A kasp molecular marker linked to a wheat stripe rust resistance gene yrzt97 and application thereof

Abstract

The present application relates to a KASP molecular marker linked to a wheat stripe rust resistance gene YrZT97 and an application thereof, the KASP molecular marker is XK7B-034; the KASP molecular marker is co-located with the wheat stripe rust resistance gene YrZT97 on the 7BL chromosome of wheat, the nucleotide sequence of the XK7B-034 is shown as SEQ ID NO.1, and the 18th base from the 5' end is a SNP site, and the polymorphism of the SNP site is G / A respectively.The KASP molecular marker of the present application is closely linked to the stripe rust resistance gene YrZT97, can accurately identify the wheat stripe rust resistance plant, and has important significance for screening of wheat germplasm resources, creation of wheat stripe rust resistance materials or molecular assisted breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of wheat gene detection, and particularly relates to a KASP molecular marker linked to a wheat stripe rust resistance gene YrZT97 and application thereof. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world, and is the staple food of 35-40% of the world's population. Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. & Henn. (Pst), is an air-borne, biotrophic and obligate fungal disease that is prevalent worldwide and seriously threatens wheat production safety in major wheat-producing countries. Long-term exploration and practice have proved that the use of wheat's own disease resistance traits, the exploration of new disease resistance gene resources, and the rational layout of resistant varieties play a huge role in preventing the occurrence and spread of wheat stripe rust. However, the promotion of resistant varieties is often accompanied by the emergence and development of new wheat stripe rust races.

[0003] With the progress of modern biotechnology and the application of molecular marker technology, 86 stripe rust resistance genes have been reported and officially named. However, most of these resistance genes have "lost" their resistance in China. Currently, only Yr5, Yr15, Yr50, Yr61, and Yr69 have good seedling resistance, but these genes are all from wheat relatives, and the introduction of these genes may be accompanied by some "linkage drag", which greatly limits their application in disease resistance breeding. Therefore, there is an urgent need to explore and utilize new disease resistance gene resources from common wheat materials to further broaden the genetic basis of disease resistance breeding materials. SUMMARY

[0004] The purpose of the present application is to overcome the defects in the prior art and provide a KASP molecular marker linked to a wheat stripe rust resistance gene YrZT97 and application thereof, which provides a basis for breeding excellent disease-resistant varieties using the wheat stripe rust resistance gene YrZT97. To achieve the above purpose, the technical solution adopted by the present application is as follows:

[0005] A wheat stripe rust resistance gene YrZT97, which is located on the 7BL chromosome of wheat and is between the physical positions of 745.3-756.7 Mb in the Chinese Spring reference genome IWGSC RefSeq v2.1 genome version.

[0006] As a further technical solution, the acquisition website of the Chinese spring reference genome IWGSC RefSeq v2.1 genome version is http: / / 202.194.139.32 / download.html.

[0007] The KASP molecular marker linked to the wheat stripe rust resistance gene YrZT97 is XK7B-034; the nucleotide sequence of XK7B-034 is shown in SEQ ID NO. 1, and the 18th base from the 5' end is a SNP site, and the polymorphism of the SNP site is G / A.

[0008] As a further technical solution, XK7B-034 is co-located with the wheat stripe rust resistance gene YrZT97 on the 7BL chromosome of wheat.

[0009] A primer set for amplifying the KASP molecular marker, comprising a susceptible site forward primer XK7B-034-FAM, a resistant site forward primer XK7B-034-HEX, and a common reverse primer XK7B-034-Common; wherein,

[0010] The nucleotide sequence of the susceptible site forward primer XK7B-034-FAM is shown in SEQ ID NO. 2;

[0011] The nucleotide sequence of the resistant site forward primer XK7B-034-HEX is shown in SEQ ID NO. 3;

[0012] The nucleotide sequence of the common reverse primer XK7B-034-Common is shown in SEQ ID NO. 4.

[0013] A wheat stripe rust resistance gene YrZT97 detection kit comprising the primer set of the KASP molecular marker.

[0014] A method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97, characterized in that it comprises the following steps: step (1), extracting the genomic DNA of the wheat material to be tested;

[0015] Step (2), using the primer set of the functional KASP molecular marker of claim 4 or the detection kit of claim 5 to perform PCR amplification on the genomic DNA of the wheat material to be tested, and obtaining an amplification product;

[0016] Step (3), moving the amplification product to a microplate reader to read fluorescence data, and then using KlusterCaller software to analyze the fluorescence data to obtain a genotype;

[0017] When the obtained genotype is a homozygous allele genotype with HEX fluorescence or a heterozygous genotype, then the wheat material to be tested contains the stripe rust resistance gene YrZT97, and when the obtained genotype is a homozygous allele genotype with FAM fluorescence, then the wheat material to be tested does not contain the stripe rust resistance gene YrZT97.

[0018] As a further technical solution, in step (2), the reaction system of the PCR amplification is: 0.2 ug of DNA, 2xKASPv4.0 Master mix 2uL, primer mixture 0.0448uL, ddH2O 1.9552uL;

[0019] In the primer mixture, the concentration of the forward primer is 12uM, and the concentration of the reverse primer is 30uM.

[0020] As a further technical solution, in step (2), the reaction program of the PCR amplification is: 94℃ pre-denaturation for 15min; 94℃ denaturation for 20s, 65℃ annealing for 60s, 10 cycles, and the annealing temperature decreases by 0.6℃ for each cycle; 94℃ denaturation for 20s, 55℃ annealing for 60s, 32 cycles.

[0021] The KASP molecular marker, the primer set or the detection kit is applied in identifying whether the wheat material contains the stripe rust resistance gene YrZT97, detecting the stripe rust resistance of wheat (detecting whether the wheat variety is resistant to stripe rust), screening germplasm resources of the stripe rust resistance gene YrZT97 of wheat, creating a stripe rust resistant wheat material or molecular assisted breeding of the stripe rust resistance of wheat.

[0022] Compared with the prior art, the beneficial effects of the present application are that:

[0023] 1, PI 660060 is a disease-resistant line created by crossing a stripe rust-resistant spring wheat variety from Spain before the 1950s with Avocet S. Through gene mapping, we found that it has an excellent disease-resistant gene YrZT97 on the 7B chromosome of wheat, which can resist CYR31, CYR32 and PST-V26, and the discovery of the gene can provide excellent disease-resistant gene resources for wheat breeding and reduce the damage of stripe rust to wheat during the whole growth and development process, which has great potential application value for improving the stripe rust resistance of wheat in the future.

[0024] 2, The present application finds a specific SNP site that co-segregates with the wheat stripe rust resistance gene YrZT97 and develops a linkage KASP molecular marker XK7B-034 of YrZT97 based on the specific SNP site, which can track the gene YrZT97 and is beneficial to the establishment of a wheat molecular marker assisted breeding system.

[0025] 3、The KASP molecular marker is used for identifying the wheat stripe rust resistance gene YrZT97 according to the application, the detection process is simple, rapid and high in flux, and the wheat stripe rust resistant plant can be accurately identified, so that the application has important significance for screening of wheat germplasm resources, creation of wheat stripe rust resistant materials or molecular assisted breeding of wheat. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The result of the initial positioning of the wheat stripe rust resistance gene YrZT97 on the 7BL chromosome in Example 1 is shown in Table 1.

[0027] Figure 2 The result of the high-density genetic and physical map of YrZT97 in Example 1 of the application is shown in Table 2.

[0028] Figure 3 The result of the genotyping of the XK7B-034 marker on the wheat materials in Example 2 of the application is shown in Table 3.

[0029] Figure 4 The result of the XK7B-034 marker detecting the disease resistance of the wheat materials containing YrZT97 and not containing YrZT97 in Example 2 of the application is shown in Table 4.

[0030] Figure 5 The result of the genotyping of the XK7B-034 marker on the genetic diversity population in Example 3 of the application is shown in Table 5. DETAILED DESCRIPTION

[0031] The technical solutions of the application will be described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0032] Example 1: Genetic positioning of YrZT97 and obtaining of closely linked KASP molecular markers

[0033] The genetic positioning of the wheat stripe rust resistance gene YrZT97 and the obtaining of closely linked KASP molecular markers include the following steps:

[0034] A recombinant inbred line (RIL) population was obtained by selfing the F2 population of the F1 hybrid of the susceptible variety Kenong 9204 and the resistant material PI660060, and was used for genetic analysis and gene mapping. A family with phenotypic segregation was selected from the RIL population as a remaining heterozygous line, and was harvested by single plant. Phenotypic identification was completed in the field inoculation nursery, and the remaining heterozygous line population was labeled for sampling, phenotypic identification and genotyping, and then recombinants were screened, so as to narrow the positioning interval.

[0035] Phenotypic identification used two evaluation indexes of reaction type (IT) and disease severity (DS). IT used a 0-9 grade identification standard (Line and Qayoum 1992), and DS used a 0, 1%, 5%, 10%, 20%, 30%, 40%, 60%, 80% and 100% identification standard (Peterson et al. 1948). Using the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) in QTL IciMapping V4.2, setting the threshold value LOD≥2.5, and using the field resistance phenotype of stripe rust, a QTL was detected and the gene was preliminarily located at the end of the 7BL chromosome. Figure 1 Afterwards, molecular markers in the corresponding interval were developed by GBW16K chip and parental resequencing data, and a secondary segregation population derived from the remaining heterozygous line was used to further narrow the positioning interval of YrZT97, and finally the gene was fine-mapped to a 0.28 cM interval, corresponding to a physical interval of 11.4 Mb. Figure 2

[0036] The KASP molecular marker XK7B-034 was derived from the resequencing data of the parents PI660060 and Kenong 9204, and was co-segregated with YrZT97. The nucleotide sequence of XK7B-034 is shown in SEQ ID NO. 1, specifically:

[0037] 5'-CACTGCGACCCATCAGC[G / A]-3';

[0038] The primer set of the KASP molecular marker XK7B-034 includes:

[0039] The forward primer XK7B-034-FAM of the susceptible site has a nucleotide sequence as shown in SEQ ID NO. 2, specifically: 5'-GAAGGTGACCAAGTTCATGCTCACTGCGACCCATCAGCG-3'; ​

[0040] Anti-disease site forward primer XK7B-034-HEX, the nucleotide sequence of which is shown in SEQ ID NO. 3, specifically: 5'-GAAGGTCGGAGTCAACGGATTCACTGCGACCCATCAGCA-3';

[0041] Common reverse primer XK7B-034-Common, the nucleotide sequence of which is shown in SEQ ID NO. 4, specifically: 5'-GACCGTAAGAGAGATGCGATGT-3'.

[0042] Example 2: Genotyping method of XK7B-034 and selection of disease-resistant wheat

[0043] The genotyping method of XK7B-034 comprises the following steps:

[0044] Step 1, extracting the genomic DNA of the wheat material to be tested by the CTAB method as the DNA amplification template;

[0045] Step 2, using the primer set of KASP molecular marker XK7B-034 obtained in Example 1, the genomic DNA of the wheat material to be tested is used as the template to perform fluorescent quantitative PCR amplification on the genomic DNA of the wheat material to be tested, and the amplification product is obtained;

[0046] The reaction system of the fluorescent quantitative PCR amplification is: 0.2 μg of DNA template, 2 μL of KASPV4.0 Master mix, 0.0448 μL of primer mixture, and 1.9552 μL of ddH2O.

[0047] In the primer mixture, the concentrations of XK7B-034-FAM and XK7B-034-HEX are both 12 μM, and the concentration of XK7B-034-Common is 30 μM;

[0048] The reaction program of the fluorescent quantitative PCR amplification is: 94℃ pre-denaturation for 15 min; 94℃

[0049] denaturation for 20 s, 65℃ annealing for 60 s, 10 cycles, and the annealing temperature decreases by 0.6℃ for each cycle; 94℃ denaturation for 20 s, 55℃ annealing for 60 s, 32 cycles.

[0050] Step 3, the amplification product is moved to a microplate reader to read the fluorescence data, and then the fluorescence data is imported into KlusterCaller software for analysis to obtain genotype data and obtain the genotype; according to the genotype result of the amplification product, it is judged whether the plant to be tested is resistant to stripe rust Figure 3 ) or not.

[0051] The homozygous allele genotype with HEX fluorescence is "Y:Y", the homozygous allele genotype with FAM fluorescence is "X:X", and the heterozygous genotype is "X:Y" or "Y:X";

[0052] If the genotype is "Y:Y" or "X:Y" or "Y:X", then the wheat material to be tested contains the stripe rust resistance gene YrZT97. If the genotype is "X:X", then the wheat material to be tested does not contain the stripe rust resistance gene YrZT97.

[0053] The wheat to be tested containing YrZT97 and the wheat to be tested not containing YrZT97 do indeed have differences in phenotype, and the resistance of the wheat containing YrZT97 is obviously higher than that of the wheat not containing YrZT97. Figure 4

[0054] Example 3: Application in a genetically diverse population

[0055] It is of guiding significance for the application of these germplasms in stripe rust resistance breeding to determine whether the wheat disease-resistant germplasm resources contain the stripe rust resistance gene YrZT97.

[0056] In this example, the KASP molecular marker XK7B-034 described in Example 1 and the method described in Example 2 are used to detect wheat stripe rust disease-resistant germplasm resources composed of domestic and foreign family varieties, lines, and some known gene carriers. The results show that a total of 6 wheat germplasms contain YrZT97 Figure 5 ), and the wheat to be tested containing YrZT97 and the wheat to be tested not containing YrZT97 do indeed have differences in phenotype, and the resistance of the wheat containing YrZT97 is obviously higher than that of the wheat not containing YrZT97; these results show that the XK7B-034 marker can be used as a functional diagnostic marker for screening of wheat stripe rust resistance gene YrZT97 germplasm resources.

[0057] The above-described embodiments are only preferred embodiments of the present application, and are not exhaustive of the feasible implementations of the present application. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present application should be considered to be included in the protection scope of the claims of the present application.​

Claims

1. A KASP molecular marker linked to the wheat stripe rust resistance gene YrZT97, characterized in that, The wheat stripe rust resistance gene YrZT97 is located on the 7BL chromosome of wheat, within the 0.19-0.47 cM interval of the Chinese Spring reference genome IWGSC RefSeq v2.1 genome version, corresponding to a physical location of 745.3-756.7 Mb; the KASP molecular marker is XK7B-034; the nucleotide sequence of XK7B-034 is shown as SEQ ID NO. 1, and the 18th base from the 5' end is a SNP site, and the polymorphism of the SNP site is G / A.

2. A primer set for amplifying the KASP molecular marker of claim 1, characterized in that, The primer set comprises the susceptible site forward primer XK7B-034-FAM, the resistant site forward primer XK7B-034-HEX, and the common reverse primer XK7B-034-Common; wherein, The nucleotide sequence of the susceptible site forward primer XK7B-034-FAM is shown as SEQ ID NO. 2; The nucleotide sequence of the resistant site forward primer XK7B-034-HEX is shown as SEQ ID NO. 3; The nucleotide sequence of the common reverse primer XK7B-034-Common is shown as SEQ ID NO.

4.

3. A detection kit of the KASP molecular marker according to claim 1, characterized in that, The primer set comprises the KASP molecular marker of claim 2.

4. A method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97, characterized in that, The wheat stripe rust resistance gene YrZT97 is located on the 7BL chromosome of wheat, within the 0.19-0.47 cM interval of the Chinese Spring reference genome IWGSC RefSeq v2.1 genome version, corresponding to a physical location of 745.3-756.7 Mb; The method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97 comprises the following steps: step (1), extracting the genomic DNA of the wheat material to be tested; Step (2), using the primer set of the KASP molecular marker of claim 2 or the detection kit of claim 3 to perform PCR amplification on the genomic DNA of the wheat material to be tested, to obtain an amplification product, using the genomic DNA of the wheat material to be tested as a template; Step (3), moving the amplification product to a microplate reader to read fluorescence data, and then using KlusterCaller software to analyze the fluorescence data to obtain a genotype; When the obtained genotype is a homozygous allele genotype with HEX fluorescence or a heterozygous genotype, it indicates that the wheat material to be tested contains the stripe rust resistance gene YrZT97, and when the obtained genotype is a homozygous allele genotype with FAM fluorescence, it indicates that the wheat material to be tested does not contain the stripe rust resistance gene YrZT97.

5. The method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97 according to claim 4, characterized in that, In step (2), the reaction system for PCR amplification is: DNA 0.2 μg, 2xKASPv4.0 Master mix 2 μL, primer mixture 0.0448 μL, and ddH2O 1.9552 μL. The concentration of the forward primer in the primer mixture is 12 μM, and the concentration of the reverse primer is 30 μM.

6. The method according to claim 4, characterized in that, In step (2), the reaction procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s, annealing at 65 ℃ for 60 s, 10 cycles, and decreasing the annealing temperature by 0.6 ℃ for each cycle; denaturation at 94 ℃ for 20 s, annealing at 55 ℃ for 60 s, 32 cycles.

7. Use of the primer set of claim 2 or the detection kit of claim 3 in identifying whether a wheat material contains the stripe rust resistance gene YrZT97, detecting a stripe rust resistance trait of wheat, screening of germplasm resources of the stripe rust resistance gene YrZT97 of wheat, or molecular-assisted breeding of stripe rust resistance of wheat, characterized in that, Genotype detection is performed on the wheat to be tested, and when the obtained genotype is a homozygous allele genotype with HEX fluorescence or a heterozygous genotype, it is determined that the wheat material to be tested contains the stripe rust resistance gene YrZT97; when the obtained genotype is a homozygous allele genotype with FAM fluorescence, it is determined that the wheat material to be tested does not contain the stripe rust resistance gene YrZT97, and the wheat containing the stripe rust resistance gene is selected for breeding.

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