be detected and comprises the following steps:
(1) extracting DNA of the tomato variety to be detected;
(2) performing PCR amplification on the DNAs by using the primer combination of the second aspect of the present invention;
(3) checking the amplification result, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
In a specific embodiment, the DNA of the tomato variety to be tested may be taken from any one of leaves, roots, stems, flowers, fruits and seeds of a tomato plant.
In one embodiment of the present invention, in the method of the fifth aspect of the present invention, the SNP typing detection on the tomato variety to be tested employs a KASP detection method, which comprises:
(1) adding a primer mixed solution and a PCR premixed solution into the leaf DNA of the tomato variety to be detected, and carrying out KASP amplification;
(2) detecting a PCR product by adopting fluorescent quantitative PCR equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group;
the primer mixture solution is composed of the primer sequences of the same primer group in the primer combination according to the second aspect of the invention.
Preferably, the PCR premix solution comprises a fluorescent probe, a quenching probe, a ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2。
Preferably, the fluorescent probe comprises a fluorescent probe A and a fluorescent probe B, and the quenching probe comprises a quenching probe A and a quenching probe B;
the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 33, wherein the 5' end is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 34, and a fluorescent group VIC or HEX is connected to the 3' end of the derivative;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 35, and the 3' end of the derivative is connected with a quenching group BHQ;
the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 36, and a quenching group BHQ is connected to the 3' end of the derivative.
Preferably, the fluorescent quantitative equipment comprises various brands of fluorescent quantitative PCR instruments, enzyme labeling instruments, high-throughput genotyping systems (automatic workstations) such as IntelliQube, GeneMatrix and the like.
According to the invention, a primer combination capable of rapidly, intuitively and effectively 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 the KASP detection method is adopted to carry out SNP typing detection, so that the haplotype (Sm/Sm, Sm/Sm or Sm/Sm) of the Sm gene segment in the detected tomato material is determined, and further the rapid and accurate transfer application of the tomato gray leaf spot resistant gene is 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: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; 0.03 mu L of primer mixed solution, wherein the final concentration of each primer is 100 pmol/L;
the reaction conditions of the PCR are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 28-32 cycles.
The method provided by the fifth aspect of the invention is simple to operate, and only needs to add the primer mixed solution and the PCR premixed solution into the PCR micropore reaction plate containing the DNA sample for PCR amplification, and then adopts fluorescent quantitative PCR equipment to detect and analyze PCR products and perform data analysis.
Different from the prior art, the invention has the following beneficial effects:
(1) according to the invention, a large amount of variation group data in the upstream and downstream adjacent regions and the gene interior of the known Sm gene are directly analyzed and identified to obtain an efficient, representative and universal SNP site combination, and the molecular marker-assisted effective selection of the target tomato disease and insect resistant gene is realized through the wide verification of different resource materials, so that the adverse linkage of the tomato disease and insect resistant gene is broken;
(2) the SNP locus combination (and respective flanking sequence information thereof) can provide powerful support help for other technical development or research such as targeted sequencing, gene chips, probes, PCR markers, gene cloning, functional research and the like;
(3) the detection substance/product (such as the primer combination, the kit and the like) developed based on the SNP locus combination can realize the rapid, accurate and high-throughput detection of the haplotype of the Sm section of the tomato gray leaf spot resistance gene, has the advantages of simple operation, low cost, automation, high flux efficiency, stable marking, safety, no toxicity, no harm and the like, can perform the resistance identification of the tomato gray leaf spot quickly, accurately and at high flux in the tomato seedling stage, reduces the workload of manual inoculation identification and field transplantation, 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 general SNP site discovery and KASP primer combination development and application for detecting tomato gray leaf spot resistance gene Sm of the invention;
FIG. 2 shows the variation group of tomato gray leaf spot resistance gene Sm (target candidate genes Solyc11g018710, Solyc11g018720 and Solyc11g018730) upstream and downstream and internal region 7 general SNP sites (SNP03 site, SNP04 site, SNP05 site, SNP07 site, SNP09 site, SNP10 site and SNP11 site) and the position information thereof on tomato reference genome (SL2.50 version); in the figure, TS-2, TS-9, Reference genome (Heinz1706) and the like are disease-susceptible genotype controls, and the rest of TS-1 to TS-39 are disease-resistant genotype controls;
FIG. 3 shows the typing of 7 KASP primer combinations developed at the loci of the tomato gray leaf spot resistance gene Sm universal SNPs in a large population; in the figure, A is primer group 01(Chr 11: 9,241,397), B is primer group 02(Chr 11: 9,246,838), C is primer group 03(Chr 11: 9,306,833), D is primer group 04(Chr 11: 9,318,930), E is primer group 05(Chr 11: 9,333,469), F is primer group 06(Chr 11: 9,334,124), G is primer group 07(Chr 11: 9,335,134), the abscissa represents FAM fluorescence signal value (dot at I, representing disease-resistant genotype), the ordinate represents HEX fluorescence signal value (dot at III, representing disease-sensitive genotype), the middle II dot represents heterozygous disease-resistant genotype, and the position close to the origin IV represents NTC negative control.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates 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 of the known resistant tomato detection materials used in the following examples, including TS series tomato germplasm resources, are published at home and abroad, and the social public can ask the agricultural genome institute of Chinese academy of agricultural sciences or other research units to repeat the following experiments, and other tomato commercial varieties can be obtained through regular commercial approaches according to the sources listed in Table 2.
In a specific embodiment, the tomato sample (DNA) to be tested can be taken from any one of leaves, roots, stems, flowers, fruits and seeds of a tomato plant. In the following examples, the leaves of tomato plants are used to extract DNA, but this is not intended to limit the scope of the invention. The PCR reagent, reaction system, platform device and amplification detection procedure used in the following examples are preferred embodiments of the present invention, and other similar reasonable reagents, equipment platforms, reaction systems and amplification procedures made in China or imported could also achieve the same detection purpose, and are not intended to limit the scope of the present invention.
FIG. 1 is a flow chart of the general SNP site discovery and KASP primer combination development and application for detecting tomato gray leaf spot resistance gene Sm of the invention. As shown in FIG. 1, in the embodiment of the present invention, the KASP detection method is used for SNP typing detection, but it is not intended to limit the scope of the present invention. The SNP locus provided by the invention can be used by the technicians in the field to carry out SNP typing detection by means of mass spectrum, chromatogram, sequencing, gene chip and other PCR technologies.
Example 1 screening of SNP site combinations
1. Experimental Material
660 representative tomato germplasm resource mutation groups of different source types in the world are selected for SNP site screening in the embodiment, wherein the tomato mutation group is mainly based on previous work of a project group of the inventor (Lin, T, Zhu, G., Zhang, J.et al. genomic analysis programs into the project of tomato breeding [ J ]. Nat Genet,2014,46: 1220-1226; Tieman D, Zhu G, resource M F R, et al. A chemical elevation map to improved tomato flavor [ J ]. Science,2017,355(6323): 391). Wherein, the genotype data of partial tomato germplasm resources and the resistance phenotype data of Sm genes are derived from the prior public database (https:// solgenomics.
2. Screening for SNP site combinations
The universal SNP site was screened using target genes Sm (Solyc11g018710, Solyc11g018720 and Solyc11g018730, database source: https:// solgenomics. net/locus/39057,39058,39059/view) within 2kb range of the upstream and downstream regions.
SNP loci with consistent difference inside or at two sides of Sm genes between resistant and sensitive materials are obtained through whole genome variation map analysis of different types of tomato with big fruits, cherry tomato, fresh-eating tomato, processing tomato and other wild disease-resistant donor germplasm resources. Solyc11g018710, Solyc11g018720 and Solyc11g018730 were selected as target candidate gene segments. TS-2(Moneymaker), TS-9(Ailsa Craig), TS-253(Heinz1706) and the like are taken as known disease-sensitive genotype (Sm/Sm) controls, TS-1(VF-36), TS-3(M-82), TS-4(Hawaii 7998), TS-7(Micro-Tom), TS-39(Cerise Gold) and the like are taken as known disease-resistant genotype (Sm/Sm) controls, wherein M-82 is processing tomato, Cerise Gold is cherry tomato, and other large-fruit cultivated tomatoes with different geographical sources or breeding stages are mostly used. Selecting according to different positions of upstream and downstream genes and inside the genes, and screening and analyzing each locus according to SNP typing results. Finally, 11 high-quality universal SNP sites (namely SNP site combinations from SNP01 site to SNP11 site) which show highly consistent stable differences in the variation groups of known anti-and sensitive genotype controls are successfully obtained from the chromosome 11. Since the 11 SNP loci show highly consistent variation among resistant and susceptible varieties of different types and sources, the remarkable association characterization effect on the tomato gray leaf spot resistance can be expected.
Considering the diversity, success rate and universality of the basic principle of different detection methods of SNPs such as sequencing, PCR amplification, gene chips and the like, and the uncertainty of cloned gene functions and molecular regulation network mechanisms, the invention further provides 11 SNP sites of the anti-gray leaf spot gene Sm region and respective flanking sequences thereof, which are used for detecting the resistance of tomato gray leaf spot, preparing related detection or auxiliary detection products, and applying tomato auxiliary breeding and germplasm resource protection and innovation.
SNP01 site-SNP 11 site and respective flanking sequences thereof are respectively shown as SEQ ID No: 1 to 11.
Example 2 primer Synthesis and kit preparation
1. Primer design and screening
According to the flanking sequences of SNP01 site to SNP11 site provided in the embodiment 1, aiming at each SNP site, according to the KASP marker design development principle (target product 80-150 bp, primer is specifically matched with target region on the reference genome and is positioned in non-SNP dense region, avoiding complex sequence region with high A \ T or G \ C content and the like), Primer3.0 software is utilized to design two forward primers at the upstream of the SNP site, and a reverse primer is designed at the downstream.
The tomato sample to be tested comprises part of germplasm resources with known genotypes and phenotypes, 21 randomly selected samples in breeding materials as resistance, infection or heterozygosis controls, and finally 3 ddH are added2O as NTC air gapWhite control, 24 parts in total.
The DNA extraction adopts a conventional CTAB method or a domestic magnetic bead kit to extract genome DNA from a leaf blade of a tomato sample, the nucleic acid concentration is measured by using Nanodrop 1000, and the concentration of a DNA template is diluted and controlled to be 10-20 ng/mu L.
The reaction system for PCR (10. mu.L) was: 5 muL of 10-20 ng/muL DNA template; 5 mu L of PCR premix; 0.14 μ L of primer mixture, wherein the final concentration of each primer is 100pmol/L (preferably, the primer mixture ratio is 12 μ L of specific typing primer each, 30 μ L of common primer, and 46 μ L of ddH2O; in other embodiments, the same detection objective can be achieved using other reasonable primer mix ratios). Wherein, the primer mixed solution consists of the primer sequences of the same primer group in the primer combination. The PCR premix solution comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 33, wherein the 5' end is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No: 34, and a fluorescent group VIC or HEX is connected to the 3' end of the derivative; the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 35, and the 3' end of the derivative is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 36, and a quenching group BHQ is connected to the 3' end of the derivative.
Editing sample and primer arrangement template according to the operation manual of a fluorescent quantitative PCR instrument (Applied Biosystems Quant Studio 3, ABI-Q3) of Thermo Fisher company, and executing an operation program { reading a fluorescent signal at 30 ℃ for 1 min; denaturation at 94 deg.C for 15 min; denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, repeating the step for 10 cycles, wherein Touch-Down temperature is set to be reduced by 0.6 ℃ in each cycle, and the final annealing temperature is reduced to 55 ℃; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and repeating the step for 28-32 cycles; the fluorescence signal was read at 30 ℃ for 1min }. Analyzing the data result, finally selecting primer combinations (comprising 7 primer groups: the primer groups 01-07, SNP information corresponding to each primer group is shown in figure 2, the size of a corresponding amplified target band and haplotype information are detailed in table 1) which are positioned at the upstream and downstream or in the target gene and have obvious aggregation and typing trends of FAM signals, VIC signals and heterozygous fluorescent signals, and carrying out follow-up verification and breeding application of the Sm region genotype or haplotype.
Primer set 01: SEQ ID No: 12-14 sequences and primers for amplifying the SNP03 locus;
primer set 02: SEQ ID No: 15-17 sequence, and primer for amplifying the SNP04 locus;
primer set 03: SEQ ID No: 18-20 sequences and primers for amplifying the SNP05 locus;
a primer set 04: SEQ ID No: 21-23 sequence, and a primer for amplifying the SNP07 site;
primer set 05: SEQ ID No: 24-26 sequences and primers for amplifying the SNP09 locus;
primer set 06: SEQ ID No: 27-29 sequence, and primer for amplifying SNP10 site;
primer set 07: SEQ ID No: 30-32 sequence and primer for amplifying SNP11 site.
TABLE 1 primer combination for detecting Sm gene of tomato and corresponding SNP locus, amplified fragment length and primer information
2. Preparation of the kit
This example applies the primer combination described above to the preparation of kits. The kit comprises a PCR premix besides a primer combination. The primer mixture consists of the primer sequences of the same primer group in the primer combination of the invention. The PCR premix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, ROX internal reference dye, KlearTaq DNA polymerase, dNTP and MgCl2. Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No: 33, wherein the 5' end is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:34, and a fluorescent group VIC or HEX is connected to the 3' end of the derivative; the nucleotide sequence of the quenching probe A is shown as SEQ ID No: 35, and the 3' end is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is shown as SEQ ID No: 36, and a quenching group BHQ is connected to the 3' end of the derivative. Three primers (each primer group) of each SNP locus are independently subpackaged and packaged together; and (5) independently packaging the PCR premix.
Example 3 verification and breeding application of tomato gray leaf spot resistant gene Sm efficient KASP marker
In this example, based on the SNP sites provided in example 1, the kit (including primer combination) provided in example 2 is used to verify and breed the tomato gray leaf spot resistance gene Sm high efficiency KASP marker.
381 parts of representative tomato samples containing mainstream commercial varieties, national resource bank core open germplasm, breeding intermediate materials, new hybrid combinations and the like are selected, and the representative tomato samples relate to cultivated tomatoes of different types and different sources such as big-fruit tomatoes, cherry tomatoes, string harvest tomatoes, fresh-eating tomatoes, processed tomatoes, farmer's species (local varieties) and the like, wherein 162 parts of Sm genotype data (comprising 15 parts of tomato germplasm resources and commercial varieties with known gray leaf spot resistance phenotypes) are obtained by using tightly linked InDel markers through a third party commercial institution, and the detailed data are shown in Table 2.
Specifically, the embodiment provides a method for detecting resistance of tomato gray leaf spot, which adopts a KASP detection method to perform SNP typing detection on a tomato variety to be detected, and comprises the following steps:
(1) extracting leaf DNA of a tomato variety to be detected;
(2) adding a primer mixture and a PCR premix into the leaf DNA of the tomato variety to be detected by using the kit provided in example 2 to perform KASP amplification (the leaf DNA is subjected to KASP amplification by using each primer group in the primer combination provided in example 2);
(3) and detecting the PCR product by adopting a fluorescent quantitative PCR instrument, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
The tomato variety to be detected is 381 parts of germplasm resources and breeding materials containing part of known genotypes and phenotypes, and the most important isAfter which 3 ddH are added2O as NTC blank, 384 parts in total.
The extraction method and conditions of the leaf DNA of the tomato variety to be tested are the same as those in example 2.
The PCR amplification reaction system (1.6. mu.L) was: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; 0.03 mu L of primer mixture, wherein the final concentration of each primer is 100pmol/L, the primer mixture ratio is that the specific typing primers are 12 mu L each, the common primer is 30 mu L, and 46 mu L ddH is added2O。
Editing a sample and a primer arrangement template according to an IntelliQube platform operation manual, and executing an operation program { pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 28-32 cycles in total }, reading fluorescence data, increasing the number of PCR cycles as appropriate, analyzing and removing data points with uncertain parts and too low or abnormal fluorescence values, finally typing the large population as shown in FIG. 3, and deriving the Excel result (shown in Table 2).
As a result, the 7 groups of KASP markers provided in example 2 obtain highly consistent and well-defined population typing effects in 381 large tomato breeding populations of different types and sources (as shown in FIG. 3), i.e., experiments prove that the SNP site combination provided in example 1 has good universality and stability in tomato variety resources of different genetic backgrounds. Further, in 15 samples to be tested with known gray leaf spot resistance phenotype, the InDel marker identification genotype and the KASP marker (i.e., primer combination) identification gene data provided by the present invention were completely consistent, i.e., the consistency P was 100% (as shown in table 2). In addition, in 162 samples to be tested with the reference of the results of the linked Sm-InDel markers, the consistency P between the test results of the 7 primer sets provided in example 2 and the commercial linked InDel markers is less than 75.0%, and the consistency P between the test results of the 7 primer sets exceeds 93.8% { the consistency calculation formula is P ═ (number of effective test samples-difference samples)/number of effective test samples 100% }. The results prove that the detection accuracy and stability of the KASP marker provided by the invention are obviously higher than those of the existing commercial InDel marker.
In addition, the present embodiment also performs Sm locus genotype detection on another 219 breeding intermediate materials, and as a result, the genotype detection result of the sample to be detected is found to be basically identical to the pedigree relationship, and the resistance of tomato gray leaf spot of offspring or father generation of the sample to be detected can be accurately predicted through the Sm locus genotype detection result of the sample to be detected.
In summary, the 7 groups of KASP markers provided in this embodiment have good versatility and stability in different types, sources, or genetic background of cultivated tomato variety resources, the detection results of the markers are substantially consistent, the accuracy and stability of the detection of each group of markers are significantly better than those of the existing commercial InDel markers, and each group can be independently applied to molecular detection of tomato gray leaf spot disease resistance. By analogy, the remaining 4 SNP sites that also meet the characteristics of the aforementioned variation set can be verified by sequencing, gene chip or other PCR markers and achieve the same expected effect. Meanwhile, the KASP markers are combined together, so that the detection accuracy can be further improved, experimental errors such as false positives 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 used for the commercial application of molecular breeding of tomato gray leaf spot resistance.
TABLE 2162 tomato germplasm resources, varieties, and Gray leaf spot resistance phenotypes and marker genotypes of breeding intermediate materials
Note: the sample numbers in the table are the arrangement sequence numbers of actual detection samples, so that the sequencing is adjusted according to the source types for the convenience of result comparison analysis and visual judgment; wherein the "resistance" column represents the tomato gray leaf spot resistance phenotype, "S" represents susceptible, "R" represents resistant, "IR" represents resistant; wherein the column of "Sm-InDel" represents the detection result of Sm gene linked InDel marker by third-party commercial institution, "SS" represents homozygous susceptible genotype, "RR" represents homozygous resistant genotype, and "H" represents heterozygous genotype; in the detection results of the primer group 01 and the primer group 07, G represents homozygous disease-resistant genotype, A represents homozygous disease-sensitive genotype, and A represents heterozygous genotype; in the detection results of the primer group 02 and the primer group 04, T represents homozygous disease-resistant genotype, C represents homozygous disease-sensitive genotype, and C represents heterozygous genotype; wherein in the detection results of the primer group 03, the primer group 05 and the primer group 06, C represents homozygous disease-resistant genotype, T represents homozygous disease-sensitive genotype, and T represents heterozygous genotype; "-" represents a data miss; the "+" notation represents the result of the inconsistency between the Sm gene-linked InDel marker and the KASP marker (i.e., the primer set) provided by the present invention or between 7 sets of markers in the KASP marker (i.e., the primer set) 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 flows. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention, and without undue experimentation, the invention may be practiced in a wide range of equivalent parameters, concentrations, and conditions. It is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
While the invention has been described with reference to specific embodiments, it will be understood that it is capable of further modifications. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Shenzhen agricultural genome institute of Chinese agricultural science institute
Institute of agricultural genomics of Chinese academy of agricultural sciences
<120> SNP locus combination for detecting tomato gray leaf spot resistance and application thereof
<130> WK21-HCP-CN1-0354
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<213> Artificial sequence (Artificial)
<400> 24
gaaggtgacc aagttcatgc tctgagaacc gaagatttaa ctgac 45
<210> 25
<211> 44
<212> DNA
<213> Artificial sequence (Artificial)
<400> 25
gaaggtcgga gtcaacggat ttgagaaccg aagatttaac tgat 44
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence (Artificial)
<400> 26
tttgggaaga caattttatg gaga 24
<210> 27
<211> 47
<212> DNA
<213> Artificial sequence (Artificial)
<400> 27
gaaggtgacc aagttcatgc tgggtgttca aagttaatta aatagac 47
<210> 28
<211> 47
<212> DNA
<213> Artificial sequence (Artificial)
<400> 28
gaaggtcgga gtcaacggat tgggtgttca aagttaatta aatagat 47
<210> 29
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 29
agcagataaa ttcagatcag ga 22
<210> 30
<211> 45
<212> DNA
<213> Artificial sequence (Artificial)
<400> 30
gaaggtgacc aagttcatgc ttaatccaac agctcttcag atccg 45
<210> 31
<211> 46
<212> DNA
<213> Artificial sequence (Artificial)
<400> 31
gaaggtcgga gtcaacggat tctaatccaa cagctcttca gatcca 46
<210> 32
<211> 23
<212> DNA
<213> Artificial sequence (Artificial)
<400> 32
acctgttccc acattgtaag ttc 23
<210> 33
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 33
gaaggtgacc aagttcatgc t 21
<210> 34
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 34
gaaggtcgga gtcaacggat t 21
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 35
agcatgaact tggtcacctt c 21
<210> 36
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 36
aatccgttga ctccgacctt c 21
Claims (13)
1. The application of the SNP locus combination in detecting or assisting in detecting the resistance of tomato gray leaf spot is characterized in that the SNP locus combination comprises the following SNP loci positioned in and on two sides of a tomato gray leaf spot resistance gene Sm:
SNP03 site, located at 9241397 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP04 site, located at 9246838 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is C or T;
SNP05 site, located at 9306833 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP07, located at 9318930 th nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706, wherein the base is C or T;
SNP09 site, located at 9333469 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP10 site, located at 9334124 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706, and the base of SNP10 site is A or G; and
SNP11, located at the 9335134 th nucleotide position on chromosome SL2.50, version 11 of tomato Heinz1706, and the base of which is A or G.
2. The use of claim 1, wherein the combination of SNP sites further includes one or more of the following SNP sites:
SNP01 site, located at 9318832 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is G or A;
SNP02, located at 9243756 th nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706, wherein the base is C or T;
SNP06 site, located at 9314583 nucleotide site on SL2.50 version 11 chromosome of tomato Heinz1706 reference genome, wherein the base is T or C;
SNP08, located at the 9322852 th nucleotide site on the SL2.50 version 11 chromosome of the tomato Heinz1706 reference genome, and the base of SNP08 is G or A.
3. The use of claim 2, wherein the SNP 01-SNP 11 and the respective flanking sequences thereof are respectively as set forth in SEQ ID Nos: 1-11, wherein the SNP01 site to the SNP11 site are respectively positioned in the nucleotide sequences shown in SEQ ID No: position 102 in the sequence 1-11.
4. The application according to claim 1, wherein the application comprises:
(1) the application in the preparation of products for detecting or assisting in detecting the resistance of tomato gray leaf spot;
(2) the application in tomato gray leaf spot resistance breeding;
(3) the application in identifying and protecting tomato germplasm resources and new varieties;
(4) the application in improvement and innovation of tomato germplasm resources.
5. The application according to claim 4, characterized in that the application is carried out by adopting the following technical means:
and detecting the polymorphism or the genotype of the SNP locus in the SNP locus combination, wherein the detection method comprises one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
6. The application according to claim 4, characterized in that the application is carried out by adopting the following technical means:
developing PCR markers and/or gene chips by using sequence information of SNP sites in the SNP site combination, wherein the PCR markers comprise one or more of PCR-RFLP markers, TaqMan markers, KASP markers, AS-PCR markers and HRM markers.
7. A primer combination of SNP locus combination for detecting tomato gray leaf spot resistance, which is characterized by comprising the following primer groups:
primer set 01: SEQ ID No: 12-14 sequences;
primer set 02: SEQ ID No: 15-17 sequences;
a primer set 03: SEQ ID No: 18-20 sequences;
a primer set 04: SEQ ID No: 21-23 sequences;
primer set 05: SEQ ID No: 24-26 sequences;
primer set 06: SEQ ID No: 27-29 sequences;
primer set 07: SEQ ID No: 30-32 sequences.
8. A kit for detecting resistance to tomato gray leaf spot, comprising the primer combination of claim 7 in powder or liquid form.
9. The kit of claim 8, further comprising a PCR premix comprising fluorescent probes, quenching probes, ROX internal reference dye, KlearTaq DNA polymerase, dntps, and MgCl 2.
10. The primer combination of claim 7, or the kit of claim 8 or 9, for any one of the following applications:
(1) the application in detecting or assisting in detecting the resistance of tomato gray leaf spot;
(2) the application in the preparation of products for detecting or assisting in detecting the resistance of tomato gray leaf spot;
(3) the application in tomato gray leaf spot resistance breeding.
11. A method for detecting the resistance of tomato gray leaf spot is characterized in that SNP typing detection is carried out on a tomato variety to be detected, and the method comprises the following steps:
(1) extracting DNA of the tomato variety to be detected;
(2) performing PCR amplification on the DNAs with the primer combination of claim 6;
(3) checking the amplification result, determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group,
in the judgment standard of the method, the detection result representing the homozygous disease-resistant genotype is as follows: the detection result of the primer group 01 is G: G. the detection result of the primer group 02 is T: the detection result of the primer group 03 is that C: C. the detection result of the primer group 04 is T: t, the detection result of the primer group 05 is C: C. the detection result of the primer group 06 is C: C. the detection result of the primer group 07 is G: g; the test results representing homozygous susceptible genotypes were: the detection result of the primer group 01 is A: A. the detection result of the primer group 02 is C: C. the detection result of the primer group 03 is T: t, the detection result of the primer group 04 is C: C. the detection result of the primer group 05 was T: the detection result of the primer group 06 is T: t, the detection result of the primer group 07 is A: a; the results of the tests representing heterozygous genotypes were: the detection result of the primer group 01 is A: G. the detection result of the primer group 02 is C: the detection result of the primer group 03 is T: C. the detection result of the primer group 04 is C: the detection result of the primer group 05 is T: C. the detection result of the primer group 06 is T: C. the detection result of the primer group 07 is a: G.
12. the method of claim 11, wherein the SNP typing assay of a tomato variety to be tested employs a KASP assay comprising:
(1) adding a primer mixed solution and a PCR premixed solution into the leaf DNA of the tomato variety to be detected, and carrying out KASP amplification;
(2) detecting a PCR product by adopting fluorescent quantitative PCR equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group;
the primer mixture is the primer combination according to claim 6.
13. The method of claim 11, wherein:
the reaction system of the PCR is as follows: 0.8 mu L of template DNA with the concentration of 10-20 ng/. mu.L; 0.8 mu L of PCR premix; 0.03 mu L of primer mixed solution, wherein the final concentration of each primer is 100 pmol/L;
the reaction conditions of the PCR are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, annealing temperature reduction of 0.6 ℃ in each cycle, 10 cycles in total, and final annealing temperature reduction to 55 ℃; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 28-32 cycles.
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