CN111235295B - KaSP molecular marker of wheat powdery mildew resistance gene Pm21 derived from haynaldia villosa and application thereof - Google Patents
KaSP molecular marker of wheat powdery mildew resistance gene Pm21 derived from haynaldia villosa and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat powdery mildew resistance gene derived from haynaldia villosaPm21The KASP molecular marker of (1) and the application thereof, the KASP molecular marker is KASP-Pm21, and the primers of the KASP molecular marker KASP-Pm21 comprise a haynaldia villosa equipotential type upstream primer KASP-Pm21-F, a wheat equipotential type upstream primer KASP-Pm21-H and a common downstream primer KASP-Pm21-C; the nucleotide sequence of the Haynaldia villosa allelic upstream primer KASP-Pm21-F is shown in SEQ ID NO. 1; the nucleotide sequence of the wheat equipotential upstream primer KASP-Pm21-H is shown in SEQ ID NO. 2; the nucleotide sequence of the common downstream primer KASP-Pm21-C is shown in SEQ ID NO. 3. The wheat powdery mildew resistance gene derived from haynaldia villosa provided by the inventionPm21The KASP molecular marker KASP-Pm21 can realizePm21Efficient tracking and detection of gene, and application of gene in wheat powdery mildew resistancePm21The molecular marker of the gene assists selective breeding.
Description
Technical Field
The invention relates to the fields of biotechnology and genetic breeding, in particular to a powdery mildew resistance gene of wheat derived from haynaldia villosaPm21The KASP molecular marker and application thereof.
Background
Wheat (Triticum aestivum L., 2n = 42, AABBDD) is one of the most important food crops in China, and the maintenance of high and stable yield of wheat is very important for guaranteeing national food safety. Among the factors affecting the yield and quality of wheat, the occurrence of diseases is very critical. Wheat powdery mildew is a leaf Disease caused by powdery mildew, which generally causes 10-15% yield loss of wheat and affects wheat quality to some extent (Singh et al, disease impact on wheat yield potential and characteristics of genetic control, annu Rev Phytopathol 2016, 54, 303-322.). In order to prevent and control wheat powdery mildew, several measures such as field agricultural management, pesticide spraying, disease-resistant variety planting and the like can be generally adopted. In comparison, planting of disease-resistant varieties is undoubtedly the most economical, effective and environmentally friendly measure (Filter et al. Identification and implementation of resistance: genetics-associated use of Genetic resources for breeding against information powder mill and statistical non-nuclear block In which: tuberosa R.J., graner A.J., frison E. (eds.) genetics of Plant Genetic resources 2014, springer, dordecht.).
In the investigation of the powdery mildew resistance of wheat varieties in China, only 3.4 percent of the varieties in the main popularized varieties in China have broad-spectrum resistance, and the powdery mildew resistance of more breeding lines participating in national regional test is not optimistic (Lihongjie and the like, the resistance reaction of Chinese wheat varieties to powdery mildew and the detection of disease-resistant genes. The crop academy.2011, 37. Therefore, at present, the breeding for wheat powdery mildew resistance in China needs to explore and utilize a more broad-spectrum resistance gene on one hand, and also needs to accelerate the breeding process and shorten the breeding period so as to cope with the increasingly serious wheat powdery mildew epidemic situation in China.
At present, scientists have found 80 wheat powdery mildew resistant genes and alleles thereof at 61 sites (Pm 1-Pm65, pm8 is allogenic to Pm17, pm18= Pm1c, pm22= Pm1e, pm23= Pm4c, pm31= Pm 21) (Li et al. A specific genes-aegiensis longissima locus cancer resistant Pm66 restriction to pore tissue. Therefore, when selecting the disease-resistant gene, not only the resistance strength and the resistance spectrum width of the disease-resistant gene are considered, but also the yield of the carrier variety, the agronomic performance and other characters can meet the requirements of breeders.
In the discovered wheat powdery mildew genes, pm21 is a broad-spectrum powdery mildew resistance gene derived from haynaldia villosa 6VS chromosome, and in the 80 s of the 20 th century, pm21 is carried by distant hybridization of wheat and haynaldia villosa and transferred to a wheat genome in a 6VS chromosome arm translocation mode, so that the powdery mildew resistance of wheat is greatly improved, and the report of Pm21 pathogenic powdery mildew strains is rarely reported since the popularization of the genes is more than 20 years, so that the gene is an powdery mildew resistance gene with important breeding value. In 2018, xing et al (2018) and He et al (2018) respectively adopt a disease-resistant gene enrichment sequencing and map-based cloning method to clone a Pm21 gene, and prove that the Pm21 gene encodes a CC-NBS-LRR disease-resistant protein to endow wheat powdery mildew resistance (He et al (2018) Pm21, encoding experimental CC-NBS-LRR protein, a control broad-spectrum resistance to a broad pore hydrophobic disease Plant, mol Plant, 11.
For transformation of Pm21 gene, numerous types of 6VS chromosome arm specific molecular markers have been developed, such as RAPD marker (Qi et al, identification, mapping, and application of polymorphic DNA associated with gene Pm21 of white blood, 1996, 39: 191-197), SCAR marker (Liu Shi Yong et al (1999) molecular Identification and marker-assisted selection of wheat powdery mildew resistant gene Pm 21. Gen. 26. He et al (2017) developed an intron-based 6 VS-specific marker using a CISP (conserved-interacting primer) (He et al, genetic, physical and compatible mapping of the pore fine resistance gene Pm21 origin from DasyYvirus. Frontiers in Plant Science, 2017, 8 1914. In addition, cao et al (2011) develop CINAU15 from the Stpk-V gene and Bie et al (2015) develop MBH1 from the Pm21 gene promoter region, and are also widely used (Cao et al. Series/hormone kinase gene Stpk-V, a key member of a powder stress response gene Pm21, a related powder stress response in a powder natural Acad Sci USA, 2011, 108: 7727-7732; bie et al. Development and characterization of a rheological index MBH1 structural compliance gene Pm21 and Pm V related stress response in a powder stress modification gene Pm21 and M V related stress in a powder stress graft. However, the markers are molecular markers based on common PCR on the basis of establishing laboratory gel electrophoresis, high-throughput, automatic and platform breeding cannot be realized, and transformation efficiency of the Pm21 gene is influenced.
In recent years, with the development of molecular marker technology, especially the emergence of third-generation molecular markers based on SNP (Single nucleotide polymorphism), the selection efficiency of target genes is greatly improved. In the third generation molecular marker, KASP (Kompetitive allee specific PCR) technology is the key for realizing SNP high-efficiency typing, can realize genotyping accurately, in a high-throughput and platform manner, avoids the traditional marker display means based on gel electrophoresis, and has the characteristics of high throughput, low cost, good genetic stability and the like. Thus, KASP markers play an increasingly important role in wheat gene mapping, map-based cloning and molecular marker assisted selection breeding (Rasheet et al Development and identification of KASP assays for genes underspining key genes in broken wheat at the door Appl Gene, 2016, 129, 1843-1860). However, although Pm21 is cloned as a powdery mildew resistance gene with important breeding value, no KASP marker for breeding, especially a functional KASP marker, is reported, and the efficient utilization of the Pm21 gene in the genetic improvement of wheat is seriously influenced. Therefore, the cloned Pm21 sequence is compared with the wheat genome to develop a Pm21 function KASP marker, so that the rapid and accurate detection of the Pm21 gene can be realized, and the efficient utilization of the Pm21 gene in molecular marker-assisted selective breeding can be greatly accelerated.
Disclosure of Invention
The invention aims to provide a KASP molecular marker of wheat powdery mildew resistance gene Pm21 derived from haynaldia villosa and application thereof, which can detect and track the powdery mildew resistance gene in wheat background in a high-throughput mannerPm21Greatly acceleratePm21The gene molecular marker assists the efficiency of selective breeding.
The invention is realized by the following method: wheat powdery mildew resistance gene derived from haynaldia villosaPm21The KASP molecular marker of (1), the KASP molecular marker is KASP-Pm21, and the primers of the KASP molecular marker KASP-Pm21 comprise a haynaldia villosa equipotential type upstream primer KASP-Pm21-F, a wheat equipotential type upstream primer KASP-Pm21-H and a common downstream primer KASP-Pm21-C;
the nucleotide sequence of the Haynaldia villosa allelic upstream primer KASP-Pm21-F is shown in SEQ ID NO. 1;
the nucleotide sequence of the wheat equipotential upstream primer KASP-Pm21-H is shown in SEQ ID NO. 2;
the nucleotide sequence of the common downstream primer KASP-Pm21-C is shown in SEQ ID NO. 3.
The powdery mildew resistance gene of wheat derived from haynaldia villosaPm21The KASP molecular marker of (1) is applied to molecular marker-assisted selective breeding.
The wheat powdery mildew resistance gene derived from haynaldia villosaPm21The KASP molecular marker is used for identifying whether wheat varieties or wheat strains carry powdery mildew resistance genes of wheat derived from haynaldia villosaPm21The use of (1).
Whether wheat powdery mildew resistance gene is carried in wheat variety or strainPm21The identification method of (3), comprising the steps of:
(1) Extracting DNA of a sample to be detected as a template;
(2) Carrying out PCR amplification on the genome DNA of a sample to be detected by using the primer of the KASP molecular marker KASP-Pm21 to obtain an amplification product;
the primers of the KASP molecular marker KASP-Pm21 comprise a haynaldia villosa allelic type upstream primer KASP-Pm21-F, a wheat allelic type upstream primer KASP-Pm21-H and a common downstream primer KASP-Pm21-C; the nucleotide sequence of the Haynaldia villosa allelic upstream primer KASP-Pm21-F is shown in SEQ ID NO. 1; the nucleotide sequence of the wheat equipotential upstream primer ASP-Pm21-H is shown in SEQ ID NO. 2; the nucleotide sequence of the common downstream primer KASP-Pm21-C is shown in SEQ ID NO. 3.
(3) Analyzing the genotype of the PCR amplification product by adopting a fluorescent quantitative PCR detector; measuring a fluorescence signal at 37 ℃, and reading a typing result by a Bio-Rad CFX Manager 3.1; in the typing result, the genotype is the genotype of haynaldia villosa or/and the genotype of a vector carrying the gene Pm21 if the allelic type is Allle 1/Allle 1, the genotype is the genotype of the wheat carrying the powdery mildew resistance gene Pm21 if the allelic type is Allle 1/Allle 2, and the genotype is the genotype of the wheat not containing the powdery mildew resistance gene Pm21 if the allelic type is Allle 2/Allle 2.
Whether the wheat variety or the wheat strain carries the wheat powdery mildew resistance genePm21The reaction system of the PCR amplification is as follows: 120 mu.L of ng/. Mu.L DNA 6.0. Mu.L, 2 XKASP Maseter Mix 10.0. Mu.L, primer Mix 0.24. Mu.L, ddH 2 O3.76 μ L, 20.0 μ L overall; the concentrations of the rye allelic type upstream primer KASP-Pm21-F and the wheat allelic type upstream primer KASP-Pm21-H in the primer mixture are both 12 mu M; the concentration of the common downstream primer KASP-Pm21-C was 30. Mu.M.
Whether the wheat variety or the wheat strain carries the wheat powdery mildew resistance genePm21The method for identifying (1), wherein the PCR amplification procedure comprises: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 seconds, gradient annealing at 64 ℃ and extension for 60 seconds, 10 cycles, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle; denaturation at 94 ℃ for 20 seconds, annealing at 58 ℃ and extension for 60 seconds, 38 cycles.
The invention carries out comparison analysis by a cloned Pm21 gene sequence and a Chinese spring wheat reference genome sequence, utilizes SNP which is compared with three subgroups of Chinese wheat A, B and D in the Pm21 gene sequence to develop a functional KASP molecular marker KASP-Pm21 of the Pm21 gene for the first time, utilizes the marker to detect haynaldia villosa, a Pm21 gene carrier, a Pm21 gene-carrying 6VS translocation line variety, a Pm21 transgenic line and a Pm21 gene-free common wheat variety, can correctly type and is accurate and error-free, and can quickly, high-flux and high-efficiently detect and track the Pm21 gene in the wheat variety or strain and accelerate the high-efficiency breeding utilization of the Pm21 gene.
Specifically, the invention has the advantages over the prior art that:
1) Compared with the conventional molecular marker of Pm21 based on common PCR amplification, the KASP marker of the wheat powdery mildew resistance gene Pm21 developed by the invention can realize rapid, high-flux and platform detection and tracking of the Pm21 gene without using laboratory gel electrophoresis, thereby greatly improving the transformation utilization of the Pm 21.
2) The KASP marker developed by the invention belongs to a functional marker based on a cloned Pm21 sequence, and compared with the traditional linkage marker, the functional marker is coseparated with a target gene, has no recombination phenomenon, and is more accurate in tracking the Pm21 gene. Meanwhile, because the KASP marker developed by the invention is based on the Pm21 gene sequence, not only can the 6VS chromosome carrying Pm21 be tracked, but also more excellent genes on the 6VS chromosome can be utilized, and only small fragment translocation carrying Pm21 or Pm21 transgenic lines transferred to the wheat background can be tracked.
Drawings
FIG. 1 is a schematic view of the utilization ofPm21Gene KASP marker KASP-Pm21 for detecting Haynaldia villosa, carrierPm21The genotyping results of different samples such as 6VS translocation line wheat varieties and wheat.
Orange round points represent Haynaldia villosa and carryPm21A vector for the gene; green triangle points representing 8 carriersPm216VS translocation line wheat varieties (Yangmai 18, yangmai 21, nannong 9918, jinhe 9123, shimai 14, neumai No. 9, guomai 301 and Shizheng 816) and 2Pm21Transgenic lines 1 and 2; blue squareThe shaped points represent 20 not to carryPm21The general wheat variety (chinese spring, yannong 19, yannong 21, yannong 1212, yannong 999, jimai 20, jimai 21, jimai 22, jimai 262, jimai 44, denna 17, liangxing 66, liangxing 99, wennong 14, shannong 20, shannong 29, tainong 18, shixin 633, shixin 733, and shixin 828).
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1 wheat powdery mildew resistance GenePm21The KASP molecular marker KASP-Pm21 is obtained and applied
1. Preparation of DNA amplification template
Powdery mildew resistance of wheatPm21The gene vector is a vector constructed in He and the like (2018) for later use. The other DNA amplification templates are respectively extracted to obtain 1 haynaldia villosa and 2 haynaldia villosa by using a CTAB methodPm21Transgenic line, 8 vectorsPm216VS translocation line and 20 non-carriersPm21The genome DNA of the leaf blade of common wheat in the one-leaf stage comprises the following specific operation steps:
(1) Shearing fresh leaves, freezing and grinding the fresh leaves by using liquid nitrogen, and putting 0.5g of grinding powder in a 2mL centrifuge tube for each sample;
(2) Adding 600 μ L of CTAB extractive solution preheated at 65 deg.C into each sample, and water-bathing at 65 deg.C for 45min;
(3) Cooling the centrifugal tube to room temperature, adding chloroform-isoamylol (24;
(4) Adding 3 times volume of anhydrous ethanol pre-cooled at-20 deg.C into the supernatant centrifuge tube until flocculent DNA appears, and standing in a refrigerator at-20 deg.C for 30min;
(5) Picking out the flocculent precipitate by using a pipette tip and placing the flocculent precipitate into another 1.5mL centrifugal tube, precooling the flocculent precipitate with 70% ethanol, washing the flocculent precipitate for 2 times, and naturally drying the DNA precipitate at normal temperature;
(6) Adding 60. Mu.L of 1 XTE buffer solution to dissolve the DNA precipitate fully;
(7) The concentration and purity of the DNA solution are detected by a spectrophotometer, and the DNA working solution with the concentration of 10-30 ng/. Mu.L is prepared by ultrapure water according to the detected concentration and is used as a PCR amplification template.
2. Powdery mildew resistance of wheatPm21Development and application of gene KASP marker
Using clonedPm21Comparing the gene Sequence (He et al, 2018) with the wheat Reference gene Sequence (IWGSC Reference Sequence v 1.0) for analysis to obtainPm21SNP locus in gene sequence specific to three subgroups of wheat A, B and D, and a SNP locus is developed according to the SNP locusPm21The function KASP of (1) marks KASP-Pm21, and the primer comprises:
nucleotide sequence of Haynaldia villosa allelic type upstream primer KASP-Pm21-F labeled with FAM:
5 'gaaggtgaccaagttcattcatgctgaccagtctgaggggagc-3' as shown in SEQ ID NO: 1;
the nucleotide sequence of the upstream primer KASP-Pm21-H of the wheat allelic type marked with HEX:
5 'gaaggtcggaagtcaacggattGCTGACCGAGTCTGAGGAGT-3' as shown in SEQ ID NO: 2;
the nucleotide sequence of the common downstream primer KASP-Pm 21-C:
5 'GATACCAAAAGCATCAACTTCACC-3', and the nucleotide sequence is shown as SEQ ID NO. 3.
Carrying out PCR amplification on the genome DNA of a sample to be detected by using the primer of the KASP molecular marker KASP-Pm21 to obtain an amplification product; the samples to be tested were: 1 Haynaldia villosa, 1 vector carrying Pm21 gene and 8 vectorsPm216VS translocation lines (Yanmai 18, yanmai 21, nannong 9918, jinhe 9123, shimai 14, neuma No. 9, guomai 301 and Shizheng 816) of 2Pm21Transgenic lines 1 and 2 (He et al. Pm21, encoding a type CC-NBS-LRR Protein, transfer Broad-Spectrum Resistance to Wheat powder miniature disease. Molecular plant, 2018, 11Pm21Common wheat (Chinese spring, ninong 19, ninong 21, ninong 1212, ninong 999, jimai 2)0. Jimai 21, jimai 22, jimai 262, jimai 44, jinan 17, jixing 66, jixing 99, wennong 14, shannon 20, shannon 29, tainong 18, shixin 633, shixin 733 and Shixin 828).
The PCR amplification system is as follows: 10-30 ng/. Mu.L DNA 6.0. Mu.L, 2 XKASP Maseter Mix 10.0. Mu.L, primer mixture 0.24. Mu.L, ddH 2 O3.76 μ L, 20.0 μ L overall; the concentrations of the Haynaldia villosa equipotential upstream primer KASP-Pm21-F and the wheat equipotential upstream primer KASP-Pm21-H working solution in the primer mixed solution are both 12 mu M; the common downstream primer KASP-Pm21-C working concentration is 30 mu M;
the procedure for PCR amplification was: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 seconds, followed by gradient annealing at 64 ℃ and extension for 60 seconds, 10 cycles, and then a 0.6 ℃ reduction in annealing and extension temperature per cycle; denaturation at 94 ℃ for 20 sec, annealing at 58 ℃ and extension for 60 sec, 38 cycles.
Performing KASP genotyping according to the PCR amplification program and the reaction system, and analyzing the genotype of the PCR amplification product by using a fluorescent quantitative PCR detector; the fluorescence signal was measured at 37 ℃ and the typing results were read by Bio-Rad CFX Manager 3.1 and are shown in FIG. 1. The figure shows that: the transverse axis far end samples are 1 Haynaldia villosa and 1 Haynaldia villosaPm21The carrier of the gene, the equipotential type is 'Allole 1/Allole 1', the group fluorescence reading form is orange round point; the longitudinal axis far end sample group is 20 non-carryingPm21The common wheat of the gene has an equipotential type of 'Allle 2/Allle 2', and a fluorescent reading form of blue square points; the intermediate sample group carries 8Pm216VS translocation lines and 2 genesPm21The transgenic line, the equipotential type is "Allle 1/Allle 2", the fluorescent reading presents green triangle points, three square black points near the origin represent no template control. Powdery mildew resistance of wheatPm21The KASP molecular marker KASP-Pm21 of the gene is in Haynaldia villosa,Pm21Gene vector and carrierPm216VS metathesis system,Pm21Transgenic lines of (3) and non-carrying genesPm21The correct typing is realized in the wheat. Thus, it can be used asPm21Functional KASP molecular marker of gene.
Therefore, the wheat powdery mildew resistance gene derived from haynaldia villosa provided by the inventionPm21KASP score ofThe sub-marker KASP-Pm21 is carriedPm216VS translocation line variety,Pm21Transgenic lines and non-vectorsPm21Realizes accurate typing in gene varieties and can be used for carrying or not carrying different wheat varietiesPm21The effective identification of the gene can be realizedPm21Efficient tracking and detection of gene, and can be used for wheat powdery mildew resistance genePm21The molecular marker of (3) assists selective breeding.
While the invention has been described in detail by way of the general description and the specific embodiments, it is to be understood that modifications and improvements will readily occur to those skilled in the art. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> smoke desk university
<120> KASP molecular marker of wheat powdery mildew resistance gene Pm21 derived from haynaldia villosa and application thereof
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 41
<212> DNA
<213> FAM-labeled Haynaldia villosa allelic type upstream primer KASP-Pm21-F
<400> 1
gaaggtgacc aagttcatgc tgctgaccag tctgagggag c 41
<210> 2
<211> 41
<212> DNA
<213> HEX-labeled wheat allelic type upstream primer KASP-Pm21-H
<400> 2
gaaggtcgga gtcaacggat tgctgaccag tctgagggag t 41
<210> 3
<211> 23
<212> DNA
<213> common downstream primer KASP-Pm21-C
<400> 3
gataccaaag catcaacttc acc 23
Claims (5)
1. Wheat powdery mildew resistance gene derived from haynaldia villosaPm21The primer of the KASP molecular marker in (1), which is characterized in that the KASP molecular marker is KASP-Pm21, and the primer of the KASP molecular marker KASP-Pm21 comprises a haynaldia villosa equipotential type upstream primer KASP-Pm21-F, a wheat equipotential type upstream primer KASP-Pm21-H and a common downstream primer KASP-Pm21-C;
the nucleotide sequence of the Haynaldia villosa allelic upstream primer KASP-Pm21-F is shown in SEQ ID NO. 1;
the nucleotide sequence of the wheat equipotential upstream primer KASP-Pm21-H is shown in SEQ ID NO. 2;
the nucleotide sequence of the common downstream primer KASP-Pm21-C is shown in SEQ ID NO. 3.
2. The powdery mildew resistance gene of haynaldia villosa-derived wheat of claim 1Pm21The KASP molecular marker primer is used for identifying whether wheat varieties or strains carry powdery mildew resistance genes of wheat derived from haynaldia villosaPm21The use of (1).
3. Whether wheat powdery mildew resistance gene is carried in wheat variety or strainPm21The identification method of (2), comprising the steps of:
(1) Extracting DNA of a sample to be detected as a template;
(2) Carrying out PCR amplification on the genome DNA of a sample to be detected by using the primer of the KASP molecular marker KASP-Pm21 to obtain an amplification product; the primers of the KASP molecular marker KASP-Pm21 comprise a haynaldia villosa allelic type upstream primer KASP-Pm21-F, a wheat allelic type upstream primer KASP-Pm21-H and a common downstream primer KASP-Pm21-C; the nucleotide sequence of the Haynaldia villosa allelic upstream primer KASP-Pm21-F is shown as SEQ ID NO. 1; the nucleotide sequence of the wheat equipotential upstream primer ASP-Pm21-H is shown in SEQ ID NO. 2; the nucleotide sequence of the common downstream primer KASP-Pm21-C is shown in SEQ ID NO. 3;
(3) Analyzing the genotype of the PCR amplification product by adopting a fluorescent quantitative PCR detector; fluorescence signals were measured at 37 ℃ and the typing results were read by Bio-Rad CFX Manager 3.1.
4. Whether a wheat variety or line of claim 3 carries a wheat powdery mildew resistance genePm21The method for identifying (2), wherein the reaction system for PCR amplification in step (2) is: 120 ng/. Mu.LDNA 6.0. Mu.L, 2 XKASP Maseter Mix 10.0. Mu.L, primer Mix 0.24. Mu.L, ddH 2 O3.76 μ L, 20.0 μ L overall; the concentrations of the Haynaldia villosa equipotential upstream primer KASP-Pm21-F and the wheat equipotential upstream primer KASP-Pm21-H in the primer mixed solution are both 12 mu M; the concentration of the common downstream primer KASP-Pm21-C was 30. Mu.M.
5. Whether a wheat variety or line of claim 4 carries a wheat powdery mildew resistance genePm21The method for identifying (3), wherein the procedure of PCR amplification in step (2) is: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 seconds, gradient annealing at 64 ℃ and extension for 60 seconds, 10 cycles, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle; denaturation at 94 ℃ for 20 sec, annealing at 58 ℃ and extension for 60 sec, 38 cycles.
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| CN112410451B (en) * | 2020-11-11 | 2023-06-09 | 中国科学院成都生物研究所 | Primer for detecting diploid Haynaldia villosa 3V chromosome specific KASP marker, detection method and application |
| CN113249511B (en) * | 2021-04-16 | 2022-11-08 | 烟台大学 | Functional KASP molecular marker of wheat powdery mildew resistance gene PmV and application thereof |
| CN113025745B (en) * | 2021-04-25 | 2023-10-10 | 河南省作物分子育种研究院 | KASP primer for detecting powdery mildew resistance gene PmBMYD of wheat in Cheng Zhu phase and application thereof |
| CN113005222B (en) * | 2021-05-12 | 2024-01-26 | 江苏省农业科学院 | Multiplex PCR (polymerase chain reaction) labeled primer group for simultaneously detecting wheat scab resistance gene and powdery mildew resistance gene and application thereof |
| CN116640768B (en) * | 2023-04-11 | 2024-02-13 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Wheat powdery mildew resistance gene PmTR1 and application thereof |
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| CN109468409A (en) * | 2019-01-04 | 2019-03-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | A kind of powdery mildew resistance gene in wheat Pm2b high throughput detection label and its application |
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