CN113699272B - Molecular marker for detecting wheat fusarium seedling blight QTL Qfsb.hbaas-6BL and application thereof - Google Patents
Molecular marker for detecting wheat fusarium seedling blight QTL Qfsb.hbaas-6BL and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker for detecting fusarium wilt-resistant QTL Qfsb.hbas-6 BL and application thereof. The invention provides an application of a substance for detecting genotype of SNP IWA3221 locus of wheat chromosome 6BL in identification or auxiliary identification of wheat fusarium seedling blight resistance; also provides the application of the substance for detecting the genotype of SNP IWA3221 locus of wheat chromosome 6BL in preparing and identifying or assisting in identifying the fusarium wilt resistance product of wheat. The invention discovers that the fusarium wilt-resistant site Qfsb.hbaas-6BL on the long arm of the wheat 1D chromosome is obtained through whole genome association analysis (GWAS), and further converts the associated SNP IWA3221 into a common PCR marker KASP-Fsb6BL, wherein the typing consistency rate of the two materials for 240 parts is 97.5 percent. The marker can be used for detecting the genotype of the fusarium wilt resistant QTL Qfsb.hbaas-6BL and for molecular breeding of fusarium wilt resistant.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker for detecting wheat fusarium wilt resistant QTL Qfsb.hbas-6 BL and a using method thereof.
Background
Wheat (Triticum aestivum L.) is one of the most important food crops in the world, and has important significance for guaranteeing the food safety. Fusarium seedling blight (Fusarium seedling blight, FSB) and scab (Fusarium headblight, FHB) are primarily caused by fusarium pathogens, are destructive diseases of wheat and other small grain crops worldwide. Wheat seedling blight can cause damage to growing seedlings or rot later in the growing season, resulting in reduced emergence and retarded growth and development, and thus yield loss. In addition, wheat seedling blight can provide a source of pathogen for subsequent wheat scab infection, resulting in reduced wheat yields and food safety hazards. In recent years, wheat seedling blight and scab often reach epidemic levels due to changes in global climate and cultivation regimes, and create huge yield losses of millions of hectares in the global wheat-producing area. In addition, wheat seedling blight and scab produce various mycotoxins during infection, which are highly toxic to humans and domestic animals, threatening human health.
The cultivation of disease-resistant wheat variety is the most economical and effective means for reducing the harm of wheat seedling blight. Molecular marker assisted breeding is an effective means for disease-resistant breeding, and positioning and development of related molecular markers are important. Compared with wheat scab, wheat seedling blight is not much concerned, and only a small amount of research at home and abroad is concerned about the QTL of wheat seedling blight resistance at present. The former identified a QTL on the 5B chromosome that controlled wheat seedling blight resistance using a Double Haploid (DH) population containing 91 parts of material, the linkage marker WMC75 interpreted 13.8% phenotypic variation. Ren et AL (2016) utilized a single major QTL comprising 110 parts of a DH population of material, locating wheat seedling blight resistance caused by pathogens Microdochium nivale and Microdochium majus on chromosomes 1AL and 2BS, respectively. The above studies are all performed by using genetic groups (DH groups) for molecular marker localization, and no reliable markers suitable for large-scale detection and capable of being used for molecular marker assisted breeding have been developed.
Disclosure of Invention
In order to excavate the fusarium wilt-resistant site of wheat and develop the molecular marker thereof, one purpose of the invention is to detect the use of genotype substances of SNP IWA3221 site of wheat chromosome 6BL.
The invention provides an application of a substance for detecting genotype of SNP IWA3221 locus of wheat chromosome 6BL in identification or auxiliary identification of wheat fusarium seedling blight resistance;
and/or, the invention provides the application of a substance for detecting the genotype of SNP IWA3221 locus of wheat chromosome 6BL in the preparation of a product for identifying or assisting in identifying wheat fusarium wilt resistance;
and/or, the invention also provides the application of the substance for detecting the genotype of SNP IWA3221 locus of wheat chromosome 6BL in breeding wheat resistant to fusarium wilt;
and/or the invention also provides application of the substance for detecting the genotype of the SNP IWA3221 locus of the wheat chromosome 6BL in preparing and breeding fusarium wilt-resistant wheat products.
And/or, the invention also provides the application of the substance for detecting the genotype of SNP IWA3221 locus of wheat chromosome 6BL in wheat breeding for resisting fusarium wilt;
and/or the invention also provides application of a substance for detecting the genotype of the SNP IWA3221 locus of the wheat chromosome 6BL in preparing a wheat breeding product for resisting fusarium wilt.
In the application, the SNP IWA3221 site is a site positioned on a wheat chromosome 6BL and the physical position is 668.0 Mb;
or, the genotype of the SNP IWA3221 site is TT or CC.
In the application, the substance for detecting the genotype of the SNP IWA3221 locus of the wheat chromosome 6BL is a specific primer group and comprises a single-stranded DNA molecule or a derivative thereof shown in a sequence 1 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table.
The derivative of the single-stranded DNA molecule shown in the sequence 1 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 1 is connected with a fluorescent sequence;
the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 2 is connected with another fluorescent sequence.
The fluorescent sequence is a fluorescent sequence FAM or a fluorescent sequence HEX.
The substance has at least one of the following functions 1) or 2) or 3):
1) Identifying or assisting in identifying fusarium seedling blight resistance;
2) Wheat breeding for resisting fusarium wilt;
3) And breeding wheat with fusarium wilt resistance.
It is another object of the invention to provide a product.
The product provided by the invention is the substance for detecting the genotype of SNP IWA3221 locus of wheat chromosome 6BL, and specifically comprises
The primer set comprises a single-stranded DNA molecule or a derivative thereof shown in a sequence 1 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table;
or, a PCR reagent containing the set of primers;
or a kit containing the set of primers or the PCR reagent.
The product has at least one of the following functions 1) or 2) or 3):
1) Identifying or assisting in identifying fusarium seedling blight resistance;
2) Selecting fusarium seedling blight wheat;
3) Wheat breeding for resisting fusarium wilt.
The invention aims at providing a method for identifying or assisting in identifying fusarium wilt resistance of wheat.
In the method provided by the invention, in order to detect whether the genotype of SNP IWA3221 locus of the chromosome 6BL of the wheat to be detected is CC or TT, the fusarium wilt resistance of the wheat to be detected is judged according to the genotype;
the resistance of the wheat fusarium seedling blight to be detected with the genotype of the SNP IWA3221 locus as CC is higher than that of the wheat to be detected with the genotype of the SNP IWA3221 locus as TT.
The 4 th object of the invention is to provide a method for breeding wheat with fusarium wilt resistance.
According to the method provided by the invention, in order to detect whether the genotype of SNP IW A3221 locus of wheat chromosome 6BL is CC or TT, wheat to be detected with the genotype of CC is bred, and wheat for resisting fusarium wilt is obtained.
In the above method, the method for detecting whether the genotype of SNP IW A3221 site of wheat chromosome 6BL to be detected is CC or TT is A) or B) as follows:
a) Analyzing a 90K SNP chip;
b) And (3) performing KASP reaction on the genome DNA of the wheat to be detected by using the substance for detecting the genotype of the SNP IW A3221 locus of the wheat chromosome 6BL, and genotyping the product.
The genotyping method comprises the following steps: irradiating a product by using a fluorescence enzyme-labeled instrument, wherein if the product only shows the color of the DNA molecule 5' end shown in the sequence 1 connected with a fluorescent sequence, the genotype of the SNP locus IWA3221 of the wheat to be detected is TT; if the product only shows the color of the fluorescent sequence connected to the 5' end of the DNA molecule shown in the sequence 2, the genotype of the SNP locus IWA3221 of the wheat to be detected is CC.
Any of the above wheat can be, but is not limited to, 240 parts wheat varieties (lines) shown in table 1 of the examples section.
The study identified a wheat seedling blight resistance consisting of 240 Chinese cultivars (lines) as the main group under indoor incubator conditions, and used wheat 90K Single Nucleotide Polymorphism (SNP) chips for genotyping. A stable wheat seedling blight-resistant site QTL Qfsb.hbaas-6BL was located on wheat 6BL and KASP markers were developed that could be used for large scale detection.
The patent finds that the fusarium wilt-resistant site Qfsb.hbas-6 BL on the wheat 6BL chromosome through whole genome association analysis (GWAS) accounts for 6.5% of explained phenotype variation, and the average lesion length of the disease-resistant allele-containing material is 29.1% -37.1% lower than that of the disease-resistant allele-containing material in 3 repetitions. The invention further converts the associated SNP IWA3221 into KASP (kompetitive allele specific PCR) labeled KASP-Fsb6BL, and the typing consistency rate of the two materials for 240 parts is 97.5 percent. The marker can be used for detecting the genotype of the fusarium wilt resistant QTL Qfsb.hbaas-6BL and for molecular breeding of fusarium wilt resistant.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Fusarium pathogenic bacteria Huanggang No. 1: reference is made to: zhu Zhanwang, yang Lijun, hanwen, et al, analysis of scab resistance of wheat variety (line) from Hubei province [ J ]. Proc. Wheat crops, 2014,34 (1): 137-142. Public are available from the institute of food crops, academy of agricultural sciences from Hubei province.
Example 1, discovery of Qfsb.hbas-6 BL and SNP markers associated therewith
Test material: the natural population of 240 wheat varieties (lines) at home and abroad is shown in table 1. The materials used are described in the literature: zhu Zhanwang, xu Dengan, chengjinghe, et al, identification of the wheat scab resistance gene Fhb1 in China, proc. J. Proc. Crop, 2018,44 (4): 473-482. Public are available from the national academy of agricultural sciences, hubei province, grain crop research.
TABLE 1 240 varieties (lines) and sources and genotypes thereof
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a The typing result of the SNP chip is 90K; b labeling the detection result for KASP; c fusarium Seedling Blight (FSB) spot length (cm) was repeated 3 times, and "NN" was the absence of chip data.
1. Discovery of Qfsb.hbas-6 BL and SNP markers associated therewith
1. Identification of fusarium seedling blight resistance
And (3) continuously inoculating and identifying fusarium wilt in an indoor incubator for 3 times, wherein the pathogenic bacterial strain is yellow oka No. 1. 3 replicates were each set, 20 replicates each. The coleoptile inoculation method is adopted for inoculation, the length of the disease spots is investigated 7 days after inoculation, and the longer the disease spot length is, the worse the resistance of wheat fusarium seedling blight is. The average value of each repetition was taken as the result of the authentication of the repetition, and then the average value of 3 repetitions was calculated as the result of the authentication.
2. Genotyping
The natural population is subjected to genotype analysis by using a 90K SNP chip, 22922 SNP with good typing result are selected for subsequent analysis, markers with deletion rate more than 20% and minimum allele frequency less than 5% are removed, and the total of 19803 SNP are used for GWAS.
3. GWAS analysis
Correlation analysis was performed using a mixed linear model of Tassel v5.0 software kineship (K) +pca method. When P.ltoreq.0.001, the marker is considered to be significantly associated with the trait.
4. Qfsb.hbas-6 BL and SNP marker linked thereto
Correlation analysis found that the fusarium wilt-resistant site located on 6BL was significant in the first, third and average two environments, respectively explaining the phenotypic variation of 5.07%, 5.20% and 6.50%, with a representative correlation marker IWA3221, flanking sequences:
5'-atggtgtctttggcgtaacagcatcattctcggtgcctgctgtcctgaacatctggtggcagctaccattctcagcattccacaaggagacttcattgca [ C/T ] cccgcagccacaaaaacgagtggtcttccactagaagatattgagttgtgtggtggtataagcaagcacaatttctctatagggcttactgtggaatagt-3' (SEQ ID NO: 4, SNP site at 101 st position of SEQ ID NO: 4). The physical position on the wheat variety China spring reference genome sequence (IWSSC, http:// www.wheatgenome.org) was 668.0Mb (Table 2). In 4 environments, the average FSB lesion length was 29.1% -37.1% lower for the disease-resistant allele-containing material than for the disease-resistant allele-containing material (table 3).
TABLE 2 Qfsb.hbaas-6BL and SNP markers linked thereto
Note that: a a representative SNP marker is provided, b the disease-resistant alleles are shown underlined, c chinese spring reference genome physical position (IWSSC, http:// www.wheatgenome.org), d the phenotypic variation is explained.
SNP locus IWA3221 is located at a physical position of a 6BL chromosome of 668.0Mb, the polymorphism is C/T, the genotype of the SNP locus is CC or TT, and the SNP locus IWA3221 is also 101 th position of a sequence 4.
Table 3 differential resistance of Fusarium seedling blight of different genotypes of chip 90K
Note that: AB indicates that the phenotype differs significantly between the two genotype materials at the 0.01 level.
2. Design of KASP-Fsb6BL labeling special primer and establishment of method thereof
1. Genome specific primer design
The homologous sequences are obtained by comparing flanking sequences of SNP IWA3221 in Ensemblplants database (http:// plants. Ensembl. Org), and 6BL chromosome specific primers P3221 (sequence 1, sequence 2 and sequence 3) are designed, and are also called KASP-Fsb6BL markers, and are synthesized by Wuhan Tian Yihui Yuan biotechnology Co.
Identifying KASP primer of SNP locus IWA3221 as P3221 primer group, wherein the primer group comprises DNA molecule with specific fluorescent sequence FAM added at 5 'end, DNA molecule with specific fluorescent sequence HEX added at 5' end and single-stranded DNA molecule shown as sequence 3 in sequence table;
the DNA molecule with the specific fluorescent sequence FAM added at the 5 '-end is obtained by adding the specific fluorescent sequence FAM at the 5' -end of the single-stranded DNA molecule shown in the sequence 1;
the DNA molecule with the specific fluorescent sequence HEX added at the 5 'end is obtained by adding the specific fluorescent sequence HEX at the 5' end of the single-stranded DNA molecule shown in the sequence 2;
P3221A:5'-GTTTTTGTGGCTGCGGGT-3' (sequence 1);
P3221B:5'-GTTTTTGTGGCTGCGGGC-3' (SEQ ID NO: 2);
P3221C:5'-TTCTTCCCTTTACAGACTCTTCAGC-3' (SEQ ID NO: 3).
The specific fluorescent sequence FAM is 5'-GAAGGTGACCAAGTTCATGCT-3';
the specific fluorescent sequence HEX is 5'-GAAGGTCGGAGTCAACGGATT-3';
adding a single-stranded DNA molecule shown in a specific fluorescent sequence FAM sequence 1 and a single-stranded DNA molecule shown in a sequence 3 at the 5' end to amplify a fragment with SNP locus genotype TT, and carrying out fluorescent irradiation on a product obtained after PCR amplification of the FAM sequence to display blue;
and (3) adding a single-stranded DNA molecule shown in a specific fluorescent sequence HEX sequence 2 and a single-stranded DNA molecule shown in a sequence 3 at the 5' end to amplify a fragment with SNP locus genotype of CC, wherein a product obtained after PCR amplification of the sequence carrying HEX is subjected to fluorescent irradiation to display green.
2. Detection method establishment
Test material: 240 parts of wheat variety (line) at home and abroad (table 1).
Extracting genome DNA of wheat to be detected, adding ddH 2 O was diluted to a concentration of 30 ng/. Mu.L as a template, and KASP reaction was performed using a primer pair comprising primer P3221A, primer P3221B and primer P3221C.
The KASP reaction system is shown in Table 4;
TABLE 4 KASP reaction System of primer P3221
KASP reaction procedure: thermally activating at 95 ℃ for 15min; denaturation at 95℃for 20s, annealing at 65℃for 30s, 10 touchdown cycles, each cycle reduced by 0.6 ℃; then denaturation at 95 ℃ for 20s, annealing at 57 ℃ and extending for 1min, and 32 cycles are carried out; preserving at 4 ℃.
PHERAstar for KASP reaction product Plus Fluorescence was read (BMG LABECH, ortenberg, germany) and typed with KlumterCaller software (LGC Genomics, teddington, UK). The blue genotype is TT, and the green genotype is CC. The marker can distinguish the IWA3221 genotype and further distinguish the allele of the fusarium wilt resistant QTL Qfsb.hbaas-6BL, which is designated KASP-Fsb6BL.
The consistency rate of the detection result of the marker in the natural population and the typing result of SNP IWA3221 is 97.5%, which indicates that the marker is successfully transformed.
Therefore, the SNP IWA3221 marker can be used for assisting in detecting whether the wheat to be detected is resistant to fusarium wilt and is used for molecular breeding of the fusarium wilt resistance; the specific method comprises the following steps:
detecting the genotype of SNP locus IWA3221 in the genome of the wheat to be detected, and judging according to the following method:
the fusarium seedling blight resistance of the wheat to be detected with the genotype of IWA3221 being CC of the SNP locus is higher than or the candidate is higher than that of the wheat to be detected with the genotype of IWA3221 being TT.
The method for detecting the genotype of the SNP locus IWA3221 in the genome of the wheat to be detected comprises the following steps:
1) Analyzing a 90K SNP chip;
2) KASP reaction was performed with primer P3221A, primer P3221B and primer P3221C using PHERAstar Plus Detecting the product, and carrying out data analysis by using KlumterCaller software; if the color is blue, the genotype of the SNP locus IWA3221 of the wheat to be detected is TT; if the sample is green, the genotype of the SNP locus IWA3221 of the wheat to be detected is CC.
Example 2 actual sample detection
Test material: natural population varieties shown in table 1.
1. Identification of fusarium seedling blight resistance
As in example 1.
2. KASP-Fsb6BL label detection
KASP reaction was performed on 240 varieties shown in table 1 using KASP-Fsb6BL markers in accordance with the method in example 1, and KASP reaction products were detected; by PHERAstar Plus Detecting the product, and carrying out data analysis by using KlumterCaller software; if the color is blue, the genotype of the SNP locus IWA3221 of the wheat to be detected is TT; if the sample is green, the genotype of the SNP locus IWA3221 of the wheat to be detected is CC.
The test results of 240 wheat variety markers and the average value of the FSB lesion length are shown in Table 1. The results of table 1 were statistically analyzed to obtain the results of table 5.
TABLE 5 fusarium seedling blight resistance differences between KASP-Fsb6BL different genotypes
From the above, it can be seen that the fusarium wilt resistance of the wheat to be tested with the CC genotype at the SNP locus IWA3221 is higher than or candidate to be higher than the wheat to be tested with the genotype at the SNP locus IWA3221 TT.
Sequence listing
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Claims (9)
1. A substance for detecting the genotype of SNP IWA3221 site of wheat chromosome 6BL, said substance having at least one of the following functions 1) or 2) or 3):
1) Identifying or assisting in identifying fusarium seedling blight resistance;
2) Wheat breeding for resisting fusarium wilt;
3) Selecting wheat with fusarium wilt resistance;
the substance is a specific primer group and comprises a single-stranded DNA molecule or a derivative thereof shown in a sequence 1 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table;
the SNPIWA3221The locus is a locus which is positioned on wheat chromosome 6BL and has a physical position of 668.0 Mb; the SNPIWA3221The locus corresponds to the 101 th position of the sequence 4, the SNPIWA3221The genotype of the locus is TT or CC;
the derivative of the single-stranded DNA molecule shown in the sequence 1 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 1 is connected with a fluorescent sequence;
the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 2 is connected with another fluorescent sequence.
2. The substance according to claim 1, characterized in that the fluorescent sequence is fluorescent sequence FAM or fluorescent sequence HEX.
3. SNP for detecting wheat chromosome 6BLIWA3221The genotype of the locus, characterized in that,
the primer set comprises a single-stranded DNA molecule or a derivative thereof shown in a sequence 1 in a sequence table, a single-stranded DNA molecule or a derivative thereof shown in a sequence 2 in the sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table; the derivative of the single-stranded DNA molecule shown in the sequence 1 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 1 is connected with a fluorescent sequence; the derivative of the single-stranded DNA molecule shown in the sequence 2 in the sequence table is that the 5' end of the single-stranded DNA molecule shown in the sequence 2 is connected with another fluorescent sequence;
or, a PCR reagent containing the set of primers;
or, a kit containing the set of primers or the PCR reagent;
the SNPIWA3221The locus is a locus positioned on wheat chromosome 6BL and the physical position is 668.0Mb, and the SNPIWA3221The locus corresponds to the 101 th position of the sequence 4, the SNPIWA3221The genotype of the site is TT or CC.
4. The substance according to claim 1 or 2 or the product according to claim 3 for detecting SNP of wheat chromosome 6BLIWA3221Use of the loci, the SNPIWA3221The locus is a locus positioned on wheat chromosome 6BL and the physical position is 668.0Mb, and the SNPIWA3221The locus corresponds to the 101 th position of the sequence 4, the SNPIWA3221The genotype of the site is TT or CC.
5. Use of a substance according to claim 1 or 2 or a product according to claim 3 for the identification or assisted identification of fusarium wilt resistance in wheat;
and/or in the preparation of a product for identifying or aiding in the identification of fusarium wilt resistance in wheat;
and/or in wheat breeding against fusarium wilt;
and/or in the preparation of wheat breeding products resistant to fusarium wilt;
and/or in breeding wheat resistant to fusarium wilt;
and/or in the preparation of wheat products for breeding fusarium wilt resistance.
6. Identification or assisted identification of wheat sickle as claimed in claim 5Application of bacterial seedling blight resistance comprises detection of SNP of wheat chromosome 6BL to be detectedIWA3221The genotype of the locus is TT or CC, and the fusarium seedling blight resistance of the wheat to be detected is judged according to the genotype;
SNP IWA3221the fusarium seedling blight resistance of wheat to be detected with locus genotype CC is higher than SNPIWA3221Wheat to be tested with the genotype of the locus TT.
7. The use of the wheat of Fusarium wilt resistance according to claim 5 comprising detecting SNP of wheat chromosome 6BLIWA3221And (3) breeding wheat to be detected with the locus genotype of CC or TT, and obtaining wheat with fusarium wilt resistance.
8. Use according to claim 6 or 7, wherein the method for detecting whether the genotype of the SNP IWA3221 site of wheat chromosome 6BL is CC or TT is a) or B) as follows:
a) Analyzing a 90K SNP chip;
b) Performing a KASP reaction on the genomic DNA of the wheat to be tested by using the product for detecting the genotype of SNP IWA3221 site of wheat chromosome 6BL according to claim 4, and genotyping the product.
9. The use according to claim 8, further comprising a genotyping method as follows: irradiating a product by using a fluorescence enzyme-labeled instrument, wherein if the product only shows the color of the DNA molecule 5' end shown in the sequence 1 connected with a fluorescent sequence, the genotype of the SNP locus IWA3221 of the wheat to be detected is TT; if the product only shows the color of the fluorescent sequence connected to the 5' end of the DNA molecule shown in the sequence 2, the genotype of the SNP locus IWA3221 of the wheat to be detected is CC.
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CA2553757A1 (en) * | 2004-01-22 | 2005-08-04 | Japan Science And Technology Agency | Gene markers linked to fusarium head blight-resistance factor and utilization thereof |
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CN111575400A (en) * | 2020-06-28 | 2020-08-25 | 湖北省农业科学院粮食作物研究所 | Wheat stripe rust resistant QTL molecular marker IWB12253 and application thereof |
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