KASP molecular marker linked with wheat stripe rust resistance gene YrZT97 and application of KASP molecular marker

Abstract

The invention relates to a KASP molecular marker linked with a wheat stripe rust resistance gene YrZT97 and application of the KASP molecular marker. The KASP molecular marker is XK7B-034; the KASP molecular marker and a wheat stripe rust resistance gene YrZT97 are co-localized on a wheat 7BL chromosome, the nucleotide sequence of XK7B-034 is as shown in SEQ ID NO.1, the 18th basic group from the 5'end of the nucleotide sequence is an SNP site, and the polymorphism of the SNP site is G / A. The KASP molecular marker provided by the invention is closely linked with the stripe rust resistant gene YrZT97, can accurately identify wheat stripe rust resistant plants, and is of great significance to wheat germplasm resource stripe rust resistant gene screening, wheat stripe rust resistant material creation or molecular assisted breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat gene detection, and in particular relates to a KASP molecular marker linked to a wheat stripe rust resistance gene YrZT97 and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world and the staple food of 35%-40% of the world's population. Wheat stripe rust is caused by the wheat-specific type of Puccinia striiformis West.f.sp.triticiEriks.&Henn. (Pst). It is an airborne, living, obligately parasitic fungal disease that is prevalent worldwide and seriously threatens the safety of wheat production in the world's major wheat-producing countries. Long-term exploration and practice have proved that utilizing wheat's own disease resistance traits, discovering new disease-resistant gene resources, and rationally arranging resistant varieties have a huge effect on preventing and controlling the occurrence and spread of wheat stripe rust. However, the promotion of resistant varieties is often accompanied by the generation and development of new physiological races of wheat stripe rust.

[0003] With the advancement of modern biotechnology and the application of molecular marker technology, 86 stripe rust resistance genes have been reported and officially named. However, despite this, most stripe rust resistance genes have "lost" resistance in my country. At present, the only genes that still have good seedling resistance are Yr5, Yr15, Yr50, Yr61, Yr69, etc., but the above genes are all from closely related species of wheat. When introducing disease-resistant genes, there may often be some "linkage drag", which greatly limits the application of the above genes in disease-resistant breeding. Therefore, there is an urgent need to discover and utilize new disease-resistant gene resources from common wheat materials to further broaden the genetic basis of disease-resistant breeding materials. Summary of the invention

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

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

[0006] As a further technical solution, the Chinese Spring reference genome IWGSC RefSeq v2.1 genome version is available at http: / / 202.194.139.32 / download.html.

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

[0008] As a further technical solution, the XK7B-034 and the wheat stripe rust resistance gene YrZT97 are co-localized on the wheat chromosome 7BL.

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

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

[0011] The nucleotide sequence of the disease resistance 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 detection kit for wheat stripe rust gene YrZT97 comprises a primer set of the KASP molecular marker.

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

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

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

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

[0018] As a further technical solution, in step (2), the reaction system for PCR amplification is: 0.2 μg DNA, 2 μL 2×KASPv4.0 Master mix, 0.0448 μL primer mixture, ddH 2 O 1.9552 μL;

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

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

[0021] The KASP molecular marker, the primer set or the detection kit are used in identifying whether wheat materials contain the stripe rust resistance gene YrZT97, detecting wheat stripe rust resistance traits (detecting whether wheat varieties are resistant to stripe rust), screening germplasm resources of the wheat stripe rust resistance gene YrZT97, creating stripe rust resistance wheat materials or molecular assisted breeding of wheat stripe rust resistance.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. PI 660060 is a disease-resistant line created by hybridizing 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 on wheat chromosome 7B that is resistant to CYR31, CYR32, and PST-V26, and named it YrZT97. Its discovery can provide excellent disease-resistant gene resources for wheat disease-resistant breeding, reduce the damage of stripe rust in the entire growth and development process of wheat, and has great potential application value for improving wheat stripe rust resistance in the future.

[0024] 2. Through research, the present invention discovered a specific SNP site that is co-segregated with the wheat stripe rust resistance gene YrZT97 and developed a linkage KASP molecular marker XK7B-034 of YrZT97 based on the specific SNP site, which can track the gene YrZT97 and is conducive to the establishment of a wheat molecular marker-assisted breeding system.

[0025] 3. The present invention uses KASP molecular markers to identify the wheat stripe rust resistance gene YrZT97. The detection process is simple, rapid, and high-throughput, and can accurately identify plants resistant to wheat stripe rust. It is of great significance for screening wheat germplasm resources for stripe rust resistance genes, creating wheat stripe rust resistance materials, or wheat molecular-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preliminary localization result of wheat stripe rust resistance gene YrZT97 on chromosome 7BL in Example 1;

[0027] Figure 2 The high-density genetic and physical map results of YrZT97 in Example 1 of the present invention;

[0028] Figure 3 The genotyping results of wheat materials using the marker XK7B-034 in Example 2 of the present invention;

[0029] Figure 4 The results of detecting disease resistance of wheat materials containing YrZT97 and not containing YrZT97 by using the XK7B-034 marker in Example 2 of the present invention;

[0030] Figure 5 This is the genotyping result of the XK7B-034 marker on the genetic diversity population in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1: Genetic localization of YrZT97 and acquisition of tightly linked KASP molecular markers

[0033] A method for genetically locating a wheat stripe rust resistance gene YrZT97 and obtaining a tightly linked KASP molecular marker comprises the following steps:

[0034] The F cultivar was obtained by crossing Kenong 9204, a cultivar susceptible to stripe rust, with PI660060, a resistant cultivar, as the male parent. 1 , F 1 Self-pollination to obtain F 2 , F 2The single plant is continuously self-pollinated and the recombinant inbred line (RIL) is obtained by the "single seed transmission" method for genetic analysis and gene positioning. The families with phenotype separation are selected from the recombinant inbred line population as the remaining heterozygous lines, and the single plant is harvested. The phenotypic identification is completed in the field seed nursery, and the remaining heterozygous line population is sampled, phenotypic identification and genotyping are carried out to screen the recombinants, thereby narrowing the positioning interval.

[0035] Phenotypic identification used two evaluation indicators: infection type (IT) and disease severity (DS). IT used a 0-9 level 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). The Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTLIciMapping V4.2 was used to set the threshold LOD ≥ 2.5, and the field resistance phenotype of stripe rust was used to detect QTL. The gene was preliminarily located at the end of chromosome 7BL ( Figure 1 ). Then, molecular markers in the corresponding interval were developed using GBW16K chip and parental resequencing data, and the secondary segregation population derived from the remaining heterozygous lines was used to further narrow the positioning interval of YrZT97. Finally, the gene was finely located within the 0.28cM interval, corresponding to a physical interval of 11.4Mb ( Figure 2 ).

[0036] KASP molecular marker XK7B-034 was derived from the resequencing data of the parent 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 KASP molecular marker XK7B-034 includes:

[0039] The nucleotide sequence of the forward primer XK7B-034-FAM of the susceptible locus is shown in SEQ ID NO.2, specifically: 5′-GAAGGTGACCAAGTTCATGCTCACTGCGACCCATCAGCG-3′;

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

[0041] The common reverse primer XK7B-034-Common has a nucleotide sequence as 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: Using the CTAB method to extract the genomic DNA of the wheat material to be tested as a template for DNA amplification;

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

[0046] The reaction system for fluorescence quantitative PCR amplification was: 0.2 μg DNA template, 2 μL 2×KASPV4.0 Master mix, 0.0448 μL primer mixture, ddH 2 O 1.9552 μL.

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

[0048] The reaction procedure of fluorescence quantitative PCR amplification was as follows: pre-denaturation at 94°C for 15 min;

[0049] Denaturation for 20 s, annealing at 65°C for 60 s, 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 32 cycles.

[0050] Step 3: Move the amplified product to an ELISA reader to read the fluorescence data, and then import the fluorescence data into KlusterCaller software for analysis to obtain genotype data and obtain the genotype; determine whether the plant to be tested is resistant to stripe rust according to the genotype result of the amplified product ( Figure 3 ).

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

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

[0053] The tested wheat containing YrZT97 and the tested wheat without YrZT97 did have phenotypic differences. The resistance of the wheat containing YrZT97 was significantly higher than that of the wheat without YrZT97 ( Figure 4 ).

[0054] Example 3: Application in genetically diverse populations

[0055] Clarifying whether disease-resistant wheat germplasm resources contain the stripe rust resistance gene YrZT97 will have guiding significance for the application of these germplasms in stripe rust resistance breeding.

[0056] In this example, the KASP molecular marker XK7B-034 described in Example 1 and the method described in Example 2 were used to perform molecular detection on wheat stripe rust resistant germplasm resources consisting of domestic and foreign farmer species, varieties (lines), and carrier varieties (lines) of some known genes. The results showed that a total of 6 wheat germplasms contained YrZT97 ( Figure 5 ), and there were indeed phenotypic differences between the tested wheat containing YrZT97 and the tested wheat without YrZT97. The resistance of the wheat containing YrZT97 was significantly higher than that of the wheat without YrZT97. These results showed that the XK7B-034 marker could be used as a functional diagnostic marker in the germplasm screening of the wheat stripe rust resistance gene YrZT97.

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

Claims

1. A wheat stripe rust resistance gene YrZT97, characterized in that: The wheat stripe rust resistance gene YrZT97 is located on the wheat chromosome 7BL, within the 0.19-0.47 cM interval of the Chinese Spring reference genome IWGSC RefSeq v2.1 genome version, and the corresponding physical position is between 745.3-756.7 Mb.

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

3. The KASP molecular marker according to claim 2, characterized in that: The XK7B-034 and the wheat stripe rust resistance gene YrZT97 are co-localized on the wheat chromosome 7BL.

4. A primer set for amplifying the KASP molecular marker as claimed in claim 3, characterized in that: It includes a forward primer XK7B-034-FAM for the susceptible site, a forward primer XK7B-034-HEX for the resistant site, and a common reverse primer XK7B-034-Common; wherein, The nucleotide sequence of the forward primer XK7B-034-FAM of the susceptible locus is shown in SEQ ID NO.2; The nucleotide sequence of the disease resistance site forward primer XK7B-034-HEX is shown in SEQ ID NO.3; The nucleotide sequence of the common reverse primer XK7B-034-Common is shown in SEQ ID NO.

4.

5. A detection kit for wheat stripe rust gene YrZT97, characterized in that: A primer set comprising the KASP molecular marker according to claim 3.

6. A method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97, characterized in that: The method comprises the following steps: step (1), extracting genomic DNA of the wheat material to be tested; Step (2), using the genomic DNA of the wheat material to be tested as a template, and using the primer set of the KASP molecular marker described in claim 4 or the detection kit described in claim 5 to perform PCR amplification on the genomic DNA of the wheat material to be tested to obtain an amplified product; Step (3), moving the amplified product to a microplate reader to read the fluorescence data, and then using KlusterCaller software to analyze the fluorescence data to obtain the genotype; When the obtained genotype is a homozygous allele genotype with HEX fluorescence or a heterozygous genotype, the tested wheat material contains the stripe rust resistance gene YrZT97; when the obtained genotype is a homozygous allele genotype with FAM fluorescence, the tested wheat material does not contain the stripe rust resistance gene YrZT97.

7. The method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97 according to claim 6, characterized in that: In step (2), the reaction system for PCR amplification is: 0.2 μg DNA, 2 μL 2×KASPv4.0 Master mix, 0.0448 μL primer mixture, 1.9552 μL ddH2O; In the primer mixture, the forward primer concentration is 12 μM, and the reverse primer concentration is 30 μM.

8. The method for identifying whether a wheat material contains the stripe rust resistance gene YrZT97 according to claim 6, characterized in that: In step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 65°C for 60 s, 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, 32 cycles.

9. Use of the KASP molecular marker according to claim 2 or 3, the primer set according to claim 4 or the detection kit according to claim 4 in identifying whether wheat materials contain the stripe rust resistance gene YrZT97, detecting wheat stripe rust resistance traits, screening germplasm resources of the wheat stripe rust resistance gene YrZT97, creating stripe rust resistance wheat materials or molecular assisted breeding of wheat stripe rust resistance.

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