KASP molecular marker linked with stripe rust resistance gene QYr.CM62-2BL of Chuanmai 62 and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker linked with a stripe rust resistance gene QYr.CM62-2BL of Chuan wheat 62 and application of the KASP molecular marker. The KASP molecular marker comprises KASP-AX-109507945 and KASP-AX-109844182, wherein the nucleotide sequences of the KASP-AX-109507945 and the KASP-AX-109844182 are respectively shown as SEQ ID NO.1 and SEQ ID NO.2. The invention further discloses a preparation method of the KASP molecular marker. According to the invention, a recombinant inbred line group constructed by Chuanmai 62 and a susceptible variety Avock S is combined with SNP (Single Nucleotide Polymorphism) chip genotyping and multi-environment adult-plant-stage stripe rust resistance identification, QTL (Quantitative Trait Loci) positioning analysis is carried out, and a stable major stripe rust resistance new locus QYr.CM62-2BL in multiple environments is identified. According to the present invention, the primer is developed by using the QYr.CM62-2BL close linkage KASP molecular marker, and the rapid and accurate genotype identification is performed on the disease-resistant site;

Description

Technical Field

[0001] This invention relates to a molecular marker linked to a stripe rust resistance gene, specifically a marker linked to the stripe rust resistance gene of Chuanmai 62. QYr.CM62-2BL Linked KASP molecular markers and their applications. Background Technology

[0002] Wheat is one of the world's most important food crops, and its safe production is crucial to ensuring global food security. It is produced by *Stripetracus rust* (…). Puccinia striiformis f. sp. tritici , Pst Wheat stripe rust, caused by an airborne fungus, is a devastating disease that can lead to a 10-30% reduction in wheat yield in epidemic years, and in severe cases, even total crop failure, posing a continuous threat to wheat production.

[0003] Currently, the control of wheat stripe rust mainly relies on chemical pesticides and the planting of disease-resistant varieties. However, the long-term and large-scale use of chemical pesticides not only increases production costs but also leads to environmental pollution and pesticide residues. Therefore, breeding and promoting disease-resistant varieties is widely recognized as the most economical, effective, and environmentally friendly control strategy.

[0004] Wheat resistance to stripe rust is mainly divided into two categories: all-stage resistance (also known as seedling resistance / major gene resistance) and adult-plant resistance (also known as durable resistance / minor gene resistance). All-stage resistance (ASR) is usually controlled by major resistance genes, exhibiting high resistance or immunity. However, its resistance is easily "lost" due to variations in the physiological races of the pathogen; that is, resistant varieties may become susceptible after several years of promotion due to the emergence of new virulent races, resulting in poor resistance durability. In contrast, adult-plant resistance (APR) typically manifests in the mid-to-late stages of plant growth (mature stage), is mostly controlled by multiple minor genes, and has quantitative inheritance characteristics. Although the resistance effect of a single gene in adult-plant resistance is relatively weak, the aggregation of multiple genes can provide durable, broad-spectrum horizontal resistance, which is not easily lost due to changes in pathogen races, and is key to achieving sustained and effective control of wheat stripe rust.

[0005] Despite the significant advantages of resistance at the adult stage, its application in traditional breeding faces substantial challenges. First, phenotypic identification of resistance at the adult stage must be conducted in the field at this stage, and is significantly affected by environmental conditions (such as temperature and humidity). This process is time-consuming, labor-intensive, and requires significant land, with accuracy difficult to guarantee. Second, in hybrid offspring, superior resistance genes at the adult stage are easily lost in segregating populations, making them difficult to effectively track and aggregate through conventional phenotypic selection. These factors severely restrict the breeding efficiency of high-yielding, long-lasting disease-resistant wheat varieties.

[0006] Marker-assisted selection (MAS) technology provides a powerful tool for overcoming the bottlenecks of traditional breeding. MAS technology enables rapid, accurate, and environmentally unaffected genotyping of breeding materials at early stages of crop development (such as the seedling stage) using DNA molecular markers closely linked to target genes, thereby achieving direct selection of target genes and greatly improving breeding efficiency.

[0007] Currently, several molecular markers linked to wheat stripe rust resistance genes (including some adult-stage resistance genes) have been developed, such as SSR (simple sequence repeat) markers and STS (sequence tag site) markers based on gel electrophoresis. However, these markers have certain limitations in practical applications:

[0008] (1) Low throughput and low efficiency: For example, SSR markers usually require gel electrophoresis for detection, which is complicated and has limited throughput, making it difficult to meet the needs of large-scale breeding population screening.

[0009] (2) Limited polymorphism: In some breeding materials with similar genetic backgrounds, existing markers may lack polymorphism and cannot effectively distinguish different genotypes.

[0010] (3) Insufficient linkage with the target gene: Some reported markers have a certain genetic distance from the target resistance gene. During the breeding process, the marker may separate from the target gene due to chromosome exchange (recombination), leading to selection errors.

[0011] Single nucleotide polymorphism (SNP) markers, as third-generation molecular markers, possess significant advantages such as abundant quantity, genome-wide distribution, high detection throughput, and ease of automation. In particular, SNP markers based on competitive allele-specific PCR (KASP) technology have become the mainstream MAS technology in modern molecular breeding. KASP markers are characterized by accurate genotyping, relatively low cost, and compatibility with high-throughput platforms, making them highly suitable for large-scale application in breeding projects. Therefore, there is an urgent need in the current technological field to discover SNP markers closely linked to new and important adult-stage resistance genes, especially to develop KASP markers that can stably, accurately, and efficiently track and aggregate these superior resistance genes, in order to accelerate the breeding process of new wheat varieties with durable and broad-spectrum resistance to stripe rust.

[0012] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0013] The purpose of this invention is to provide a gene for resistance to stripe rust in Chuanmai 62 wheat. QYr.CM62-2BL Linked KASP molecular markers and their applications address the problems of low marker throughput, limited polymorphism, and insufficient linkage with target genes in existing technologies. This invention can efficiently and accurately detect markers containing stripe rust resistance loci. QYr.CM62-2BL This marker can also be used for high-throughput molecular marker-assisted selection of wheat lines, effectively improving breeding efficiency and serving molecular breeding for wheat disease resistance. It has important practical significance in molecular marker-assisted selection breeding of stripe rust resistance genes.

[0014] To achieve the above objectives, this invention provides a gene for resistance to stripe rust in Chuanmai 62 wheat. QYr.CM62-2BL Linked KASP molecular markers, comprising KASP-AX-109507945 and KASP-AX-109844182; the nucleotide sequence of KASP-AX-109507945 is shown in SEQ ID NO.1; the nucleotide sequence of KASP-AX-109844182 is shown in SEQ ID NO.2.

[0015] The second objective of this invention is to provide a gene for resistance to stripe rust in Chuanmai 62 wheat. QYr.CM62-2BL A primer set linked to the KASP molecular marker, comprising: a primer for the molecular marker KASP-AX-109507945 and a primer for the molecular marker KASP-AX-109844182; the primer sequences for the molecular marker KASP-AX-109507945 are shown in SEQ ID NO. 3-5; and the primer sequences for the molecular marker KASP-AX-109844182 are shown in SEQ ID NO. 6-8.

[0016] A third objective of this invention is to provide a gene for resistance to stripe rust in Chuanmai 62 wheat. QYr.CM62-2BL A kit for linked KASP molecular markers, the kit containing the primer combination described above.

[0017] A fourth objective of this invention is to provide the application of the KASP molecular marker, the primer combination, or the kit in screening for stripe rust resistant lines or in assisting molecular breeding for wheat resistance to stripe rust.

[0018] The fifth objective of this invention is to provide a screening method for products containing the stripe rust resistance gene of Chuanmai 62. QYr.CM62-2BL A method for determining wheat varieties, the method comprising: Using genomic DNA from the plant samples as templates, quantitative real-time PCR amplification was performed using primers as described in claim 2 for the molecular marker KASP-AX-109507945 and for the molecular marker KASP-AX-109844182, respectively. Genotyping of the wheat samples was then performed based on the PCR amplification results. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are GG and TT, then the plant sample to be tested is a stripe rust resistant strain. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are TT and CC, then the plant sample to be tested is a stripe rust susceptible strain.

[0019] Preferably, the fluorescence quantitative PCR amplification system comprises: DNA template, 2×KASP Master Mix, primer mixture and ultrapure water; wherein, the primer mixture is a mixture of primers with sequences as shown in SEQ ID NO. 3~5 or a mixture of primers with sequences as shown in SEQ ID NO. 6~8.

[0020] More preferably, the fluorescence quantitative PCR amplification system comprises: 2.0 μL of DNA template with a concentration of 50 ng / μL, 4.5 μL of 2×KASP Master Mix, 2.0 μL of primer mixture, and 1.5 μL of ultrapure water; wherein, in the primer mixture, the volume ratio of the upstream primer containing FAM, the upstream primer containing HEX, the downstream primer, and the ultrapure water is 6: 6: 15: 23; wherein, the concentration of each primer is 100 μM.

[0021] Preferably, the procedure for the real-time PCR amplification is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, annealing and extension at 61-55℃ for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.6℃ per cycle; 95℃ denaturation for 20 s, 55℃ annealing and extension for 40 s, 35 cycles.

[0022] The present invention relates to the stripe rust resistance gene of Chuanmai 62. QYr.CM62-2BL Linked KASP molecular markers and their applications solve the problems of low marker throughput, limited polymorphism, and insufficient linkage with target genes in existing technologies, and have the following advantages: (1) This invention utilizes a recombinant inbred line population constructed from Chuanmai 62 and the susceptible variety Avocet S, combined with 55K SNP chip genotyping and multi-environment adult stripe rust resistance identification, to conduct QTL mapping analysis, identifying a new stable major stripe rust resistance locus in Chuanmai 62 under multiple environments. QYr.CM62-2BLThis locus is located on the long arm of chromosome 2B, in the region between 151.67 and 154.06 cM. This invention utilizes... QYr.CM62-2BL Primers were developed using tightly linked KASP molecular markers to enable rapid and accurate genotyping of this resistance locus.

[0023] (2) The technology of the present invention can accurately screen target genes in wheat seedling stage, avoiding the defects of traditional field identification which is time-consuming, labor-intensive and easily affected by environmental interference. Through the high-throughput KASP detection platform, the breeding efficiency is significantly improved. Attached Figure Description

[0024] Figure 1 The figure shows the genotyping of the parental varieties Chuanmai 62 and Avocet S and their F9 populations using the KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182. In the figure, blue indicates the genotype consistent with the stripe rust resistant variety Chuanmai 62; red indicates the genotype consistent with the stripe rust susceptible variety Avocet S; and black indicates the blank control.

[0025] Figure 2 The genotyping diagrams of 95 varieties / lines developed in the middle and upper reaches of the Yangtze River were obtained using the KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182 for this invention. In the diagrams, red indicates the genotype consistent with the stripe rust resistant variety Chuanmai 62; blue indicates the genotype consistent with the stripe rust susceptible variety Avocet S; and black indicates the blank control. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0028] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0029] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0030] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0031] Chuanmai 62 is a wheat variety bred by the Crop Research Institute of the Sichuan Academy of Agricultural Sciences and approved by Sichuan Province for its long-lasting resistance to stripe rust. This invention utilizes a recombinant inbred line population (RIL, n=281) constructed from Chuanmai 62 and the susceptible variety Avocet S. Combined with 55K SNP chip genotyping and stripe rust resistance identification at the adult stage in multiple environments (Xindu, Pixian, Mianyang, Leshan, Jintang, and Xichang), QTL mapping analysis was conducted, identifying a novel, stable, major stripe rust resistance locus in Chuanmai 62 under multiple environments. QYr.CM62-2BL This locus is located on the long arm of chromosome 2B, in the region between 151.67 and 154.06 cM. Currently, there is no information regarding... QYr.CM62-2BL Reports of tightly linked KASP molecular markers have hindered rapid and efficient selection of this resistance site, severely limiting its application in wheat disease resistance breeding. This invention utilizes... QYr.CM62-2BL Primers were developed using tightly linked KASP molecular markers to enable rapid and accurate genotyping of this disease resistance locus. These primers, designed based on competitive allele-specific PCR (KASP) technology, offer advantages such as accurate genotyping, high throughput, and relatively low cost, enabling genotyping at the wheat seedling stage. QYr.CM62-2BL Precise detection of the site eliminates the need to wait for the resistance phenotype to appear in mature plants, thus greatly shortening the breeding cycle and improving breeding efficiency.

[0032] The following examples illustrate the interaction between the stripe rust resistance gene provided by this invention and the Chuanmai 62 wheat variety. QYr.CM62-2BL The linkage of KASP molecular markers and their applications are explained in detail.

[0033] Example 1 and the stripe rust resistance site of Chuanmai 62 QYr.CM62-2BL Development of linked KASP molecular markers 1. Materials and Methods The disease-resistant wheat variety Chuanmai 62 was used as the female parent and the disease-susceptible variety Avocet S was used as the male parent to obtain the F1 hybrid generation. After self-pollination of the F1 generation, the F2 generation was obtained. The single-seed propagation method was used to continuously increase the generation to the F9 generation, and finally a recombinant inbred line (RIL) population consisting of 281 individual plants was constructed, which was used as a genetic mapping population.

[0034] 2. Identification of stripe rust resistance in mature plants Field identification of stripe rust resistance at the mature plant stage was conducted in the stripe rust identification nursery from 2016 to 2022 in Xindu (16XD, 18XD, 19XD, 20XD, 21XD, and 22XD, with year + location abbreviations representing different environments), 2021 in Jintang (21JT), 2021 in Leshan (21LS), 2021 in Mianyang (21MY), 2021 in Pixian (21PX), 2021 in Xichong (21XC), 2022 in Jintang (22JT), and 2022 in Xichong (22XC). Two replicates were planted for each environment, with a row length of 1m, row spacing of 0.3m, and plant spacing of 0.1m. A row of susceptible material Avocet S was planted every 20 rows as an inducer, and susceptible materials Chuanmai 24 and SY95-71 were planted around the perimeter as protection rows. Before the wheat jointing stage, apply an appropriate amount of mixed bacterial strains (such as Strip 32, Strip 33, and Strip 34) to the induced and protected row materials using the smear method. When the susceptible control is fully infected (Avocet S flag leaf severity reaches 60%–100%), starting in mid-to-late March, during the peak period of stripe rust, investigate the severity and reactivity of the tested materials.

[0035] Disease severity (DS) (%) refers to the percentage of the area of ​​uredinia on diseased leaves relative to the total leaf area, and is categorized as follows: 0%, 5%, 10%, 20%, 40%, 60%, 80%, and 100%. The severity assessment standards are based on GB / T 15795-2011 "Technical Specification for Monitoring and Forecasting of Wheat Stripe Rust," where a DS of less than 60% indicates resistance (R), and a DS of greater than 60% indicates susceptibility (S).

[0036] 3. Wheat 55K SNP chip typing and QTL positioning Genomic DNA was extracted from the Chuanmai 62, Avocet S, and RIL populations using a modified CTAB method (Murray and Thompson, 1980). Genotyping analysis of the sample DNA was performed by Zhongyu Jin Marker (Beijing) Biotechnology Co., Ltd. Genetic maps were constructed based on the genotyping results from the wheat 55K SNP chip using QTL IciMapping 4.2 software.

[0037] Based on the disease resistance phenotype data of the adult plants in various environments, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping 4.2 was used. With a LOD threshold of ≥3, QTL detection was performed in 13 environments from 2016 to 2022, locating a stripe rust resistance locus in Chuanmai 62. QYr.CM62-2BL This site is located on the long arm of chromosome 2B in the range of 151.67-154.06 cM.

[0038] 4. Resistance to stripe rust sites QYr.CM62-2BL Development of chain tags Based on the wheat 55K SNP microarray data localization results, KASP molecular markers were designed using PolyMarker software (https: / / www.polymarker.info / ) according to the polymorphic sites of SNP markers, and primer specificity was detected using the EnsemblPlants website (https: / / plants.ensembl.org / ).

[0039] KASP markers were used to amplify genomic DNA from the resistant parent Chuanmai 62, the susceptible parent Avocet S, and the RIL population. The resistant and susceptible materials were clearly genotyped, and the two molecular markers were named KASP-AX-109507945 and KASP-AX-109844182, respectively. Primers were designed based on the sequences of KASP-AX-109507945 (SEQ ID NO.1) and KASP-AX-109844182 (SEQ ID NO.2).

[0040] The nucleotide sequence of KASP-AX-109507945 (SEQ ID NO.1): 5'-GCTGGAATGGTGTTGCGGGATGACACAAGAGCAG[G / T]TGTCTTTCCTCCGGTTGCTCCTCCGTTGGAGG-3'.

[0041] The nucleotide sequence of KASP-AX-109844182 (SEQ ID NO.2): 5'-GAACGTCAGCAAATGACGAGACCCATATCCTTGA[C / T]TGTATCTGTCATTAACAATGTCTCTATCTCCACCT-3'.

[0042] The KASP molecular marker primer set for KASP-AX-109507945 includes three primers: KASP-AX-109507945-FAM, KASP-AX-109507945-HEX, and KASP-AX-109507945-C, as shown in Table 1.

[0043] The KASP molecular marker primer set for KASP-AX-109844182 includes three primers: KASP-AX-109844182-FAM, KASP-AX-109844182-HEX, and KASP-AX-109844182-C, as shown in Table 1.

[0044] Table 1 Primer Sequences

[0045] Example 2: Detection Method of Tightly Linked Molecular Markers for Resistance Sites Screening for disease sites resistant to stripe rust QYr.CM62-2BL Methods for processing wheat include: Using genomic DNA from the plant samples as templates, PCR amplification was performed on the templates using the primer set of the KASP molecular marker linked to the wheat stripe rust resistance QTL developed in Example 1; genotyping was performed using the amplification results. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are GG and TT, then the plant sample to be tested is a stripe rust resistant strain. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are TT and CC, then the plant sample to be tested is a stripe rust susceptible strain.

[0046] The PCR amplification system for primers KASP-AX-109507945 and KASP-AX-109844182 is consistent. The PCR amplification system for both is: 2.0 μL DNA template (50 ng / μL), 4.5 μL 2×KASP Master Mix, 2.0 μL primer mixture, and 1.5 μL ultrapure water. The primer mixture was prepared by mixing 12.0 μL of the FAM-containing upstream primer, 12.0 μL of the HEX-containing upstream primer, 30.0 μL of the downstream primer, and 46.0 μL of ultrapure water; the concentration of each primer was 100 μM.

[0047] The specific PCR amplification procedure is as follows: (1) Pre-denaturation at 95℃ for 10 min; (2) 95℃ denaturation for 20s, 61~55℃ annealing and extension for 60s, 10 cycles, each cycle reducing the annealing and extension by 0.6℃; (3) 95℃ denaturation for 20s, 55℃ annealing and extension for 40s, 35 cycles; (4) Read the fluorescence signal at 30℃ for 30s.

[0048] In this embodiment, the KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182 were used to detect the genotyping patterns of the parental Chuanmai 62, Avocet S, and their RIL populations, as shown in the figure below. Figure 1 As shown.

[0049] like Figure 1 As shown, the genotyping is as follows: red (Chuanmai 62) represents the stripe rust resistant strain, blue (Avocet S) represents the stripe rust susceptible strain, and XX and X represent the blank control.

[0050] Based on marker-based genotyping results, 114 lines with genotypes identical to Chuanmai 62 (GG and TT) amplified at the KASP-AX-109507945 (GG) and KASP-AX-109844182 (TT) loci were selected. These lines all carried disease resistance loci from Chuanmai 62. QYr.CM62-2BL The 114 lines were then subjected to stripe rust resistance identification using the same method as in Example 1. The genotypes of each line and their phenotypic identification at the adult stage are shown in Example 1. The results showed that all 114 lines contained stripe rust resistance loci. QYr.CM62-2BL The average severity of the strains was less than 60%, and all showed resistance to stripe rust at the adult stage. The genotyping and phenotypic results of some RIL strains are shown in Table 2.

[0051] Table 2. Genotyping results of some lines in the Chuanmai 62×Avocet S RIL population using KASP-AX-109507945 and KASP-AX-109844182 markers.

[0052] Example 3: Application of tightly linked molecular markers at resistance sites This embodiment investigated the stripe rust resistance loci of Chuanmai 62. QYr.CM62-2BL Distribution of linked KASP molecular markers in bred varieties. KASP marker analysis was performed on 95 bred varieties / lines from the middle and upper reaches of the Yangtze River using the KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182, which are linked to this QTL segment.

[0053] Leaf DNA was extracted as a template according to the method described in Example 2, and amplified using the fluorescent primer set corresponding to the KASP label provided in Example 1. The PCR amplification system, amplification procedure, and genotyping steps were consistent with those in Example 2.

[0054] In this embodiment, the KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182 were used to detect the genotyping of 95 varieties / lines developed in the middle and upper reaches of the Yangtze River, as shown in the figure. Figure 2 As shown.

[0055] like Figure 2 As shown in Table 3, based on the marker genotyping results, a total of 17 materials were screened, and their amplified genotypes at the KASP-AX-109507945 and KASP-AX-109844182 loci were consistent with those of Chuanmai 62 (17.8% of the tested materials). This result indicates that... QYr.CM62-2BL The disease resistance locus has a low distribution frequency in existing bred varieties. Its closely linked markers KASP-AX-109507945 and KASP-AX-109844182 can be used to further promote the conversion of this disease resistance locus into bred varieties.

[0056] Table 3 QYr.CM62-2BL Distribution of varieties / strains bred in the middle and upper reaches of the Yangtze River

[0057] In summary, the site developed in this invention is related to resistance to stripe rust. QYr.CM62-2BL The tightly linked KASP molecular markers KASP-AX-109507945 and KASP-AX-109844182 enable high-throughput, accurate detection of this QTL and can be used for stripe rust resistance sites. QYr.CM62-2BL Tracking and identification. All carrying QYr.CM62-2BLThe lines at these loci all exhibited good resistance to stripe rust. Therefore, these two KASP molecular markers have important application value in marker-assisted selection breeding of stripe rust resistance genes.

[0058] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Related to the stripe rust resistance gene of Chuanmai 62 QYr.CM62-2BL Linked KASP molecular markers, characterized by, The KASP molecular markers include: KASP-AX-109507945 and KASP-AX-109844182; The nucleotide sequence of KASP-AX-109507945 is shown in SEQ ID NO.1; The nucleotide sequence of KASP-AX-109844182 is shown in SEQ ID NO.

2.

2. The stripe rust resistance gene of Chuanmai 62 QYr.CM62-2BL A primer combination linked to KASP molecular markers, characterized in that, The primer set contains primers for the molecular marker KASP-AX-109507945 and primers for the molecular marker KASP-AX-109844182; The primer sequences for the molecular marker KASP-AX-109507945 are shown in SEQ ID NO.3~5; The primer sequences for the molecular marker KASP-AX-109844182 are shown in SEQ ID NO.6~8.

3. The stripe rust resistance gene of Chuanmai 62 QYr.CM62-2BL A kit for linked KASP molecular markers, characterized in that, The kit contains the primer combination as described in claim 2.

4. The application of the KASP molecular marker as described in claim 1, or the primer combination as described in claim 2, or the kit as described in claim 3 in screening for stripe rust resistant lines or in assisting molecular breeding for wheat resistance to stripe rust.

5. A screening method for products containing the stripe rust resistance gene of Chuanmai 62 QYr.CM62-2BL The method for wheat varieties is characterized by, The method includes: Using genomic DNA from the plant samples as templates, quantitative real-time PCR amplification was performed using primers as described in claim 2 for the molecular marker KASP-AX-109507945 and for the molecular marker KASP-AX-109844182, respectively. Genotyping of the wheat samples was then performed based on the PCR amplification results. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are GG and TT, then the plant sample to be tested is a stripe rust resistant strain. If the genotyping results of the molecular markers KASP-AX-109507945 and KASP-AX-109844182 are TT and CC, then the plant sample to be tested is a stripe rust susceptible strain.

6. The method according to claim 5, characterized in that, The system for quantitative real-time PCR amplification includes: DNA template, 2×KASP Master Mix, primer mixture, and ultrapure water; The primer mixture is a mixture of primers with sequences as shown in SEQ ID NO. 3~5 or a mixture of primers with sequences as shown in SEQ ID NO. 6~8.

7. The method according to claim 6, characterized in that, The system for quantitative real-time PCR amplification comprises: 2.0 μL of DNA template at a concentration of 50 ng / μL, 4.5 μL of 2×KASP Master Mix, 2.0 μL of primer mixture, and 1.5 μL of ultrapure water; The primer mixture contains an upstream primer containing FAM, an upstream primer containing HEX, a downstream primer, and ultrapure water in a volume ratio of 6:6:15:23; and each primer has a concentration of 100 μM.

8. The method according to any one of claims 5 to 7, characterized in that, The procedure for quantitative real-time PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing and extension at 61–55℃ for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.6℃ per cycle; denaturation at 95℃ for 20 s, annealing and extension at 55℃ for 40 s, 35 cycles.

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