CN113897457B - KASP molecular marker linked with wheat stripe rust resistance QTL and application - Google Patents

KASP molecular marker linked with wheat stripe rust resistance QTL and application Download PDF

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CN113897457B
CN113897457B CN202111448029.2A CN202111448029A CN113897457B CN 113897457 B CN113897457 B CN 113897457B CN 202111448029 A CN202111448029 A CN 202111448029A CN 113897457 B CN113897457 B CN 113897457B
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nucleotide sequence
wheat
stripe rust
molecular marker
primer set
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CN113897457A (en
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伍碧华
王宇凡
黄林
胡燕灵
龚方仪
贺靖舒
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Sichuan Agricultural University
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Abstract

The invention belongs to the technical field of crop molecular genetic breeding, and discloses a KASP molecular marker linked with wheat stripe rust resistance QTL and application thereof, wherein the nucleotide sequence of the KASP molecular marker comprises a first nucleotide sequence and a second nucleotide sequence; the first nucleotide sequence is SEQ ID NO:1, the second nucleotide sequence is SEQ ID NO:2. the molecular marker which is positioned on the wheat 2A chromosome and is interlocked with wheat stripe rust resistance is a flanking marker of stripe rust resistance site QYr.sicau-2AS on a short arm of the wheat 2A chromosome, and the linkage degree is high. The marker provided by the invention can be used for detecting a stripe rust resistance locus QYr.sicau-2AS on a wheat 2A chromosome, and rapidly screening plants with the locus, thereby improving the disease resistance breeding efficiency of the wheat.

Description

KASP molecular marker linked with wheat stripe rust resistance QTL and application
Technical Field
The invention belongs to the technical field of crop molecular genetic breeding, and particularly relates to a KASP molecular marker linked with wheat stripe rust resistance QTL and application thereof.
Background
Currently, wheat stripe rust is an aerofax disease caused by rust Puccinia striiformis f.sp.tritici (Pst), which severely jeopardizes wheat production. The development, identification and utilization of effective disease-resistant genes, the development of molecular markers closely linked with the effective disease-resistant genes and the application of the effective disease-resistant genes in cultivating durable stripe rust-resistant wheat varieties are the most effective measures for controlling the hazard of stripe rust.
To date, there are 83 stripe rust resistance genes (Yr 1-Yr 83) that have been formally named. As the pathogenic variation of the stripe rust bacteria is fast, new toxic minispecies appear and are popular, most stripe rust resistance genes lose resistance successively, and the disease resistance breeding utilization value is lost. Therefore, it is extremely important to continuously excavate new sources of stripe rust resistance and apply them to wheat production for breeding of disease resistant varieties. The molecular marker can realize rapid development and positioning of disease-resistant genes, and the molecular marker associated with disease-resistant characters can be used for molecular marker assisted selection, so that rapid detection of disease-resistant genes and effective breeding utilization are realized. However, the conventional molecular marker has low detection flux and low efficiency, and cannot realize large-scale screening. In recent years, with the development of gene chips and high throughput sequencing technologies, single base polymorphism (Single nucleotidepolymorphism, SNP) markers based on genomic sequences between biological individuals have been successfully developed. The third generation KASP (Kompetitive allele specific PCR) molecular marker based on SNP has the advantages of high flux, rich sites, low cost, good stability and the like, and plays an important role in crop gene positioning, cloning and molecular marker assisted selection breeding.
At present, a large number of QTL sites against wheat stripe rust have been reported internationally. These disease-resistant sites are distributed on 21 chromosomes of wheat. However, most stripe rust resistant QTLs are minor sites, which are detected only in part of the experimental environment, and QTLs have large linkage regions on the chromosome and part of the sites are non-repeatable. In addition, most reported QTL sites lack molecular markers that can be used for closely linked high-throughput detection of breeding selection. Therefore, the novel site of wheat for mainly resisting stripe rust is continuously discovered, and the high-flux detection mark closely matched with the novel site is developed, so that the novel site has important significance for the cultivation of the antipathogenic varieties.
A stripe rust resistance site QYr.sicau-2AS is identified from wheat variety Shumai 126 bred from wheat of Sichuan agricultural university by a QTL positioning method in the early stage and is located in a 7.58cM region on a short arm of a 2A chromosome. However, no report on QYr.sicau-2AS closely linked KASP molecular markers exists at present, and rapid and efficient selection of the disease resistance site cannot be realized, so that the utilization of the disease resistance site in wheat disease resistance breeding is greatly limited.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a KASP molecular marker linked with wheat stripe rust resistance QTL and application thereof, in particular to a specific KASP molecular marker linked with wheat stripe rust resistance site QYr.sicau-2AS and application thereof. The molecular marker is tightly linked with the stripe rust resistance site QYr.sicau-2AS, can be used for carrying out molecular marker assisted selection on the site, has high selection accuracy, and can remarkably improve the selection and identification efficiency of stripe rust resistance wheat varieties in different environments.
Based on the purposes, the invention utilizes the wheat 55K SNP chip to evaluate the diversity of wheat local germplasm stripe rust resistance and yield related characters in the middle and downstream wheat regions in the Yangtze river from the literature [ Cheng Yukun.2019 ], and the QTL positioning [ D ]]Doctor graduation paper]F obtained by hybridization of a bench length 29 (susceptible parent) of the plant protection institute of Gansu agricultural sciences and a Sichuan province approval wheat variety Shumai 126 (resistant parent) bred by Sichuan agricultural university wheat 6 QTL analysis of the stripe rust resistance locus was performed on the generation recombinant inbred population. The wheat 55K SNP chip is an economic medium-density SNP chip developed on the basis of the wheat 660K SNP chip. The chip contains about 55,000 wheat SNP markers, which are uniformly distributed on 21 chromosomes, wherein 2,600 markers are uniformly distributed on each chromosome, the average genetic distance between the markers is about 0.1cM, and the average physical distance is less than 300Kb, so that the chip is suitable for common germplasm resource diversity analysis, genetic mapping and new gene discovery, comparative genome analysis, variety registration and identification (fingerprint analysis).
Genetic maps were constructed using JoinMap4.0 based on 55K SNP chip data. Combining disease resistance phenotype data of a population, positioning a stripe rust resistance QTL site by using a QTL Icimapping4.1, positioning a main effective stripe rust resistance site QYr.sicau-2AS which is stably expressed in all detection environments in a 7.58cM interval on a short arm of a 2A chromosome, developing molecular markers of the obtained polymorphic SNP site, and finally obtaining 2 pairs of KASP molecular marker nucleotide sequences, wherein the first nucleotide sequence and the second nucleotide sequence are tightly linked with the stripe rust resistance site QYr.sicau-2AS.
The molecular marker provided by the invention can not only efficiently and accurately detect the wheat strain containing the stripe rust resistance locus QYr.sicau-2AS, but also use the marker for high-flux molecular marker assisted selection, thereby effectively improving the breeding efficiency and being used for breeding wheat disease-resistant molecules.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a KASP molecular marker linked to a wheat stripe rust resistance QTL, the nucleotide sequence of the KASP molecular marker linked to a wheat stripe rust resistance QTL comprising a first nucleotide sequence and a second nucleotide sequence;
the first nucleotide sequence is SEQ ID NO:1, the second nucleotide sequence is SEQ ID NO:2.
another object of the present invention is to provide a primer set using the KASP molecular marker linked to a wheat stripe rust resistance QTL and a KASP molecular marker linked to a wheat stripe rust resistance QTL, where the primer set of the KASP molecular marker linked to a wheat stripe rust resistance QTL includes a primer set of a first nucleotide sequence and a primer set of a second nucleotide sequence.
Further, the primer set of the first nucleotide sequence includes a primer set having a nucleotide sequence of SEQ ID NO:3, the nucleotide sequence of the primer group 1 is SEQ ID NO:4 and the nucleotide sequence of the primer group 2 is shown as SEQ ID NO:5, and a primer set 3.
Further, the primer set of the second nucleotide sequence includes a primer set having a nucleotide sequence of SEQ ID NO:6, the nucleotide sequence of the primer group 4 is SEQ ID NO:7 and the nucleotide sequence of the primer group 5 is SEQ ID NO:8, and a primer set 6 of 8.
Another object of the present invention is to provide a method for screening a wheat strain containing a stripe rust resistance site QYr.sicau-2AS using the primer set of KASP molecular marker linked to a stripe rust resistance QTL of wheat, which comprises the steps of:
taking genomic DNA of a plant sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using the primer group of the KASP molecular marker linked with the wheat stripe rust resistance QTL, and carrying out genotyping by using an amplification result.
Specifically, in one embodiment of the present invention, the above application includes the following steps:
1. extracting plant genome DNA;
2. taking plant genome DNA to be detected as a template, and carrying out PCR amplification reaction and reading in a CFX96 Real-Time System instrument by using the primer group of the molecular marker;
further, the reaction system of the fluorescent quantitative PCR is as follows: mu.L SYBR Green, 1.4. Mu.L mixed primer, 5ng template DNA, double distilled water was added to a total of 10. Mu.L.
Further, the reaction procedure of the fluorescent quantitative PCR is as follows: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 20s, annealing extension at 61-55 ℃ for 40s,10 cycles, wherein each cycle of annealing extension is 0.6 ℃ lower; denaturation at 95℃for 20s and annealing at 55℃for 60s,30 cycles.
The invention also aims to provide an application of the KASP molecular marker linked with the wheat stripe rust resistance QTL in crop molecular breeding and screening of wheat varieties or strains with stripe rust resistance.
The invention also aims to provide an application of the primer group of KASP molecular marker linked with wheat stripe rust resistance QTL in crop molecular breeding and screening of wheat varieties or strains with stripe rust resistance.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a KASP molecular marker linked with wheat stripe rust resistance QTL and application thereof, and discloses a fluorescent quantitative PCR platform-based molecular marker for accurately detecting wheat stripe rust resistance sites QYr.sicau-2AS, wherein the nucleotide sequence comprises a first nucleotide sequence and a second nucleotide sequence, is co-dominant, and has the advantages of accurate and efficient detection, and convenient and stable amplification. The disclosed KASP molecular marker and stripe rust resistance locus QYr.sicau-2AS show closely linked marker characteristics, and the accuracy of molecular marker assisted selection is high, so that the selection and identification efficiency of disease-resistant wheat varieties can be remarkably improved.
The invention provides a nucleotide sequence of a molecular marker which is positioned on a wheat 2A chromosome and is linked with wheat stripe rust resistance, the molecular marker comprises a first nucleotide sequence and a second nucleotide sequence, the molecular marker is a flanking marker of a stripe rust resistance site QYr.sicau-2AS on a short arm of the wheat 2A chromosome, and the linkage degree is high. The marker provided by the invention can be used for detecting a stripe rust resistance locus QYr.sicau-2AS on a wheat 2A chromosome, and rapidly screening plants with the locus, thereby improving the disease resistance breeding efficiency of the wheat.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for screening wheat strains containing a site QYr.sicau-2AS against stripe rust according to an embodiment of the present invention.
FIG. 2 is a genotyping diagram of a KASP molecular tagged first nucleotide sequence detection parent Shumai 126, bench length 29 and recombinant inbred populations thereof provided in an embodiment of the invention;
in the figure: the dotted line frame is a parting chart with the same table length 29 of the stripe rust variety; the solid line box is a parting chart of the stripe rust resistant variety Shumai 126; black is a blank.
FIG. 3 is a typing chart of a KASP molecular tagged second nucleotide sequence detection parent Shumai 126, bench length 29 and recombinant inbred populations thereof provided by an embodiment of the invention;
in the figure: the dotted line box is a parting consistent with the stripe rust variety bench length 29; the solid line box is the parting consistent with the stripe rust resistant variety Shumai 126; black is a blank.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a KASP molecular marker linked with a wheat stripe rust resistance QTL and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in FIG. 1, the method for screening wheat strains containing a site QYr.sicau-2AS for stripe rust resistance provided by the embodiment of the invention comprises the following steps:
s101, taking genome DNA of a plant sample to be detected as a template;
s102, performing fluorescent quantitative PCR amplification on the template by using the primer group of the KASP molecular marker linked with the wheat stripe rust resistance QTL;
s103, genotyping by using the amplification result.
The technical scheme of the invention is further described below with reference to specific embodiments.
Example 1: obtaining of wheat stripe rust resistance locus QYr.sicau-2AS linkage KASP molecular marker
1. Test material
Hybridization is carried out by taking wheat disease-resistant variety bench length 29 as male parent and wheat disease-resistant variety Shumai 126 as female parent to obtain hybrid F 1 ，F 1 Obtaining F by substitution selfing 2 Using single particle transfer to F 6 The generation, the recombination inbred line containing 154 single plants is obtained to form genetic mapping population.
2. Identification of stripe rust resistance phenotype in seedling stage and adult stage
Identification of seedling stage resistance: in the seedling stage 2 leaf stage, the current epidemic rust physiological race CYR34 (from the plant protection institute of the national academy of Gansu province) is utilized to inoculate the rust on the Shumai 126, the bench length 29 and 154 recombinant inbred lines by adopting a spray method. When the induced disease strain SY95-71 (bred by wheat of Sichuan agricultural university) is completely ill (14-18 days after inoculation), the stripe rust resistance is identified, and the identification standard adopts an Infection Type (IT) value to refer to 0-9 grades [ Linear Qayoum1992.Virulence, aggressive, evolution and distribution of races of Puccinia striiformis (the causes of stripe rust of wheat) in North America,1968-1987.Tech. Bull.1788:42-46], wherein 0-2 grades are high resistance, 3-5 grades are medium resistance, 6 grades are medium-sensitive and 7-9 grades are infected. Disease Severity (DS) values were investigated, and DS value standards reference the modified Cobbscale method (Peterson et al 1948.Can. J. Res. 26:496-500).
Identification of field stripe rust resistance: the fields were inoculated with mixed stock (CYR 32, CYR33, CYR34, zhong4, and hy 46) using a painting method. The mixed species are from the plant protection institute of the national academy of science in Gansu province. Phenotype identification of stripe rust resistance was performed when the induced material SY95-71 was sufficiently developed. IT and Disease Severity (DS) values were investigated separately.
3.55K SNP chip analysis
Parent durum 126, bench length 29 and recombinant inbred line population plant DNA were extracted with plant DNA extraction kit (tiangen biotech co.ltd, beijin, china). The genotype analysis of the amphiphile mapping population in the present invention was performed by 55K SNP chips developed by the Cooperation of the Jade gold Biotechnology Co-Ltd (http:// www.cgmb.com.cn /) and Gu Jizeng subject group in Beijing, which are commercially available. Genetic maps were constructed using JoinMap4.0 based on 55K SNP chip data. Combining disease resistance phenotype data of the population, detecting the QTL by using a complete interval mapping method (Inclusive Composite Interval Mapping-ADD, ICIM-ADD) in QTL Icimapping4.1 under the condition that a threshold LOD is more than or equal to 2.5, and detecting the QTL by using MDS values of 7 environments in 6 adult periods and 1 seedling period of 2020-2021, so AS to position a wheat stripe rust resistance site QYr.sicau-2AS.
4. Development of stripe rust resistance site QYr.sicau-2AS linkage marker
And designing a KASP molecular marker according to the polymorphism site of the SNP marker by utilizing the data positioning result of the 55K SNP chip, and detecting the specificity of the primer by utilizing a wheat family multiple-study data website (http:// 202.194.139.32 /).
The KASP marker is used for carrying out KASP amplification reaction on genome DNA of the antipathogenic parent Shumai 126, the infectious parent bench length 29 and the recombinant inbred line population, and the antipathogenic material can be obviously typed, and a first nucleotide sequence (SEQ ID NO: 1) and a second nucleotide sequence (SEQ ID NO: 2) can be obtained.
The nucleotide sequence of the first nucleotide sequence (see SEQ ID NO: 1) is as follows:
5'-TATACTACAACAATTGTGGTACATCCTCATTTGGT[C/T]GTTTTCCAAGAACTACAAAGACCCAAGTAGCTAGG-3'；
the nucleotide sequence of the second nucleotide sequence (see SEQ ID NO: 2) is as follows:
5'-CTGACGTAAGACGTGTTTCTTCTGTTTGCCGTGTA[C/T]ATTTTGATGCACGCTCGATGTATCCTCTCGTAAGC-3'。
the set of KASP molecular tagged primers (plus fluorescent sequence) used to amplify the first nucleotide sequence included 3 primers: primer set 1, primer set 2 and primer set 3.
The nucleotide sequence of primer set 1 (see SEQ ID NO: 3) is as follows:
5'-GAAGGTGACCAAGTTCATGCTtgtggtacatcctcatttggtc-3'；
the nucleotide sequence of primer set 2 (see SEQ ID NO: 4) is as follows:
5'-GAAGGTCGGAGTCAACGGATTtgtggtacatcctcatttggtt-3'；
the nucleotide sequence of primer set 3 (see SEQ ID NO:5, shared by downstream primer C) is as follows:
5'-cctagctacttgggtctttgt-3'；
the set of KASP molecular tagged primers (plus fluorescent sequence) used to amplify the second nucleotide sequence included 3 primers: primer set 4, primer set 5 and primer set 6.
The nucleotide sequence of primer set 4 (see SEQ ID NO: 6) is as follows:
5'-GAAGGTGACCAAGTTCATGCTtgtttcttctgtttgccgtgtat-3'；
the nucleotide sequence of primer set 5 (see SEQ ID NO: 7) is as follows:
5'-GAAGGTCGGAGTCAACGGATTtgtttcttctgtttgccgtgtac-3'；
the nucleotide sequence of primer set 6 (see SEQ ID NO:8, shared by downstream primer C) is as follows:
5'-ttgcgacaagtattagcgga-3'；
the primer of the first nucleotide sequence and the primer of the second nucleotide sequence are identical in PCR amplification system. The PCR amplification systems are as follows: 5. Mu.L SYBR Green (TaKaRa), 0.5. Mu.L genomic DNA (100 ng/. Mu.L), 1.4. Mu.L primer mix and 3.1. Mu.L ddH 2 O。
The PCR amplification procedure is as follows:
(1) Pre-denaturation at 95℃for 10min;
(2) Denaturation at 95 ℃ for 20s, annealing extension at 61-55 ℃ for 40s,10 cycles, wherein each cycle of annealing extension is 0.6 ℃ lower;
(3) Denaturation at 95℃for 20s and annealing at 55℃for 60s,30 cycles.
The genotyping diagram of the KASP molecular marker first nucleotide sequence detection parent hollyhock 126, the bench length 29 and the recombinant inbred line population thereof provided by the embodiment of the invention is shown in fig. 2, and the genotyping diagram of the KASP molecular marker second nucleotide sequence detection parent hollyhock 126, the bench length 29 and the recombinant inbred line population thereof provided by the embodiment of the invention is shown in fig. 3.
As a result of PCR amplification, genotyping was performed with a dotted line (bench length 29) as a stripe rust-sensitive strain and a solid line (Shumai 126) as a stripe rust-resistant strain; black is a blank.
Example 2: application of wheat stripe rust resistance site QYr.sicau-2AS linkage KASP molecular marker
KASP marker detection was performed on 154 recombinant inbred individuals obtained by hybridization of bench length 29 and durum 126 using KASP molecular marker first and second nucleotide sequences linked to qyr.sicau-2AS segments. Leaf DNA was extracted as a template according to the method of example 1, and amplification was performed using the fluorescent primer set for KASP molecular markers of the first nucleotide sequence and the second nucleotide sequence provided by the present invention, as shown in example 1. The PCR amplification system, the PCR amplification procedure and the genotyping were the same as in example 1.
By labeling the typing result, 30 strains with genotypes consistent with the hollyhock 126 were selected for amplification at the first nucleotide sequence and the second nucleotide sequence. These lines obtained carry the hollyhock 126 disease resistance locus qyr.sicau-2AS segment. The 30 lines were subjected to the identification of the stripe rust resistance, and the identification method of the stripe rust resistance was the same as in example 1. The genotypes and seedling stage and adult stage phenotype identification values for each line are shown in Table 1. The test results show that the 30 strains containing the site QYr.sicau-2AS for stripe rust show resistance to stripe rust in both seedling stage and adult stage.
In summary, the first nucleotide sequence and the second nucleotide sequence of KASP molecular markers which are linked with the stripe rust resistance locus QYr.sicau-2AS and are developed by the invention can realize high-flux accurate detection of the stripe rust resistance locus QYr.sicau-2AS and are used for tracking and identifying the stripe rust resistance locus QTL QYr.sicau-2AS. Strains containing the QYr.sicau-2AS site all exhibited resistance to stripe rust. Therefore, the KASP molecular marker first nucleotide sequence and the KASP molecular marker second nucleotide sequence which are closely linked with the stripe rust resistance locus QYr.sicau-2AS are developed by the invention, and have important practical significance in molecular marker-assisted selection breeding of stripe rust resistance genes.
TABLE 1 results of genotyping and phenotyping of 29 XShumai 126 recombinant inbred lines
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
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Claims (9)

1. A KASP molecular marker linked to a wheat stripe rust resistance QTL, wherein the nucleotide sequence of the KASP molecular marker linked to a wheat stripe rust resistance QTL comprises a first nucleotide sequence and a second nucleotide sequence;
the first nucleotide sequence is SEQ ID NO:1, the second nucleotide sequence is SEQ ID NO:2.
2. a primer set for use in a KASP molecular marker linked to a wheat stripe rust resistant QTL according to claim 1, wherein the primer set comprises a primer set of a first nucleotide sequence and a primer set of a second nucleotide sequence.
3. The primer set of claim 2 wherein the primer set of the first nucleotide sequence comprises a nucleotide sequence of SEQ ID NO:3, the nucleotide sequence of the primer group 1 is SEQ ID NO:4 and the nucleotide sequence of the primer group 2 is shown as SEQ ID NO:5, and a primer set 3.
4. The primer set of claim 2 wherein the primer set of the second nucleotide sequence comprises a nucleotide sequence of SEQ ID NO:6, the nucleotide sequence of the primer group 4 is SEQ ID NO:7 and the nucleotide sequence of the primer group 5 is SEQ ID NO:8, and a primer set 6 of 8.
5. A method for screening a wheat strain containing a site QYr.sicau-2AS against stripe rust using the primer set according to any one of claims 2 to 4, wherein the method for screening a wheat strain containing a site QYr.sicau-2AS against stripe rust comprises: taking genomic DNA of a plant sample to be detected as a template, carrying out fluorescent quantitative PCR amplification on the template by using the primer group of the KASP molecular marker linked with the wheat stripe rust resistance QTL, and carrying out genotyping by using an amplification result.
6. The method for screening wheat strains containing a site QYr.sicau-2AS for stripe rust resistance according to claim 5, wherein the reaction system of the fluorescent quantitative PCR is: 5. Mu.L of LSYBRGreen, 1.4. Mu.L of mixed primer, 5ng of template DNA, double distilled water was added to a total of 10. Mu.L.
7. The method of claim 5, wherein the fluorescent quantitative PCR is performed by: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 20s, annealing extension at 61-55 ℃ for 40s,10 cycles, wherein each cycle of annealing extension is 0.6 ℃ lower; denaturation at 95℃for 20s and annealing at 55℃for 60s,30 cycles.
8. Use of a KASP molecular marker linked to a wheat stripe rust resistance QTL according to claim 1 for screening varieties or lines of wheat resistant to stripe rust.
9. Use of a primer set according to any one of claims 2 to 4 for screening varieties or lines of wheat resistant to stripe rust.
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