KASP molecular marker related to wheat stripe rust resistance character, primer pair and application of KASP molecular marker

Abstract

The invention relates to the field of biomolecular detection, in particular to a KASP molecular marker related to wheat stripe rust resistance characters, a primer pair and application of the KASP molecular marker and the primer pair. According to the invention, 559 parts of common wheat germplasm resources are subjected to whole genome association analysis, a stable stripe rust resistance site QYr.hzau-3B. 1 is identified on a wheat 3B chromosome long arm, and a KASP molecular marker (KASP molecular marker KASP 3B2800) is developed aiming at the site and is used for screening large-scale materials. The KASP molecular marker provided by the invention provides a new site and a new marker for wheat stripe rust resistance breeding; meanwhile, the KASP molecular marker can be used for rapidly screening wheat germplasm containing the site, so that the time cost is effectively saved, and the progress of wheat resistance germplasm genetic background detection and wheat disease-resistant gene-assisted selective breeding is accelerated.

Description

Technical Field

[0001] The invention relates to the field of biological molecule detection, and in particular to a KASP molecular marker related to wheat stripe rust resistance traits, a primer pair and an application thereof. Background Art

[0002] Wheat stripe rust is a fungal airborne disease caused by Puccinia striiformis f.sp.tritici. When the disease is severe, it can cause a 25% or more reduction in wheat yield, or even total failure. In recent years, due to environmental and climate changes and the continuous mutation of wheat stripe rust strains, wheat stripe rust has become prevalent in many wheat-growing areas in my country, threatening wheat production safety.

[0003] Although chemical agents can effectively control stripe rust, they are costly and extremely unfriendly to the environment, humans and animals. Therefore, using disease-resistant genes to breed disease-resistant varieties is the most environmentally friendly, economical and effective measure to prevent and control wheat stripe rust. In view of the increasingly serious damage caused by wheat stripe rust in my country due to global climate change, there are only a handful of effective stripe rust resistance genes with durable resistance in wheat production. Therefore, discovering more wheat stripe rust resistance loci can enrich the wheat stripe rust resistance gene resources and is of great significance for breeding new wheat varieties with durable resistance. However, there are currently no reports on molecular markers associated with the wheat stripe rust resistance locus QYr.hzau-3B.1. Summary of the invention

[0004] The purpose of the present invention is to provide a KASP molecular marker, a primer pair and an application thereof related to the wheat stripe rust resistance trait, so as to solve the problems existing in the above-mentioned prior art. The KASP molecular marker provided by the present invention is closely related to the trait of stripe rust resistance, and the KASP molecular marker can accurately, efficiently and at low cost identify the wheat stripe rust resistance trait.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a KASP molecular marker related to wheat stripe rust resistance trait. The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.1, and there is a deletion at the 18th and 19th bases of the sequence.

[0007] The present invention provides a KASP primer pair for detecting the above-mentioned KASP molecular marker, wherein the KASP primer pair comprises a first upstream primer having a nucleotide sequence as shown in SEQ ID NO.2, a second upstream primer having a nucleotide sequence as shown in SEQ ID NO.3, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.4.

[0008] The present invention provides a detection product for the above-mentioned KASP molecular marker, and the detection product comprises the above-mentioned KASP primer pair.

[0009] Preferably, the detection product includes a detection reagent, a detection kit or a detection chip.

[0010] The present invention provides the use of the above-mentioned KASP molecular marker, the above-mentioned KASP primer pair or the above-mentioned detection product in any of the following items:

[0011] (1) Application in identification of wheat stripe rust resistance traits;

[0012] (2) Application in screening wheat varieties or strains with stripe rust resistance traits;

[0013] (3) Application in screening improved wheat varieties or strains with stripe rust resistance traits;

[0014] (4) Application of molecular marker-assisted breeding in wheat.

[0015] The present invention provides a method for identifying wheat stripe rust resistance traits, comprising the following steps:

[0016] The genomic DNA of the wheat sample to be tested is used as a template, and the template is PCR amplified using the above-mentioned KASP primer pair; if the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the first upstream primer in the KASP primer pair, the wheat sample to be tested is a plant containing a susceptible genotype (Hap1); if the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the second upstream primer in the KASP primer pair, the wheat sample to be tested is a plant containing a disease-resistant genotype (Hap2).

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses 559 common wheat germplasm resources to conduct genome-wide association analysis, and identifies a stable stripe rust resistance site, QYr.hzau-3B.1, on the long arm of chromosome 3B. The candidate genes in the site positioning interval are further analyzed, and the results show that the candidate gene TraesCS3B03G1242800 has insertion / deletion (InDel) sequence variation in the resistant material, which causes the gene to produce frameshift mutation and premature termination of coding in the disease-resistant material. In the specific embodiment of the present invention, InDel is used to develop a KASP molecular marker (KASP molecular marker KASP3B2800) and screen large-scale materials, and the developed KASP molecular marker is verified in natural populations to be closely related to disease resistance in all environments. In summary, the present invention identified the wheat stripe rust adult plant resistance locus QYr.hzau-3B.1, and designed a tightly linked marker KASP molecular marker KASP3B2800 for the locus, providing a new locus and a new marker for wheat stripe rust resistance breeding; at the same time, the KASP marker KASP3B2800 can quickly screen wheat germplasm containing the locus, effectively saving time and cost, and accelerating the process of genetic background detection of wheat resistance germplasm and wheat disease resistance gene assisted selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The frequency distribution diagram of the final severity of wheat stripe rust of 559 samples (A) and the correlation coefficient between different environments (B); among them, EZ2020-EZ2022 are the frequency distribution diagrams of the final severity of wheat stripe rust of Ezhou Experimental Base in 2020-2023, and GS2021-GS2022 are the frequency distribution diagrams of the final severity of wheat stripe rust of Gansu Experimental Base in 2021-2022;

[0021] Figure 2 The results of association analysis of wheat stripe rust resistance; A is the Manhattan plot of association analysis of wheat stripe rust resistance in adult plants, with the arrow indicating the QYr.hzau-3B.1 locus; B is the SNP / InDel cluster at the QYr.hzau-3B.1 locus; C is the high-confidence gene in the physical interval of the QYr.hzau-3B.1 locus; D is the sequence variation information on the candidate gene TraesCS3B03G1242800 and causes a frameshift mutation;

[0022] Figure 3Genotyping of KASP molecular markers in subpopulations (A) and single marker analysis results in Gansu environment in 2021 (B). DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Identification and analysis of wheat stripe rust phenotype

[0030] The 559 wheat materials used in this example include 394 cultivated varieties, 86 local varieties, 39 artificially synthesized wheat, 16 colored wheat and 24 hexaploid wheat.

[0031] In this example, these 559 wheat germplasms were used for six field wheat stripe rust identifications at the Ezhou Experimental Base (EZ, 2020-2023) and the Gansu Experimental Base (GS, 2021-2022).

[0032] The field experiment was designed with a row length of 1.5m and a row width of 20cm. One disease-inducing row was planted every approximately 1m in the vertical direction between two rows. The experimental materials were repeated twice, with a randomized block design and sowing in strips.

[0033] At the jointing stage of wheat, a mixed local stripe rust species (mainly including CYR33 and CYR34) was selected to artificially inoculate the susceptible wheat material. The inoculation method was spray inoculation or powder shaking inoculation of light mineral oil or corn starch mixed with stripe rust.

[0034] Field disease surveys include two trait indicators, namely disease severity and host response type; disease severity is obtained by estimating the percentage of wheat leaf area occupied by wheat stripe rust spore piles (Peterson et al 1948); host response type is divided according to the symptoms shown in Table 1 (Roelfs et al 1992); when the disease severity of susceptible materials or susceptible parents reaches 60%-80%, the first field phenotypic record is started, and the second survey is conducted at an interval of 7 days, and the area under the disease development curve (AUDPC) is calculated; that is, the field wheat stripe rust disease severity of 559 wheat samples was investigated twice, 0 means that the plant is completely resistant to stripe rust, and 100% means that the plant is completely susceptible to stripe rust. The final severity was statistically analyzed, and the results are as follows Figure 1 The results show that the severity of wheat disease presents a continuous distribution, which is a typical quantitative trait ( Figure 1 The severity range is 0-100%, and the severity correlation coefficient under different environments is between 0.58-0.85 ( Figure 1 B), the broad-sense heritability is 0.93, and the population contains a sufficient level of genetic variation.

[0035] Table 1 Response types and corresponding symptoms of wheat stripe rust in the adult stage in the field

[0036] Host response type Host response type Leaf symptoms R High resistance No necrosis, or no or small uredia in the necrotic area MR Zhongkang There are a few summer spores around the chlorotic or necrotic area M Moderate resistance-moderate infection Combination of MR and MS MS Medium Moderate amount of uredia, no necrosis, no or slight chlorosis S High Sensitivity Lots of summer spores, no necrosis, no chlorosis

[0037] 2. SNP / InDel marker screening

[0038] CTAB method was used to extract DNA from 559 wheat germplasms, and the next generation sequencing was performed on the Illumina sequencing platform. The sequencing depth of each sample was about 2×. After quality control, the data was aligned to the Chinese Spring IWGSCRef2.1 genome using BWA-mem software, and SNP / InDel calling was performed using GATK software, and marker filtering was performed according to the --geno 0.05--maf0.05 parameters. Finally, more than 8.7 million SNP / InDel markers were obtained for subsequent analysis.

[0039] 3. Association analysis of wheat stripe rust resistance

[0040] Fastlmm software was used to perform association analysis on phenotypic data and filtered high-quality SNP / InDel markers using -log 10 (p)>5 was used as the threshold, and a total of 2788 SNPs / InDels were identified to be significantly associated with adult resistance to stripe rust. Among them, a major QTL locus significantly associated with adult resistance to stripe rust was identified on chromosome 3BL, named QYr.hzau-3B.1, which was identified in all five environments and could explain 9.23%-16.55% of the phenotypic variation (Table 2, Figure 2 A in the figure). This site has a SNP / InDel cluster within the 1.18Mb physical interval, and there are 6 high-confidence genes within the interval. Among them, TraesCS3B03G1242800 has a 2bp InDel variation between the resistant and susceptible materials, which leads to a frameshift mutation, resulting in premature termination of coding ( Figure 2 in BD).

[0041] Table 2 Wheat stripe rust adult resistance loci identified by genome-wide association analysis

[0042]

[0043]

[0044] Note: a -LOG of peak SNP of QTL in different environments 10 (P); b Environmental variation that can be explained by QTL under different environments; c Different environments are named in the form of "location" + "year", and "1", "2" and "AUDPC" represent the first survey result, the second survey result and the area under the disease development curve, respectively.

[0045] 4. KASP molecular marker development

[0046] The InDel site on the wheat stripe rust adult plant resistance site QYr.hzau-3B.1 was developed into a KASP molecular marker KASP3B2800, whose nucleotide sequence is shown in SEQ ID NO.1, and there is a 2 bp deletion at the 18th and 19th bases of the sequence.

[0047]

[0048] The bold italic parts in the sequence are the missing bases.

[0049] In summary, this example uses single nucleotide polymorphism (SNP) and insertion / deletion (InDel) markers obtained by whole genome resequencing to perform genome-wide association study (GWAS) on the field stripe rust phenotypes of 559 wheat germplasms; inoculation-induced material is used for field inoculation, and the disease severity and response type of natural population materials are statistically analyzed; Fastlmm software is used to perform genome-wide association analysis on genotypes and phenotypes, using -log 10 (p)>5 was used as the threshold, and a total of 12 stable QTL loci were identified, which were located on chromosomes 1B, 3A, 3B, 5A, 5B, 6B, 7A, 7B and 7D, respectively. Among them, chromosome 7B had the most QTLs, with a total of 3. The locus QYr.hzau-3B.1 on chromosome 3BL was identified in all five environments, which could explain 9.23%-16.55% of the phenotypic variation. The candidate gene TraesCS3B03G1242800 in the positioning interval of this locus had a conservative 2bp InDel variation between resistant and susceptible materials, and caused a frameshift mutation. Based on the variation information, the KASP molecular marker was developed, and the marker detection was performed on the population. The results showed that the marker can be used to identify resistant germplasm at the QYr.hzau-3B.1 locus.

[0050] 5. Development of KASP markers and amplification methods

[0051] The KASP marker was designed based on the 2 bp InDel variation on the candidate gene TraesCS3B03G1242800 at the QYr.hzau-3B.1 locus. The KASP marker information is shown in Table 3.

[0052] Table 3 Sequence information of KASP marker

[0053]

[0054] PCR amplification system (5 μL system), as follows:

[0055] DNA template (50-100 ng / μL): 1 μL;

[0056] K2800-F1 (10 μM): 0.1 μL;

[0057] K2800-F2 (10 μM): 0.1 μL;

[0058] K2800-R (10 μM): 0.3 μL;

[0059] FLU-ARMS2×for KASPPCRMix: 2.5μL;

[0060] dH 2 O: 1 μL.

[0061] The PCR amplification procedure is:

[0062] 1 95.0℃, 10min;

[0063] 2 95.0℃, 15s;

[0064] 3 65.0℃, 30s(-0.6℃ / cycle);

[0065] 4Go to 2, 10 more times;

[0066] 5 95.0℃, 15s;

[0067] 6 59.0℃, 30s;

[0068] 7Goto 5, 35more times;

[0069] 8 30.0℃, 1min+Plate Read;

[0070] PCR was performed on a Bio-Rad qRTPCR instrument (model: C1000 Touch Thermal Cycler).

[0071] 6. Application of KASP Mark

[0072] The KASP markers developed above were used to genotype the wheat materials. The PCR amplification system and procedure were the same as above. The results are shown in Figure 3 As shown in A. The susceptible materials YM18 and ND183 are a group of haplotypes (Hap1, there is a 2bp deletion at the 12th and 13th bases of the sequence shown in SEQ ID NO.1, lacking 2bp), and the resistant materials ZM9023 and JM21 are a group of haplotypes (Hap2, there is no 2bp deletion at the 18th and 19th bases of the sequence shown in SEQ ID NO.1, not lacking 2bp).

[0073] Example 2 Application of KASP markers developed in Example 1

[0074] In order to eliminate the influence of known strong stripe rust resistance genes or loci on QYr.hzau-3B.1, the functional molecular markers or tightly linked markers of these known genes or loci (Yr5, Yr10, Yr15, Yr18, Yr27, Yr29, Yr30, Yr36 and Yr46) were used to detect 559 wheat germplasms and remove materials containing the above genes or loci. The remaining materials were genotyped using the KASP marker developed in Example 2. The PCR amplification system and procedure were the same as in Example 2. The results are shown in Table 4. At the same time, the single marker results in the Gansu environment in 2021 were statistically analyzed. The results are shown in Table 4. Figure 3 As shown in B.

[0075] Table 4 Genotype identification results of the remaining materials

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Single marker analysis of KASP markers in the remaining materials showed that the severity of the disease in germplasm containing Hap2 was significantly lower than that in germplasm containing Hap1 ( Figure 3 B and Table 4). This indicates that the KASP marker can be used to identify resistant germplasm at the QYr.hzau-3B.1 locus.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A KASP molecular marker associated with wheat stripe rust resistance, characterized in that: The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.1, and there are deletions at the 18th and 19th bases of the sequence.

2. A KASP primer pair for detecting the KASP molecular marker according to claim 1, characterized in that: The KASP primer pair includes a first upstream primer having a nucleotide sequence as shown in SEQ ID NO.2, a second upstream primer having a nucleotide sequence as shown in SEQ ID NO.3, and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

4.

3. A KASP molecular marker detection product according to claim 1, characterized in that: The detection product comprises the KASP primer pair described in claim 2.

4. The detection product according to claim 3, characterized in that: The detection product includes a detection reagent, a detection kit or a detection chip.

5. Use of the KASP molecular marker according to claim 1, the KASP primer pair according to claim 2, or the detection product according to claim 3 or 4 in any of the following: (1) Application in identification of wheat stripe rust resistance traits; (2) Application in screening wheat varieties or strains with stripe rust resistance traits; (3) Application in screening improved wheat varieties or strains with stripe rust resistance traits; (4) Application of molecular marker-assisted breeding in wheat.

6. A method for identifying and / or assisting in identifying wheat stripe rust resistance traits, characterized in that: The following steps are involved: Using the genomic DNA of the wheat sample to be tested as a template, and using the KASP primer pair described in claim 2 to perform PCR amplification on the template; If the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent connector of the first upstream primer of the KASP primer pair, the wheat sample to be tested is wheat containing a susceptible genotype; if the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent connector of the second upstream primer in the KASP primer pair, the wheat sample to be tested is wheat containing a disease-resistant genotype.

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