A kasp molecular marker, primer pair and application thereof related to a wheat stripe rust resistance trait

Abstract

The present application relates to the field of biomolecule detection, in particular to a KASP molecular marker related to the resistance trait of wheat stripe rust, a primer pair and application thereof. The present application carries out whole genome association analysis on 559 common wheat germplasm resources, identifies a stable stripe rust resistance locus on the long arm of wheat 3B chromosome, namely QYr.hzau-3B.1, and develops a KASP molecular marker (KASP molecular marker KASP3B2800) for the locus for screening large-scale materials. The KASP molecular marker provided by the present application provides a new locus and new marker for wheat stripe rust resistance breeding; at the same time, the KASP molecular marker can quickly screen wheat germplasm containing the locus, effectively save time cost, and accelerate the process of wheat resistance germplasm genetic background detection and wheat disease resistance gene assisted selection breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomolecule detection, in particular to a KASP molecular marker related to a wheat stripe rust resistance trait, a primer pair and application thereof. BACKGROUND

[0002] Wheat stripe rust is a fungal airborne disease caused by Puccinia striiformis f.sp.tritici, which can cause more than 25% reduction in yield or even absolute yield when the disease is severe. In recent years, due to environmental climate change and continuous variation of wheat stripe rust strains, wheat stripe rust has become prevalent in many wheat-growing areas in China, threatening the safety of wheat production.

[0003] Chemical agents can effectively control stripe rust, but the cost is high and is 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. Given that global climate change has led to increasingly severe wheat stripe rust damage in China, and there are only a few effective genes with persistent resistance to wheat stripe rust in wheat production. Therefore, exploring more wheat stripe rust resistance sites can enrich wheat stripe rust resistance gene resources and is of great significance for breeding new wheat varieties with persistent resistance. However, there is no report on molecular markers associated with the wheat stripe rust resistance site QYr.hzau-3B.1. SUMMARY

[0004] The present application provides a KASP molecular marker related to a wheat stripe rust resistance trait, a primer pair and application thereof, to solve the problems existing in the prior art. The KASP molecular marker provided by the present application 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-mentioned purpose, the present application provides the following solutions.

[0006] The present application provides a KASP molecular marker related to a 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 application provides a KASP primer pair for detecting the above-mentioned KASP molecular marker, the KASP primer pair comprises a first upstream primer with a nucleotide sequence as shown in SEQ ID NO. 2, a second upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3 and a downstream primer with 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:

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

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

[0013] (3) Application in screening improved wheat varieties or lines 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 from 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 from 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 resistant genotype (Hap2).

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

[0018] The present invention conducted a genome-wide association analysis using 559 common wheat germplasm resources and identified a stable stripe rust resistance locus, QYr.hzau-3B.1, on the long arm of chromosome 3B. Further analysis of candidate genes within the locus mapping interval revealed that the candidate gene, TraesCS3B03G1242800, exhibited insertion / deletion (InDel) sequence variation in resistant and susceptible materials, leading to a frameshift mutation and premature termination of coding in the resistant materials. In a specific embodiment of the present invention, a KASP molecular marker (KASP molecular marker KASP3B2800) was developed using InDel and screened across a large number of materials. The developed KASP molecular marker was verified in natural populations to be closely associated with disease resistance under all conditions. In summary, the present invention identified the wheat stripe rust adult plant resistance locus QYr.hzau-3B.1 and designed a tightly linked marker for this locus, the KASP molecular marker KASP3B2800, providing a new locus and new marker for wheat stripe rust resistance breeding. At the same time, the KASP marker KASP3B2800 can quickly screen wheat germplasm containing this locus, effectively saving time and costs, and accelerating the process of genetic background detection of wheat resistant 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0020] Figure 1 Frequency distribution of final severity of wheat stripe rust for 559 samples (A) and correlation coefficients between different environments (B); EZ2020-EZ2022 are frequency distributions of final severity of wheat stripe rust at the Ezhou Experimental Base from 2020 to 2023, and GS2021-GS2022 are frequency distributions of final severity of wheat stripe rust at the Gansu Experimental Base from 2021 to 2022.

[0021] Figure 2 Figure 5 is the result 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 within the physical interval of the QYr.hzau-3B.1 locus; D is the sequence variation information of the candidate gene TraesCS3B03G1242800, which leads to 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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. Phenotypic identification and analysis of wheat stripe rust

[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 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 1m in the vertical direction between two rows. The experimental materials were repeated twice, with a randomized block design and sowing in rows.

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

[0034] Field disease surveys included two trait indicators: disease severity and host response type. Disease severity was estimated by estimating the percentage of wheat leaf area occupied by wheat stripe rust spores (Peterson et al. 1948). Host response type was classified according to the symptoms shown in Table 1 (Roelfs et al. 1992). When the disease severity of susceptible materials or susceptible parents reached 60%-80%, the first field phenotypic record was started. A second survey was conducted 7 days later, and the area under the disease progression curve (AUDPC) was calculated. In other words, the field disease severity of wheat stripe rust was surveyed twice for 559 wheat accessions, with 0 indicating complete resistance to stripe rust and 100% indicating complete susceptibility. The final severity was statistically analyzed, and the results are shown in Figure 2. Figure 1 The results showed that the severity of wheat disease showed a continuous distribution, which was 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 Reaction 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 A small number of uredia around the chlorotic or necrotic area M Moderate resistance-moderate cold Combination of MR and MS MS Neutral Moderate amount of uredia, no necrosis, no or slight chlorosis S High sensitivity A large number of summer spores, no necrosis, no chlorosis

[0037] 2. SNP / InDel marker screening

[0038] DNA from 559 wheat germplasm samples was extracted using the CTAB method and sequenced using the Illumina platform for next-generation sequencing, with each sample sequencing at a depth of approximately 2×. After quality control, the data were aligned to the Chinese Spring IWGSCRef2.1 genome using BWA-mem software. SNP / InDel calling was performed using GATK software, with marker filtering using the --geno 0.05 --maf 0.05 parameters. Over 8.7 million SNP / InDel markers were identified 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 Using a threshold of (p)>5, a total of 2788 SNPs / InDels were identified that were 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. It was identified in all five environments and explained 9.23%-16.55% of the phenotypic variation (Table 2, Figure 2 A in the figure). This site has a SNP / InDel cluster within a 1.18Mb physical interval, with six 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 BD in ).

[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 explained by QTL in different environments; c Different environments are named in the form of "location" + "year", "1", "2" and "AUDPC" represent the first differential survey result, the second differential 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 used single nucleotide polymorphism (SNP) and insertion / deletion (InDel) markers obtained by whole genome resequencing to conduct genome-wide association study (GWAS) on the field stripe rust phenotypes of 559 wheat germplasms; inoculated materials were used for field inoculation, and the disease severity and response type of natural population materials were statistically analyzed; Fast1mm software was used to perform genome-wide association analysis on genotypes and phenotypes, with -log 10 Using a threshold of (p)>5, 12 stable QTLs were identified, located on chromosomes 1B, 3A, 3B, 5A, 5B, 6B, 7A, 7B, and 7D. Chromosome 7B had the most QTLs, with three. The QYr.hzau-3B.1 locus on chromosome 3BL was identified in all five environments, explaining 9.23%-16.55% of the phenotypic variation. Within the mapping interval of this locus, the candidate gene TraesCS3B03G1242800 exhibited a conserved 2-bp indel variant between resistant and susceptible accessions, resulting in a frameshift mutation. Based on this variant, a KASP molecular marker was developed and tested in a population. The results showed that this 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] The PCR amplification system (5 μL system) is 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] ddH2O: 1μL.

[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] 4Goto 2, 10more 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 were as follows: Figure 3 As shown in A in FIG. The susceptible materials YM18 and ND183 are a group of haplotypes (Hap1, there is a 2 bp deletion at the 12th and 13th bases of the sequence shown in SEQ ID NO. 1, and 2 bp are missing), and the resistant materials ZM9023 and JM21 are a group of haplotypes (Hap2, there is no 2 bp deletion at the 18th and 19th bases of the sequence shown in SEQ ID NO. 1, and 2 bp are not missing).

[0073] Example 2 Application of the KASP marker developed in Example 1

[0074] To eliminate the influence of known strong stripe rust resistance genes or loci on QYr.hzau-3B.1, 559 wheat germplasms were tested using functional molecular markers or tightly linked markers of these known genes or loci (Yr5, Yr10, Yr15, Yr18, Yr27, Yr29, Yr30, Yr36 and Yr46) and materials containing the above genes or loci were removed. 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 germplasms containing Hap2 was significantly lower than that in germplasms 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a KASP molecular marker associated with wheat stripe rust resistance, a KASP primer pair for detecting the KASP molecular marker, or a detection product containing the KASP primer pair in any of the following: (1) Application in identification of wheat stripe rust resistance traits; (2) Application in screening wheat varieties or lines with stripe rust resistance traits; (3) Application in screening improved wheat varieties or lines with stripe rust resistance traits; (4) Application in molecular marker-assisted breeding of wheat; the breeding trait is resistance to wheat stripe rust; The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.1, and there is a deletion at the 18th and 19th bases of the sequence; the KASP primer pair includes a first upstream primer with a nucleotide sequence shown in SEQ ID NO.2, a second upstream primer with a nucleotide sequence shown in SEQ ID NO.3, and a downstream primer with a nucleotide sequence shown in SEQ ID NO.

4.

2. 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, PCR amplification is performed on the template using the KASP primer pair described in claim 1; 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 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 linker of the second upstream primer in the KASP primer pair, the wheat sample to be tested is wheat containing a resistant genotype.

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