CN113699273B - SNP locus combination for detecting resistance of tomato root-knot nematode and application thereof - Google Patents
SNP locus combination for detecting resistance of tomato root-knot nematode 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 the resistance of tomato root-knot nematodes and application thereof. Based on the above, the invention develops a primer combination, a kit and a detection method which can rapidly, intuitively and effectively identify the genotype state of the target SNP locus. The method can realize the rapid, accurate and high-flux detection of the haplotype of the tomato root-knot nematode resistance gene Mi-1 section, has the advantages of simple operation, low cost, automation, high flux efficiency, stable marking, safety, no toxicity, no harm and the like, can rapidly, accurately and high-flux identify the tomato root-knot nematode resistance in the tomato seedling stage, reduce the workload of artificial inoculation identification and field transplantation, improve the breeding efficiency, reduce the breeding cost, 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 root-knot nematode and application thereof.
Background
Tomatoes are important vegetable economic crops in the world and have important production application and basic research values. With the gradual expansion of the planting area of tomatoes all over the world, the influence of tomato plant diseases and insect pests is increasing day by day. Root-knot nematodes are a very serious pest of the species, and have a serious impact on tomato yield and quality. The insect pests are distributed widely, and are serious in many areas in temperate and tropical zones, especially in protected cultivation conditions such as greenhouses. Root-knot nematodes infect roots of plants, particularly lateral roots, so that normal absorption functions of the plants are influenced, the plants are severely limited in growth, leaves are withered, the fruit yield is reduced, the plants are easily infected by other germs, and then a plurality of complications are caused. Therefore, the development of effective molecular markers for detecting the resistance of the tomato root-knot nematodes is very important and urgent for the efficient auxiliary breeding of new species of root-knot nematodes.
To date, 9 meloidogyne resistance genes have been identified in tomato, of which Mi-1 located on the short arm of Chr.06 was the first to be discovered and utilized as a meloidogyne gene with a wide resistance range (capable of resisting against all three types of meloidogyne incognita, meloidogyne arachidicola and meloidogyne javanica within a certain resistance range), and thus has been widely utilized in both scientific research and production practice.
Molecular markers currently developed for detecting tomato root-knot nematode resistance gene Mi-1 are mainly CAPS (clean Amplified polymorphic Sequences) markers such as REX-1 and Cf-2 closely linked to Mi-1 gene, and co-dominant SCAR (Sequence-sequenced Amplified Region) markers on both sides of some target genes such as Mint-1, mi23, pmi and PM3Fb/PM3Rb, and InMi1-R and InMi2-R. Because the Mi-1 gene is linked with a plurality of undesirable traits, the undesirable traits are introduced in the breeding process by utilizing a close linkage marker, and the quality, the yield and the like of a new tomato line are influenced. In addition, the CAPS marker needs complicated enzyme digestion process of a PCR product, and most SCAR markers need manual operation, are easy to make mistakes and are not suitable for high-throughput automatic 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 tomato root knot nematode resistance gene Mi-1, but the selection of target SNPs 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 effectiveness, universality and universality in tomato resources and commercial breeding under 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 tomato root knot nematode resistance and the breeding efficiency of new insect-resistant varieties are limited.
Therefore, the efficient, representative and universal SNP locus combination and high-throughput detection application technical scheme capable of quickly and effectively detecting the resistance of the tomato root-knot nematode are developed, and the practical and theoretical significance is very important for the commercial breeding application and the genetic research of tomatoes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an SNP locus combination for detecting the resistance of tomato root-knot nematode and application thereof. A plurality of SNP loci capable of rapidly and effectively detecting the resistance of tomato root-knot nematodes are obtained by analyzing and identifying a large amount of mutation group data in upstream and downstream adjacent regions and genes of known Mi-1 genes, and accurate identification and selection of target gene regions can be realized, so that the problems that in the prior art, the universality of SNP loci is insufficient, or target gene selection is invalid due to the fact that the SNP loci are not functional molecular markers (only linkage markers) directly related to target characters/genes, or other non-target adverse gene fragments are caused by linkage drag and the like are solved.
In order to achieve the above objects, the present invention provides a SNP site combination for detecting resistance of tomato root-knot nematode, wherein the SNP site combination comprises one or more of the following SNP sites located inside and at two sides of the resistance gene Mi-1 of tomato root-knot nematode:
the SNP01 site is located at the 2359017 th nucleotide site on the SL2.50 version 06 chromosome of the tomato Heinz 1706, and the base of the SNP01 site is T or C;
the SNP02 site is located at 2359427 nucleotide site on SL2.50 version 06 chromosome of tomato Heinz 1706, and the base of the SNP02 site is C or T;
the SNP03 site is located at the 2357937 nucleotide site on the SL2.50 version 06 chromosome of the tomato Heinz 1706 reference genome, and the base of the SNP03 site is C or G;
SNP04 site, located on chromosome 2357725 of tomato Heinz 1706 reference genome SL2.50 version 06, with base C or G;
SNP05 site, located on chromosome 2356533 of tomato Heinz 1706 reference genome SL2.50 version 06, with base C or G;
the SNP06 locus is located at the 2356449 th nucleotide locus on the SL2.50 version 06 chromosome of the tomato Heinz 1706 reference genome, and the base of the SNP06 locus is C or G;
SNP07 site, located on chromosome 2355234 of tomato Heinz 1706 reference genome SL2.50 version 06, with base C or G;
SNP08 site, located at 2354549 nucleotide site on SL2.50 version 06 chromosome of tomato Heinz 1706, and the base of the SNP08 site is C or G; and
SNP09, located at 2354363 nucleotide site on chromosome SL2.50 version 06 of tomato Heinz 1706, whose base is C or G.
The genome information corresponding to the SNP locus combination in the first aspect of the invention is derived from a database https:// solgenomics. Net/focus/25630/view, and the sequence and SNP physical position information of the corresponding tomato root-knot nematode resistance gene Mi-1 (Solyc 06g 008450) correspond to the reference genome SL2.50 version of tomato (Heinz 1706).
Based on the SNP locus combination of the first aspect of the invention, the high-throughput SNP typing detection of the resistance of the tomato root-knot nematode 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 SNP01 site to SNP09 site and their respective flanking sequences are respectively as set forth in SEQ ID nos: 1-9, and the SNP01 site to the SNP09 site are respectively positioned in the SEQ ID No: position 102 in the sequence 1-9.
In one embodiment of the present invention, the SNP site combination includes one or more of the SNP01 site, the SNP04 site, and the SNP08 site.
The second aspect of the present invention provides a primer combination for amplifying the above-mentioned SNP site combination, wherein the primer combination comprises one or more of the following primer sets 01 to 03:
primer set 01: SEQ ID No: 10-12 sequences, and a primer for amplifying the SNP01 site;
primer set 02: SEQ ID No: 13-15 sequence, primer for amplifying the SNP04 site;
primer set 03: SEQ ID No: 16-18 sequence and primer for amplifying SNP08 site.
In a third aspect, the present invention provides a kit for detecting resistance to tomato root knot nematode, which comprises one or more primer sets in the primer combination according to the second aspect of the present invention in a powdered or liquid state.
In one embodiment of the present invention, the kit of the third aspect of the present invention further comprises a PCR premix comprising a fluorescent probe, a quenching probe, a ROX internal 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:19, the 5' end of the derivative is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:20, 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:21, 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:22, and the 3' end is connected with a quenching group BHQ.
In a fourth aspect, the present invention provides any one of the following applications of the SNP site 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 root-knot nematode;
(2) The application in the preparation of products for detecting or assisting in detecting the resistance of tomato root-knot nematodes;
(3) The application in tomato root-knot nematode resistance breeding;
(4) The application in identifying and protecting tomato germplasm resources and new varieties;
(5) The application in improvement and innovation of tomato germplasm resources.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
detecting the polymorphism or genotype of one or more SNP sites in the SNP site combination provided by the first aspect of the invention, wherein the detection method comprises one or more of flight mass spectrometry, liquid chromatography, resequencing, targeted sequencing and multiplex PCR sequencing.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
the sequence information of one or more SNP sites in the SNP site combination provided by the first aspect of the invention is utilized to develop PCR markers and/or gene chips, wherein the PCR markers comprise one or more of PCR-RFLP markers, taqMan markers, KASP markers, AS-PCR markers and HRM markers.
Preferably, the application provided by the fourth aspect of the present invention is performed by the following technical means:
molecular breeding improvement and germplasm resource innovation of tomato root-knot nematode 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.
According to the needs, 1 or several or all of the 9 SNP sites of the SNP site combination of the first aspect of the invention can be selected for SNP site polymorphism or genotype detection. In some embodiments, the detection of 1 SNP locus therein is used to identify the presence or absence of Mi-1 gene in the tomato variety to be tested and/or to identify the Mi-1 gene segment haplotype in the tomato variety to be tested (homozygous tomato Mi-1/Mi-1 resistant to root knot nematode, heterozygous tomato Mi-1/Mi-1 resistant to root knot nematode, or homozygous tomato Mi-1/Mi-1 susceptible to root knot nematode). In other embodiments, the presence or absence of the Mi-1 gene in the tomato variety to be tested is identified by detecting 2 or more than 2 or all of the SNP sites in the tomato variety to be tested and/or the haplotype of the Mi-1 gene segment in the tomato variety to be tested is identified. Preferably, the method is used for identifying whether the tomato variety to be detected contains the Mi-1 gene and/or identifying the Mi-1 gene segment haplotype in the tomato variety to be detected by detecting 1 or more of SNP01 locus, SNP04 locus and SNP08 locus in the SNP locus combination.
The fifth aspect of the invention provides a method for detecting the resistance of tomato root-knot nematodes, which is used for carrying 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 of 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 the PCR product by adopting fluorescent quantitative equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group;
the primer mixture solution is composed of the primer sequences of the same primer group in the primer combination according to the second aspect of the invention.
Preferably, the PCR premix comprises a fluorescent probe, a quenching probe, a ROX internal reference dye, klearTaq DNA polymerase, dNTP and 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:19, the 5' end of the derivative is connected with a fluorescent group FAM;
the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:20, the 3' end of the derivative is connected with a fluorescent group VIC or HEX;
the nucleotide sequence of the quenching probe A is shown as SEQ ID No:21, wherein 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:22, 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.
The invention successfully develops a primer combination which can quickly, intuitively and effectively identify and distinguish the genotype state of the target SNP locus based on the SNP locus combination provided by the first aspect of the invention, and carries out SNP typing detection by adopting a KASP detection method, thereby judging the haplotype (Mi-1/Mi-1, mi-1/Mi-1 or Mi-1/Mi-1) of the Mi-1 gene section in the detected tomato material, and further assisting in developing the application of quickly and accurately transferring the tomato root knot nematode resistant gene.
In one embodiment of the present invention, according to the method of the fifth aspect of the present invention, the reaction system of the PCR is: 0.8 mu L of template DNA with the concentration of 10-20 ng/. Mu.L; 0.8 mu L of PCR premix; the primer mixture is 0.03 mu L, 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 extension at 61 ℃ for 60s, wherein the annealing temperature of each cycle is reduced by 0.6 ℃, the annealing temperature is reduced to 55 ℃ for 10 cycles in total, and the final annealing temperature is reduced to 55 ℃; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 28-32 cycles in total.
The method provided by the fifth aspect of the invention is simple to operate, and only needs to add the primer mixed solution and the PCR premixed solution into the PCR micropore reaction plate containing the DNA sample for PCR amplification, and then adopts fluorescent quantitative equipment to detect and analyze the PCR product and perform data analysis.
Different from the prior art, the invention has the following beneficial effects:
(1) According to the invention, a large amount of variation group data in the upstream and downstream adjacent regions and the interior of the known Mi-1 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, PCR markers, gene cloning, functional research and the like;
(3) The detection substance/product (such as the primer combination, the kit and the like) developed based on the SNP locus combination can realize the rapid, accurate and high-flux detection of the haplotype of the tomato root knot nematode resistance gene Mi-1 section, has the advantages of simple operation, low cost, automation, high flux efficiency, stable marking, safety, no toxicity, no harm and the like, can rapidly, accurately and high-flux identify the resistance of the tomato root knot nematode in the tomato seedling stage, reduce the workload of artificial inoculation identification and field transplantation, improve the breeding efficiency, reduce the breeding cost, accelerate the breeding process and is very suitable for modern commercial breeding application and large-scale genetic improvement research.
Drawings
FIG. 1 is a flow chart of the general SNP site discovery and KASP primer combination development and application for detecting tomato root knot nematode resistant gene Mi-1 of the present invention;
FIG. 2 shows the variation set of 3 universal SNP sites (SNP 01, SNP04, SNP 08) in the upstream and downstream and internal regions of tomato root-knot nematode resistant gene Mi-1 and the position information thereof on tomato genome (SL 2.50 version); in the figure, TS-2, TS-3, reference genome (Heinz 1706) and the like are taken as disease-sensitive genotype controls, and the rest TS-11 to TS319 are taken as disease-resistant genotype controls;
FIG. 3 shows the typing of 3 KASP primer combinations developed at the locus of the common SNPs of the tomato root knot nematode resistant gene Mi-1 in a large population; in the figure, A is a primer group 01 (Chr 06: 2359017), B is a primer group 02 (Chr 06: 2357725), C is a primer group 03 (Chr 06: 2354549), the abscissa represents the FAM fluorescence signal value (dot at I, representing the disease-resistant genotype), the ordinate represents the HEX fluorescence signal value (dot at III, representing the disease-sensitive genotype), the dot at the middle II represents the heterozygous disease-resistant genotype, and the dot near the origin IV represents the NTC negative control.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. 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 public can ask the agriculture 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, said tomato sample (DNA) to be tested may 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 root knot nematode resistant gene Mi-1. 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 skilled in the art can perform SNP typing detection by means of mass spectrometry, chromatography, sequencing, gene chip and other PCR technologies based on the SNP locus provided by the invention.
Example 1 screening of SNP site combinations
1. Experimental Material
660 representative tomato germplasm resources in different source types worldwide are selected for SNP site screening in the embodiment, and the source of the tomato mutation group is mainly based on the previous work of the project group of the inventor (Lin, T., zhu, G., zhang, J.et al. Genomic analysis programs into the history of tomato breeding [ J ]. Nat Genet,2014,46, 1220-1226 Tieman D, zhu G, resend M F R, et al. A chemical genetic code to improved genetic flag J ]. Science,2017,355 (6323): 391. Wherein, the genotype data of part of tomato germplasm resources and the resistance phenotype data of Mi-1 gene are derived from the prior public database (https:// solgenomics.
2. Screening for SNP site combinations
The universal SNP locus is screened in the range of 2kb in the internal region and the upstream and downstream regions of a target gene Mi-1 (Solyc 06g 008450).
Through the analysis of the whole genome variation maps of different types of tomato with big fruit, cherry tomato, fresh tomato, processed tomato and other wild disease-resistant donor germplasm resources, SNP loci with consistent differences in Mi-1 genes or on both sides between resistant and sensitive materials are obtained. Tomato germplasm resources TS-2 (Moneymaker), TS-3 (M-82), a reference genome (Heinz 1706) and the like are taken as known homozygous susceptible genotypes (Mi-1/Mi-1) for comparison, TS-11-TS 319 and the like are taken as known homozygous disease-resistant genotypes (Mi-1/Mi-1) for comparison, wherein TS-40 is cherry tomatoes, and TS-2, TS-3, TS-11, TS-151, TS-210, TS-314 and the like are large-fruit cultivated tomatoes with different geographical sources. And 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, 9 high-quality universal SNP sites (namely the SNP site combination of the invention: SNP01 site to SNP09 site) which show highly consistent stable differences in the variation groups of known resistance and sensitivity genotype controls are successfully obtained from the No. 6 chromosome. Since the 9 SNP loci show highly uniform variation among resistant and susceptible varieties of different types and sources, the remarkable association characterization effect on the resistance of the tomato root-knot nematode can be expected. And a group of KASP primers { development of anti-susceptible parental SNP (T: C) at 9173bp by using tomato root knot nematode resistance gene Mi-1.2 }, wherein the variation does not show consistent variation in 660 tomato variation groups, so that SNP locus screening is not passed, and meanwhile, the kit is directly used for detecting and typing of a large tomato population, and an obvious typing result cannot be obtained.
Considering the diversity, success rate and universality of basic principles of different detection methods of SNPs such as sequencing, PCR amplification, gene chips and the like, and the uncertainty of cloned gene functions and molecular regulation network mechanisms, the invention further provides 9 SNP loci in the Mi-1 region of the root knot nematode resistant gene and respective flanking sequences thereof, which are used for detecting the resistance of the tomato root knot nematode, preparing related detection or auxiliary detection products, and applying tomato auxiliary breeding and germplasm resource protection and innovation.
SNP01 site to SNP09 site and respective flanking sequences thereof are respectively shown as SEQ ID No:1 to 9.
Example 2 primer Synthesis and kit preparation
1. Primer design and screening
According to the flanking sequences of SNP01 site to SNP09 site provided by the embodiment 1, aiming at each SNP site, two forward primers are designed at the upstream of the SNP site by using Primer3.0 software according to the KASP marker design development principle (target product 80-150 bp, primer is specifically matched with a target region on a reference genome and is positioned in a non-SNP dense region, and a complex sequence region with high A \ T or G \ C content and the like is avoided), and a reverse primer is designed at the downstream.
The tomato sample to be detected comprises part of germplasm resources with known genotypes and root knot nematode resistance phenotypes, 21 randomly selected samples in breeding materials as resistance, infection or heterozygous controls, and finally 3 ddH are added 2 O as NTC blank, 24 parts in total.
DNA extraction adopts a conventional CTAB method or a domestic magnetic bead kit to extract genome DNA from leaves of a tomato sample, utilizes Nanodrop 1000 to measure the concentration of nucleic acid, dilutes and controls the concentration of a DNA template to be 10-20 ng/mu L.
The reaction system for PCR (10. Mu.L) was: 5 mul of DNA template with the concentration of 10-20 ng/. Mu.L; 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 ddH 2 O; 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 MgCl 2 . Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:19, the 5' end of the derivative is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:20, the 3' end of the derivative is connected with a fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown as SEQ ID No:21, wherein 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:22, 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 fluorescent signals at 30 ℃ for 1min; denaturation at 94 deg.C for 15min; denaturation at 94 ℃ for 20s and 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 and annealing at 55 ℃ for 60s, and repeating the step for 28-32 cycles; reading the fluorescent signal for 1min at 30 ℃, analyzing the data result, finally selecting primer combinations (comprising 3 primer groups, namely a primer group 01, a primer group 02 and a primer group 03, wherein SNP information corresponding to each primer group is shown in figure 2, and the size and haplotype information of a corresponding amplified target band 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 the FAM signal, the VIC signal and the heterozygous fluorescent signal, and carrying out follow-up verification and breeding application of the Mi-1 region genotype or haplotype of the large population.
Primer set 01: a forward specific primer A (SEQ ID No: 10), a forward specific primer B (SEQ ID No: 11) and a reverse common primer (SEQ ID No: 12), a primer for amplifying the SNP01 site;
primer set 02: a forward specific primer A (SEQ ID No: 13), a forward specific primer B (SEQ ID No: 14) and a reverse common primer (SEQ ID No: 15), and a primer for amplifying the SNP04 site;
a primer set 03: a forward specific primer A (SEQ ID No: 16), a forward specific primer B (SEQ ID No: 17) and a reverse common primer (SEQ ID No: 18), and a primer for amplifying the SNP08 site.
TABLE 1 primer combination for detecting tomato Mi-1 gene and corresponding SNP site, 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 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 MgCl 2 . Wherein, the nucleotide sequence of the fluorescent probe A is shown as SEQ ID No:19, the 5' end of the derivative is connected with a fluorescent group FAM; the nucleotide sequence of the fluorescent probe B is shown as SEQ ID No:20, the 3' end of the derivative is connected with a fluorescent group VIC or HEX; the nucleotide sequence of the quenching probe A is shown as SEQ ID No:21, wherein 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:22, 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 root-knot nematode resistant gene Mi-1 efficient KASP marker
In this example, based on the SNP sites provided in example 1, the kit (including primer combination) provided in example 2 was used to verify and breed the tomato anti-nematode gene Mi-1 efficient KASP marker.
381 parts of representative tomato samples containing mainstream commercial varieties, national resource library core open germplasm, breeding intermediate materials, new hybridization 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, bunch-harvested tomatoes, fresh-eating tomatoes, processed tomatoes, farmed families (local varieties) and the like, wherein 200 parts of cultivated tomatoes obtain Mi-1 genotype data (comprising 36 parts of tomato germplasm resources and commercial varieties with known root-knot nematode resistance phenotypes, and detailed in Table 2) by utilizing closely-linked SCAR markers through third-party commercial institutions.
Specifically, the embodiment provides a method for detecting resistance of tomato root-knot nematode, which adopts a KASP detection method to perform SNP typing detection on a tomato variety to be detected, and comprises the following steps:
(1) Extracting leaf DNA of a tomato variety to be detected;
(2) Adding a primer mixture and a PCR premix into the leaf DNA of the tomato variety to be detected by using the kit provided in example 2 to perform KASP amplification (the leaf DNA is subjected to KASP amplification by using each primer group in the primer combination provided in example 2);
(3) And detecting the PCR product by adopting a fluorescent quantitative PCR instrument, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
The tomato variety to be detected is 381 parts of germplasm resources and breeding materials containing part of known genotypes and phenotypes, and finally 3 ddH are added 2 O 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 added 2 O。
Editing a sample and primer arrangement template according to an IntelliQube platform operation manual, and executing an operation program {95 ℃ pre-denaturation for 10min; denaturation at 95 ℃ for 20s, annealing extension at 61 ℃ for 60s, wherein the annealing temperature of each cycle is reduced by 0.6 ℃, the annealing temperature is reduced to 55 ℃ for 10 cycles in total, and the final annealing temperature is reduced to 55 ℃; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 28-32 cycles in total }, reading fluorescence data, increasing the number of PCR cycles as appropriate, analyzing and removing partially uncertain data points with too low or abnormal fluorescence values, finally typing large populations as shown in FIG. 3, and deriving the Excel result (as shown in Table 2).
As a result, the 3 sets of KASP markers provided in example 2 obtain highly consistent and well-defined population typing effects in 381 large populations of tomato breeding with different types and sources (as shown in FIG. 3), that is, experiments prove that the SNP site combinations provided in example 1 have good universality and stability in tomato variety resources with different genetic backgrounds. Further, in 36 samples to be tested with known meloidogyne resistance phenotype, the SCAR marker identification genotype and the KASP marker (i.e., primer combination) identification gene data provided by the present invention were completely identical, i.e., identity P =100% (as shown in table 2). In addition, in 200 samples to be tested with reference to the results of linked scarr markers, the identities of the detection results of the 3 primer sets provided in example 2 and the commercial linked scarr markers are P =91.7%, 89.9% and 91.7%, respectively, and the identities P among the 3 primer sets exceed 98% { identity calculation formula is P = (effective detection sample number-difference sample number)/effective detection sample number 100% }. Further analysis shows that the detection results of the same repeated material samples in the SCAR marker detection results are inconsistent (for example, numbers Ta114 and Ta295, ta127 and Ta128, and Ta125 and Ta294 are three groups of same repeated materials, and intentional test design is repeated), but the detection results of the 3 primer groups provided by the invention do not have the phenomenon. The results prove that the detection accuracy and stability of the KASP marker provided by the invention are obviously higher than those of the existing commercial SCAR marker.
In addition, the embodiment also carries out Mi-1 locus genotype detection on the other 181 breeding intermediate materials, and the results show that the genotype detection results of the samples to be detected are basically identical with the pedigree relationship, so that the resistance of the tomato root-knot nematodes of the offspring or the parents of the samples to be detected can be accurately predicted according to the genotype detection results of the Mi-1 loci of the samples to be detected.
In conclusion, the 3 groups of KASP markers provided by the embodiment have good universality and stability in tomato variety resources of different types, sources or genetic backgrounds, the detection results of the markers are basically consistent, the detection accuracy and stability of each group of markers are remarkably superior to those of the existing commercial scarr markers, and each group of markers can be independently applied to molecular detection of tomato root-knot nematode resistance. By analogy, the remaining 6 SNP sites that also meet the characteristics of the aforementioned variation set can be verified by sequencing, gene chips or other PCR markers and achieve the same expected effect. Meanwhile, the KASP markers are combined together, so that the detection accuracy can be further improved, the resistance identification errors caused by experimental errors such as false positives and genetic variation factors such as incomplete selection or partial loss of a target gene region can be avoided, and a better detection and judgment effect is obtained. Therefore, the high-efficiency KASP marker provided by the invention can be directly used for the commercial application of tomato root knot nematode resistant molecular breeding.
TABLE 2 root knot nematode resistance phenotype and marker genotype of 200 tomato germplasm resources, varieties and 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 column "resistant" represents the tomato root knot nematode resistant phenotype, "S" represents susceptible, "R" represents resistant, "IR" represents resistant; wherein, the column of SCAR represents the detection result of Mi-1 gene linkage SCAR marker by third-party commercial institution, "SS" represents homozygous susceptible genotype, "RR" represents homozygous resistant genotype, and "H" represents heterozygous genotype; wherein in the detection result of the primer group 01, T represents homozygous susceptible genotype, C represents homozygous disease-resistant genotype, and C represents heterozygous genotype; in the detection result of the primer group 02, G represents homozygous susceptible genotype, C represents homozygous disease-resistant genotype, and C represents heterozygous genotype; in the detection result of the primer group 03, A: A represents a homozygous susceptible genotype, G: G represents a homozygous disease-resistant genotype, and G: A represents a heterozygous genotype; "-" represents a data miss; the "" -notation represents the inconsistent results between the Mi-1 gene-linked scarr marker and the KASP markers provided herein (i.e., primer combinations) or between different markers within the KASP markers provided herein.
While the preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts and related principles. Therefore, it is intended that the appended claims be interpreted as including 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 made possible within the scope of the claims attached below.
Sequence listing
<110> Shenzhen agricultural genome institute of Chinese academy of agricultural sciences
Institute of agricultural genomics of Chinese academy of agricultural sciences
<120> SNP locus combination for detecting tomato root-knot nematode resistance and application thereof
<130> WK21-HCP-CN1-0352
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Claims (12)
1. A kind ofThe application of the reagent in detecting or assisting in detecting the resistance of the tomato root-knot nematode is characterized in that the reagent is used for detecting the genotype of SNP (single nucleotide polymorphism) sites or SNP site combinations, and the SNP sites or SNP site combinations comprise resistance genes positioned in the tomato root-knot nematodeMi-1One or more of the following SNP01 sites, SNP04 sites and SNP08 sites inside and on both sides:
the SNP01 site is located at 2359017 th nucleotide site on SL2.50 version 06 chromosome of a tomato Heinz 1706 reference genome, and the basic group of the SNP01 site is T or C, wherein T is an infection genotype and C is a disease-resistant genotype;
the SNP04 locus is located at 2357725 th nucleotide locus on SL2.50 version 06 chromosome of tomato Heinz 1706 reference genome, and the basic group of the SNP04 locus is C or G, wherein C is an infection genotype and G is a disease-resistant genotype;
the SNP08 site is located at 2354549 nucleotide site on SL2.50 version 06 chromosome of tomato Heinz 1706 reference genome, and the base of the SNP08 site is A or G, wherein A is a susceptible genotype and G is a disease-resistant genotype.
2. The use of claim 1, wherein the combination of SNP sites further includes one or more of the following SNP sites:
the SNP02 site is located at 2359427 nucleotide site on SL2.50 version 06 chromosome of tomato Heinz 1706, and the base of the SNP02 site is C or T;
the SNP03 site is located at the 2357937 nucleotide site on the SL2.50 version 06 chromosome of the tomato Heinz 1706 reference genome, and the base of the SNP03 site is C or G;
a SNP05 site, which is located at the 2356533 th nucleotide site on the SL2.50 version 06 chromosome of the tomato Heinz 1706 reference genome, and the base of the SNP05 site is A or T;
the SNP06 locus is located at the 2356449 th nucleotide locus on the SL2.50 version 06 chromosome of the tomato Heinz 1706 reference genome, and the base of the SNP06 locus is A or T;
a SNP07 site which is located at 2355234 nucleotide site on chromosome SL2.50 version 06 of tomato Heinz 1706, and the base of the SNP site is A or G;
SNP09, located at 2354363 th nucleotide site on SL2.50 version 06 chromosome of tomato Heinz 1706 reference genome, and the base of the SNP is G or A.
3. The use of claim 2, wherein the SNP01 site to SNP09 site and the respective flanking sequences thereof are respectively set forth in SEQ ID Nos: 1-9, and the SNP01 site to the SNP09 site are respectively positioned in the SEQ ID No: position 102 in the sequence 1-9.
4. The application according to claim 1, characterized in that it comprises:
(1) The application of the compound in preparing products for detecting or assisting in detecting the resistance of tomato root-knot nematodes;
(2) The application in tomato root-knot nematode resistance breeding;
(3) Application in identifying tomato germplasm resources and new varieties.
5. The application according to claim 4, characterized in that it is carried out by means of the following technical measures:
and detecting the polymorphism or the genotype of one or more SNP sites in the SNP site combination by 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 it is carried out by means of the following technical measures:
developing PCR markers and/or gene chips by using sequence information of one or more 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. Use of a primer combination for detecting or assisting in detecting resistance to tomato root knot nematode, or for preparing a product for detecting or assisting in detecting resistance to tomato root knot nematode, or for breeding for resistance to tomato root knot nematode, wherein the primer combination is used for amplifying the SNP site combination of claim 1, and the primer combination comprises one or more of the following primer sets 01-03:
primer set 01: SEQ ID No: 10-12 sequence, primer for amplifying the SNP01 site;
primer set 02: SEQ ID No: 13-15 sequence, primer for amplifying said SNP04 site;
primer set 03: SEQ ID No: 16-18 sequence and primer for amplifying SNP08 site.
8. Use of a kit for detecting or assisting in detecting tomato root knot nematode resistance, or for preparing a product for detecting or assisting in detecting tomato root knot nematode resistance, or for breeding tomato root knot nematode resistance, wherein the kit comprises one or more primer sets in the primer combination of claim 7 in a powdered or liquid state.
9. The use of claim 8, further comprising a PCR premix comprising a fluorescent probe, a quenching probe, a ROX reference dye, klearTaq DNA polymerase, dNTPs, and MgCl 2 。
10. A method for detecting the resistance of tomato root-knot nematodes is characterized in that 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) Performing PCR amplification on the DNAs with the primer combination according to claim 7, respectively;
(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 amplification result, the detection result representing the homozygous disease-resistant genotype is as follows: the detection result of the primer group 01 is C: C. the detection result of the primer group 02 is C: C. the detection result of the primer group 03 is G: g; the test results representing homozygous susceptible genotypes were: the detection result of the primer group 01 is T: t, the detection result of the primer group 02 is G: G. the detection result of the primer group 03 is A: a; the results of the tests representing heterozygous genotypes were: the detection result of the primer group 01 is C: t, the detection result of the primer group 02 is C: G. the detection result of the primer group 03 is G: A.
11. the method of claim 10, wherein the SNP typing assay of the tomato variety to be tested employs a KASP assay comprising:
(1) Adding a primer mixed solution and a PCR premixed solution into the leaf DNA of the tomato variety to be detected, and carrying out KASP amplification;
(2) And detecting the PCR product by adopting fluorescent quantitative equipment, and determining the genotype of the tomato variety to be detected at the SNP site corresponding to each primer group.
12. The method of claim 10, wherein:
the reaction system of the PCR is as follows: 0.8 mu L of template DNA with the concentration of 10-20 ng/. Mu.L; 0.8 mu L of PCR premix; the primer mixture is 0.03 mu L, wherein the final concentration of each primer is 100pmol/L;
the reaction conditions of the PCR are as follows: pre-denaturation at 95 ℃ for 10min; 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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| EP3018205A4 (en) * | 2013-07-05 | 2017-04-19 | Takii & Company Limited | Root-knot nematode resistance marker for tomato plant, root-knot nematode resistant tomato plant, production method for root-knot nematode resistant tomato plant, and screening method for root-knot nematode resistant tomato plant |
| CN105463082B (en) * | 2015-12-10 | 2018-10-30 | 江苏省农业科学院 | A kind of multi-PCR detection method of tomato Mi-1 genes and ty-5 genes |
| US11608507B2 (en) * | 2020-01-06 | 2023-03-21 | Seminis Vegetable Seeds, Inc. | Tomato plants with resistance to Mi-1 resistance-breaking root-knot nematodes |
| CN112522433B (en) * | 2020-12-18 | 2022-05-03 | 北京通州国际种业科技有限公司 | SNP marker closely linked with tomato root knot nematode resistant gene Mi1 and application thereof |
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| CN1952143A (en) * | 2005-10-17 | 2007-04-25 | 华中农业大学 | Cloning of gene against meloidogyne of capsicum and application thereof |
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