CN110923352A - KASP marker of wheat powdery mildew resistance gene PmDTM and application thereof - Google Patents
KASP marker of wheat powdery mildew resistance gene PmDTM and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of crop breeding science, and relates to a powdery mildew resistance gene of wheatPmDTMAnd the KASP marker and application thereof. The KASP label HNU58900 andPmDTMthe genetic distance of the gene was 0 cM. The invention internationally obtainsPmDTMThe most closely linked molecular markers. HNU58900 is co-dominant marker, and has the advantages of high throughput, high efficiency, stable amplification, and convenient detection. Detection with marker HNU58900PmDTMGenes, can be identifiedPmDTMAnd predicting wheat powdery mildew resistance, and then rapidly screening the existence or non-existence and existence state of wheatPmDTMThe plants are used for breeding new varieties of powdery mildew resistance.
Description
Technical Field
The invention belongs to the field of crop breeding science, and relates to a powdery mildew resistance gene of wheatPmDTMAnd the KASP marker and application thereof.
Background
Wheat is the second largest food crop in the world after rice. In 2050, the total population of the world is estimated to exceed 90 hundred million, and the wheat yield needs to be increased by 1.6 percent year by year to meet the requirement of population increase. Thus, the yield of wheat at 2050 would need to be increased from the current 3 tons/hectare to 5 tons/hectare (Singh et al, 2016). From BrinellPowdery mildew (A)Blumeria graminisf. sp.tritici, Bgt) The resulting powdery mildew is a serious threat to the achievement of this stimulation goal. Powdery mildew is a worldwide disease, occurs in wheat growing areas all over the world, and is particularly more serious in wheat high-yield areas with proper climate. After wheat is infected by powdery mildew, the powdery mildew compete for the nutrition of the host and reduce the photosynthetic capacity of leaves, which can cause 5-8% of yield loss. In severe disease epidemic years, yield loss can reach more than 30% (Morgounov et al 2012). With the continuous improvement of water and fertilizer conditions, the increase of seeding density, the popularization of high-yield dwarf varieties, the variation of virulence structures of powdery mildew and the like, powdery mildew has risen from secondary diseases to main diseases, and becomes one of important factors for limiting the high yield of wheat in China (Huaet al, 2009).
The wheat powdery mildew resistant gene resource library comprises common wheat and wheat near and distant species. Among the genes that have been named, the gene,Pm6、Pm27is derived from the plant Triticum aestivum,Pm7、Pm8、Pm17、Pm20is derived from the rye or a combination thereof,Pm21、Pm55、Pm62is derived from the plant of Haynaldia villosa,Pm43from Elytrigia intermedium. The resistance of these exogenous genes is very outstanding, but their use in breeding is very cumbersome due to the effects of linkage drag. The local variety of the common wheat is a variety formed by long-term natural or artificial selection under local natural or cultivation conditions, has better adaptability to local natural environment, and has a plurality of excellent powdery mildew resistance sources.Pm2c、Pm3b、Pm3e、Pm5d、Pm5e、Pm24a、Pm24b、PmHLT、Pm47、Pm59、Pm61、Pm63The isogenes are all discovered from wheat local varieties. The local wheat variety can be freely recombined with the cultivated variety, and the disease-resistant gene is easy to transfer to the popularized variety.
Lu et al (2019) selects a local variety of barley from wheat germplasm stored in a national germplasm resource bank. The identification of the resistance of 28 strains with different toxicity in different regions in China in the seedling stage shows that the barley expresses immunity to 27 strains in the barley to high resistance. Thus, barley is a broad spectrum, excellent source of resistance to wheat powdery mildew. F constructed by hybridizing kohlrabi and susceptible variety Huixian red2And F2:3The segregation population is subjected to genetic analysis, and the result shows that the barley carries a single dominant powdery mildew resistance gene, and the barley is tentatively named asPmDTM. By mixed packet analysis (BSA) and SSR marker analysis,PmDTMlocated 7.44 cM apart on the 1DS chromosomeXgwm337–Xbarc229The physical distance is 29.6 Mb.
Competitive allelic polymorphism PCR (KASP) is a high-throughput, high-efficiency molecular marker, and can be used for accurately typing SNP (single nucleotide polymorphisms) and InDel (insertion-deletion polymorphisms) which are widely present in genomic DNA. The technology utilizes a common primer and two primers with different terminal bases to carry out PCR amplification. Two primers with different terminal bases carry fluorescent linker sequences FAM and HEX, respectively. According to different fluorescent signals carried by the amplification products, a large number of samples can be rapidly detected, the genotype of the samples can be accurately judged, and the method is very suitable for molecular marker-assisted selective breeding.
Therefore, screening out the powdery mildew resistance gene carried by the barleyPmDTMThe co-separated SNP or InDel is mutated and developed into KASP marker easy to detect, and the KASP marker is applied to wheat powdery mildew resistant molecular breeding, which has important significance for improving the yield and quality of wheat.
Disclosure of Invention
The invention provides a powdery mildew resistance gene of wheatPmDTMThe KASP marker and the application thereof solve the problem of linkage drag caused by too large introduction section in wheat backcross breeding.
The technical scheme of the invention is realized as follows:
wheat powdery mildew resistance genePmDTMThe KASP marker of (1), said KASP marker being the co-dominant molecular marker HNU 58900.
The KASP marker of the present application is obtained by:
one, F2Construction of populations andPmDTMselection of segment recombinants
(1) Performing the anti-powdery mildew on barley (female parent) and the anti-powdery mildew on Pink barley (male parent) and Binhua (male parent) of Jingxian cityHybrid F is obtained by hybridization1，F1Selfing to produce F2A large population;
(2) extracting F by SDS method2DNA of individual plants in the population; by usingPmDTMBoundary marks GWM337 and BARC229 screening F2Individuals in the population that recombine in this segment. The sequence information of the primers used is as follows:
TABLE 1 primer sequence information
II, F2Powdery mildew resistance identification of generation recombinant selfing progeny
For each F2Carrying out powdery mildew resistance identification on selfing progeny of the generation recombinant; identification was carried out in the plant growth chamber from each F2Randomly selecting 25 seeds from selfing progeny of the generation recombinant, and planting the seeds in a plug tray with 72 holes; in the first leaf and first heart stage, the epidemic physiological race E09 of powdery mildew is used for inoculation, and after 7 to 10 days, the disease-resistant reaction type of each individual plant is investigated according to the grading standard of 0 to 4 grades; the growth conditions of the wheat are set as 16 hours of illumination, 8 hours of darkness, the temperature in the illumination is 22 ℃, and the temperature in the darkness is 18 ℃.
III, molecular marker analysis
(1) Extracting disease-resistant parent barley, susceptible parent Huixian red and F by SDS method2DNA of the generation recombinant; according toPmDTMSearching SNP and InDel variation between barley and red in Hui county by using Chinese spring reference sequence information corresponding to the section and sequence information of cloned candidate genes, developing KASP markers, and performing polymorphism screening.
(2) Selection between parents and F2Polymorphic KASP markers and flanking SSR markers GWM337 and BARC229 amplification F in the population2The DNA of the recombinants was substituted, and the marker genotype data of each recombinant was obtained.
The PCR amplification method adopted by the SSR marker comprises the following steps: the PCR reaction volume was 25. mu.L, with 12.5. mu.L of 2 XTAQA PlusMaster Mix II (Vazyme, Biotech), 1.0. mu.L each of 10. mu.M primers, 100ng template DNA, and 25. mu.L of water; the PCR amplification program comprises pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 15s, annealing at 55-60 ℃ for 20s, extension at 72 ℃ for 40s, circulation for 35 times, and final extension at 72 ℃ for 5 minutes; PCR amplification is carried out on a PTC-225 amplification instrument, the amplification product is subjected to electrophoretic separation on 8% non-denaturing polyacrylamide gel, silver staining and photographing are carried out, and the result is recorded.
The KASP marker adopts a PCR amplification method: the PCR reaction volume was 10. mu.L, with 5.0. mu.L of 2 XKASP Mastermix (LGC Genomics), 0.047. mu.L of 90. mu.M common primers, 0.047. mu.L of 36. mu.M allele-specific primers each, 100ng of template DNA, and 10. mu.L of water; PCR amplification adopts touchdown program: pre-denaturation at 94 ℃ for 15 minutes, denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, 10 cycles, and annealing temperature reduction by 0.6 ℃ per cycle; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, 32 cycles; reacting at 37 ℃ for 60s, and reading a fluorescence signal; amplification was performed on a LightCycler480 II system (Roche, Indianapolis, IN, USA) fluorescent quantitative PCR instrument, and the fluorescence signal values of the amplified products were analyzed using LightCycler480 software 1.5.1.62, and the results were recorded.
Obtaining of Co-segregating molecular markers
(1) According to each F2Disease-resistant response type speculation of generation recombinant selfed progenyPmDTMGenotype: if a certain F2The selfing progeny of the generation recombinant are all resistant to diseases, and are presumed to carry powdery mildew resistant allele consistent with barleyPmDTM/PmDTM(ii) a If the selfed progeny are all susceptible, the selfed progeny are presumed to carry powdery mildew allele consistent with Huixian redpmDTM/pmDTM(ii) a If the selfed progeny has resistance and infection separation, the selfed progeny is presumed to carry heterozygous allelesPmDTM/pmDTM。
(2) Comparison of the marker genotype of each recombinant withPmDTMGenotype, calculation of marker-to-marker and marker-toPmDTMThe number of recombinations occurring in between; according to the law of chain exchange, user= {2-(4-6*n/N)1/2}/3 (N is the number of recombinant individuals, N is F2Population total individual plant number) and using a Kosambi mapping functiond= ln { (1+2r)/(1-2r) }/4 willrConversion to picture distanced(ii) a Construction Using Mapdraw 2.0PmDTMA genetic linkage map of (2) is obtainedAndPmDTMco-separated KASP markers HNU 58900.
The primers of the codominant molecular marker HNU58900 comprise a common primer HNU5890-F sequence and a specific primer pair HNU58900-FAM-R and HNU 58900-HEX-R; wherein the HNU5890-F sequence is shown as SEQ ID NO.1, the HNU58900-FAM-R sequence is shown as SEQ ID NO.2, and the HNU58900-HEX-R sequence is shown as SEQ ID NO. 3.
Identification of wheat powdery mildew resistance gene by using KASP markerPmDTMThe method comprises the following steps: using the DNA of wheat to be detected as a template, using a common primer and a specific primer pair of a molecular marker HNU58900 as primers, carrying out PCR amplification, detecting a fluorescence signal carried by an amplification product by using LightCycler480 software v1.5.1.62 software, and judging whether the wheat carries a powdery mildew resistance gene or not according to the fluorescence signalPmDTM。
The judgment of carrying of the wheat powdery mildew resistance gene is carried out by the fluorescent signalPmDTMThe method comprises the following steps: if the amplification product mainly carries FAM signals, the wheat to be detected is marked to carry disease-resistant allelesPmDTM/PmDTM(ii) a If the amplification product mainly carries HEX signal, the wheat to be detected is marked to carry disease allelepmDTM/pmDTM(ii) a If the amplification product carries two signals of FAM and HEX, the fact that the wheat to be detected carries heterozygous disease-resistant allele is markedPmDTM/pmDTM。
A method for screening powdery mildew resistant wheat by using KASP markers comprises the following steps: the method comprises the steps of carrying out PCR amplification by using DNA of wheat to be detected as a template and using a common primer and a specific primer pair of a molecular marker HNU58900 as primers, detecting a fluorescent signal carried by an amplification product by using LightCycler480 software v1.5.1.62 software, and screening the wheat to be detected, wherein the amplification product mainly carries an FAM signal, namely the wheat for resisting powdery mildew.
In the PCR amplification procedure: the PCR reaction system was 10. mu.L, 2 XKASP Master mix (LGCGenomics) 5.0. mu.L, 90. mu.M common primers 0.047. mu.L, 36. mu.M specific primers each 0.047. mu.L, template DNA 100ng, plus ddH2O to 10 μ L; the PCR amplification program is pre-denaturation at 94 ℃ for 15min, denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, and 10 cycles, wherein the annealing temperature is reduced by 0.6 ℃ in each cycle; denaturation at 94 ℃ for 20s, annealing at 55 ℃ 60s, 32 cycles; the reaction was carried out at 37 ℃ for 60 s.
The KASP marker is used for identifying powdery mildew resistance genes in wheat germplasm resourcesPmDTMThe use of (1).
The KASP marker is applied to molecular breeding for cultivating powdery mildew resistant wheat.
The invention has the following beneficial effects:
1. the invention internationally obtainsPmDTMThe co-separated KASP marker HNU58900 can accelerate powdery mildew resistance genePmDTMCan be used in wheat breeding for disease resistancePmDTMMap-based cloning of the genes. Obtain and (2)PmDTMThe co-separated KASP marker HNU58900 is helpful for effectively transferring the gene to a promoted variety, and solves the linkage drag problem caused by too large introduction section in backcross breeding.
2. HNU58900 has the advantages of high throughput, high efficiency, stable amplification, and convenient detection. Detection with KASP marker HNU58900PmDTMGenes, can be identifiedPmDTMPresence or absence and presence status of, and then rapidly screening for carriersPmDTMThe plant is used for breeding new powdery mildew resistant varieties, greatly improves the breeding selection efficiency and reduces the breeding cost.
3. HNU58900 can be used for resisting powdery mildewPmDTMCloning studies of (3). Map bit cloningPmDTMThe key to (1) is to obtain molecular markers that are closely linked to it. HNU58900 andPmDTMcoseparation, which can be directly used for screening genome library, walking to both sides after obtaining positive clone, and finally constructing overlayPmDTMClonal contigs of regions.
4. KASP tag HNU58900 of the present application andPmDTMthe genetic distance of the gene is 0 cM, and HNU58900 is a codominant marker, and has the advantages of high throughput, high efficiency, stable amplification, convenient detection and the like. Detection with marker HNU58900PmDTMGenes, can be identifiedPmDTMAnd predicting wheat powdery mildew resistance, and then rapidly screening the existence or non-existence and existence state of wheatPmDTMThe plants are used for breeding new varieties of powdery mildew resistance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
HNU58900 at F in FIG. 12Typing results in surrogate recombinants; wherein the horizontal axis represents FAM fluorescence signal values and the vertical axis represents HEX fluorescence signal values.
FIG. 2 shows HNU58900 and SSR markers GWN337 and BARC229 on both sides and anti-powdery mildew gene of wheatPmDTMA genetic linkage map of (a); the right side of the graph is the markers of the genetic linkage map, and the left data is the genetic distance between the markers.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1 wheat powdery mildew resistance GenePmDTMObtaining of Co-segregating molecular tags
(one) (Ma X Hui Xian hong) F2Construction of populations andPmDTMselection of segment recombinants
(1) Hybridizing the parent barley (female parent) resisting powdery mildew with the parent Ping Xian hong (male parent) resisting powdery mildew to obtain a hybrid F1，F1Selfing to produce F containing 1,192 individuals2A population;
(2) extracting F by SDS method2DNA of individual plants in the population; by usingPmDTMBoundary marks GWM337 and BARC229 (http:// heat. pw. usda. gov/GG2/index. shtml) from F2173 individuals were selected from the population that had recombined in this region.
(II)173F2Powdery mildew resistance identification of generation recombinant selfing progeny
Powdery mildew resistance identification was performed in the plant growth chamber from each F2Randomly selecting 25 seeds from selfing progeny of the generation recombinant, and planting the seeds in a plug tray with 72 holes; in the first leaf and first heart stage, the epidemic physiological race E09 of powdery mildew is used for inoculation, and after 7 to 10 days, the disease-resistant reaction type of each individual plant is investigated according to the grading standard of 0 to 4 grades; the growth conditions of the wheat are set as 16 hours of illumination, 8 hours of darkness, the temperature in the illumination is 22 ℃, and the temperature in the darkness is 18 ℃. The identification result shows that: 42F2The selfed progeny of the generation recombinant are all shown to be disease-resistant, 42F2The selfed progeny of the generation recombinants all appeared to be susceptible, and another 84F2The inbred progeny of the generation recombinant has resistance and infection separation.
(III) development of polymorphic KASP markers and recombinant genotyping
(1) Extracting disease-resistant parent barley, susceptible parent Huixian red and F by SDS method2DNA of the generation recombinant; according toPmDTMSearching SNP and InDel variation between barley and red in Hui county by using Chinese spring reference sequence information corresponding to the section and sequence information of cloned candidate genes, developing KASP markers, and performing polymorphism screening. The sequence alignment results show that: candidate genes in the barley genome compared to Hui county RedTraesCS1D02G058900There is a deletion of 6 bases (5 '-AAAGGA-3') in exon 5. From this 6-base InDel, 1 polymorphic KASP marker HNU58900 (FIG. 1) was developed.
(2) 173F were amplified using HNU58900 and the two-sided SSR marker GWM337 and BARC2292The DNA of the recombinants was used to obtain the marker genotype data of each recombinant (FIG. 1).
The PCR amplification method adopted by the SSR marker comprises the following steps: the PCR reaction volume was 25. mu.L, with 12.5. mu.L of 2 XTAQA PlusMaster Mix II (Vazyme, Biotech), 1.0. mu.L each of 10. mu.M primers, 100ng template DNA, and 25. mu.L of water; the PCR amplification program comprises pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 15s, annealing at 55-60 ℃ for 20s, extension at 72 ℃ for 40s, circulation for 35 times, and final extension at 72 ℃ for 5 minutes; PCR amplification is carried out on a PTC-225 amplification instrument, the amplification product is subjected to electrophoretic separation on 8% non-denaturing polyacrylamide gel, silver staining and photographing are carried out, and the result is recorded.
The KASP marker adopts a PCR amplification method: the PCR reaction volume was 10. mu.L, with 5.0. mu.L of 2 XKASP Mastermix (LGC Genomics), 0.047. mu.L of 90. mu.M co-primer (HNU 58900-F), 0.047. mu.L of 36. mu.M allele-specific primers (HNU 58900-FAM-R and HNU 58900-HEX-R), 100ng of template DNA, and 10. mu.L of water; PCR amplification adopts touchdown program: pre-denaturation at 94 ℃ for 15 minutes, denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, 10 cycles, and annealing temperature reduction by 0.6 ℃ per cycle; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, 32 cycles; reacting at 37 ℃ for 60s, and reading a fluorescence signal; amplification was performed on a LightCycler480 II system (Roche, Indianapolis, IN, USA) fluorescent quantitative PCR instrument, and the fluorescence signal values of the amplified products were analyzed using LightCycler480 software v1.5.1.62 software, and the results were recorded.
(IV) obtaining Co-segregating molecular markers
(1) According to each F2Disease-resistant response type speculation of generation recombinant selfed progenyPmDTMGenotype: if a certain F2The selfing progeny of the generation recombinant are all resistant to diseases, and are presumed to carry powdery mildew resistant allele consistent with barleyPmDTM/PmDTM(ii) a If the selfed progeny are all susceptible, the selfed progeny are presumed to carry powdery mildew allele consistent with Huixian redpmDTM/pmDTM(ii) a If the selfed progeny has resistance and infection separation, the selfed progeny is presumed to carry heterozygous allelesPmDTM/pmDTM。
(2) Comparison of the marker genotype of each recombinant withPmDTMGenotype, calculation of marker-to-marker and marker-toPmDTMThe number of recombinations occurring in between; the results show that GWM337 and GWMPmDTMBetween 78 recombinants, BARC229 andPmDTMthere were 95 recombinants in between, and HNU58900 andPmDTMno recombination occurred in between.
(3) According to the law of chain exchange, user= {2-(4-6*n/N)1/2}/3 (N is the number of recombinant individuals, N is F2Population total individual plant number) and using a Kosambi mapping functiond= ln{(1+2r)/(1-2r)}/4 willrConversion to picture distanced. The results show that GWM337 and GWMPmDTMThe genetic distance between the two is 3.37 cM, BARC229 andPmDTMthe genetic distance between them was 4.07cM, and HNU58900 andPmDTMthe genetic distance between them is 0 cM. Constructed using Mapdraw 2.0 as shown in FIG. 2PmDTMGenetic linkage maps of regions.
Example 2 wheat powdery mildew resistance GenePmDTMApplication of coseparation molecular marker
The identification of powdery mildew resistance was performed on a wheat core germplasm population containing 428 parts of material. The identification was carried out in a plant growth chamber in the same manner as in example 1. 428 parts of the genomic DNA of the material were extracted by SDS method and amplified by PCR using HNU 58900. The method of amplification of KASP marker is the same as in example 1. The amplification products were analyzed for fluorescence signal values using LightCycler480 software v1.5.1.62 software and the results were recorded.
The results show that: of the 428 parts wheat core germplasm materials, only 2 parts of materials carried powdery mildew resistant alleles consistent with those of barley, both of which showed broad-spectrum resistance to powdery mildew. Therefore, the KASP marker HNU58900 provided by the invention can accurately screen the gene containing the wheat powdery mildew resistancePmDTMThe breeding selection efficiency can be greatly improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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Claims (8)
1. Wheat powdery mildew resistance genePmDTMThe KASP marker of (1), characterized in that: the KASP marker is a co-dominant molecular marker HNU 58900.
2. The wheat powdery mildew resistance gene of claim 1PmDTMThe KASP marker of (1), characterized in that: the primers of the codominant molecular marker HNU58900 comprise a common primer HNU5890-F sequence and a specific primer pair HNU58900-FAM-R and HNU 58900-HEX-R; wherein the HNU5890-F sequence is shown as SEQ ID NO.1, the HNU58900-FAM-R sequence is shown as SEQ ID NO.2, and the HNU58900-HEX-R sequence is shown as SEQ ID NO. 3.
3. Identification of wheat powdery mildew resistance gene using KASP marker of claim 2PmDTMThe method is characterized by comprising the following steps: to stand forDetecting DNA of wheat as a template, using a common primer and a specific primer pair of a molecular marker HNU58900 as primers, carrying out PCR amplification, detecting a fluorescence signal carried by an amplification product by using LightCycler480 software v1.5.1.62 software, and judging whether the wheat anti-powdery mildew gene is carried or not by the fluorescence signalPmDTM。
4. The KASP marker of claim 3 identifying a wheat powdery mildew resistance genePmDTMThe method is characterized in that whether the wheat powdery mildew resistance gene is carried or not is judged through a fluorescent signalPmDTMThe method comprises the following steps: if the amplification product mainly carries FAM signals, the wheat to be detected is marked to carry disease-resistant allelesPmDTM/PmDTM(ii) a If the amplification product mainly carries HEX signal, the wheat to be detected is marked to carry disease allelepmDTM/pmDTM(ii) a If the amplification product carries two signals of FAM and HEX, the fact that the wheat to be detected carries heterozygous disease-resistant allele is markedPmDTM/pmDTM。
5. The method of screening powdery mildew resistant wheat using the KASP marker of claim 2, comprising the steps of: the method comprises the steps of carrying out PCR amplification by using DNA of wheat to be detected as a template and using a common primer and a specific primer pair of a molecular marker HNU58900 as primers, detecting a fluorescent signal carried by an amplification product by using LightCycler480 software v1.5.1.62 software, and screening the wheat to be detected, wherein the amplification product mainly carries an FAM signal, namely the wheat for resisting powdery mildew.
6. The method of any one of claims 3-5, wherein: in the PCR amplification procedure, the PCR reaction system is 10. mu.L, wherein 5.0. mu.L of 2 XKASP Master mix (LGC Genomics), 0.047. mu.L of 90. mu.M common primer, 0.047. mu.L of 36. mu.M specific primer, 100ng of template DNA, and ddH2O to 10 μ L; the PCR amplification program is pre-denaturation at 94 ℃ for 15min, denaturation at 94 ℃ for 20s, annealing at 61 ℃ for 60s, and 10 cycles, wherein the annealing temperature is reduced by 0.6 ℃ in each cycle; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, 32 cycles; the reaction was carried out at 37 ℃ for 60 s.
7. The KASP marker of claim 1 or 2 for identifying powdery mildew resistance genes in wheat germplasm resourcesPmDTMThe use of (1).
8. Use of a KASP marker as claimed in claim 1 or 2 in molecular breeding for the development of powdery mildew resistant wheat.
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