CN108300799B - High-throughput detection marker of wheat powdery mildew resistance gene Pm5e and application thereof in breeding - Google Patents
High-throughput detection marker of wheat powdery mildew resistance gene Pm5e and application thereof in breeding Download PDFInfo
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Abstract
The invention discloses two single nucleotide polymorphism molecular markers of a powdery mildew resistance gene Pm5e, which can be used for high-throughput marker-assisted selective breeding, and an application method thereof in breeding. Pm5e shows high resistance or immunity to powdery mildew races currently prevalent in China, and has important utilization value in breeding. The two detection markers are KASP markers, the distribution of the wheat powdery mildew resistance gene Pm5e in wheat varieties and the distribution situation of the wheat powdery mildew resistance gene Pm5e in segregation populations can be detected quickly, efficiently and in high throughput, the fine positioning and cloning of Pm5e are facilitated, the marker-assisted selection of Pm5e in breeding populations can be carried out, the powdery mildew resistance of wheat is improved by utilizing Pm5e, the influence on the yield of the wheat is reduced, and the powdery mildew resistance breeding efficiency of the wheat is improved.
Description
Technical Field
The invention relates to the technical field of crop molecular marker assisted breeding, in particular to a powdery mildew resistance gene of wheatPm5eThe high-throughput detection marker and the application thereof in breeding.
Background
Wheat (A), (B)Triticum aestivumL.) is the second crop of rice in China, the annual planting area is more than 2666.67 ten thousand mu, and the second crop accounts for about 27% of the total area of the crop. Therefore, ensuring the stable improvement of the yield and the quality of the wheat is an important factor which is related to the continuous improvement of food safety in China and the living standard of people.
Wheat powdery mildew is a worldwide disease and is distributed around the world, especially in areas with high humidity. The yield is reduced by 5 to 10 percent when the disease occurs, and the yield is reduced by more than 20 percent in seriously ill fields. In recent years, the incidence range of wheat powdery mildew is continuously expanded, the damage degree is continuously increased, and the wheat powdery mildew is a serious threat for the current wheat production. Breeding and using high-efficiency disease-resistant varieties is an economic, safe and effective measure for preventing and treating powdery mildew. The basis of powdery mildew disease-resistant breeding is high-efficiency and diversified resistance sources. The intensive research on the resistance expression and the genetic characteristics of the disease-resistant genes can help to effectively utilize the disease-resistant genes. Different wheat varieties have different resistance and different disease resistance genes. Up to now, there are 86 mass anti-powdery mildew genes that have been located and given formal nomenclature, i.e.Pm1-Pm60WhereinPm1、Pm2、Pm3、Pm4、Pm5AndPm24there are 5, 2, 17, 4, 6 and 2 alleles, respectively,Pm18=Pm1c，Pm22=Pm1e，Pm23=Pm4c，Pm31=Pm21. These 86 alleles are located at 56 chromosomal loci of 21 chromosomes. Wherein,Pm5the gene is located on 7BL chromosome, contains 6 alleles, and is respectivelyPm5a、Pm5b、Pm5c、Pm5d、Pm5eAndPm5f。
Pm5ethe gene is a recessive powdery mildew resistance gene, and the winter wheat variety rejuvenation 30 carrying the gene is obtained by systematic breeding from Jingyang 30, shows high resistance or immunity to powdery mildew races currently prevalent in China, and has important utilization value. The former people use SSR markers to locate the gene and preliminarily locate the gene on a 7BL chromosome, but the genetic distances between the two SSR markers and the target gene are 11cM and 6.6cM respectively (Huang et al 2003),on the basis of the results, 2 SSR markers are developed by Wanghonggang et al (2008), the genetic distances of the SSR markers are 5.1cM and 4.9cM respectively, and the two SSR markers shorten the genetic distance between the Pm5e gene and the marker to a certain extent, but the SSR markers still have the problems of long genetic distance, low polymorphism level, low detection efficiency and the like; then Liu Shi Yong et al (2008) screened out specific SNP locus by BSR-Seq, and transformed into specific primer, and further pairedPm5eGene mapping ofPm5eThe gene mapping between EST markers CJ729392 and CJ584170 improved primer specificity, but the level of polymorphism was still low and the genetic distance between 2 markers was large (23.5 cM).
Competitive allelic polymorphism PCR (KASP) is one of the mainstream methods for genotyping single SNP internationally at present, and the principle is to type SNP and InDel according to the Specific matching of the terminal bases of primers, wherein the SNP and InDel comprise 2 forward primers and 1 reverse primer, wherein the terminal bases of the forward primers are different (namely two SNPs), and the 5' ends of the two forward primers respectively carry fluorescent linker sequences FAM or HEX with different sequences, so the specificity of the SNP can be detected according to the difference of fluorescent signals. The KASP marker can be used for carrying out accurate biallelic gene judgment on SNP loci, can be used for detecting a large number of samples at the same time, has the advantages of good genetic stability and the like, and is a high-flux molecular marker.
Therefore, SNP related to wheat powdery mildew resistance is screened out and further developed into KASP marker suitable for high-throughput and high-efficiency detection, and the method is applied to selection of wheat powdery mildew resistant materials and has important significance in aspects of improving yield and quality of wheat and the like.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a wheat powdery mildew resistance genePm5eThe high-throughput detection marker and the application thereof in breeding.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided an SNP marker associated with wheat powdery mildew resistance, comprising a total of 2 SNP markers, marker AX-95000860 and marker AX-94638908; the markers AX-95000860 and AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of the marker AX-95000860 is 708115366, and the basic group at SNP site is C or T; the physical position of the marker AX-94638908 is 721220492, and the base at the SNP site is C or T.
The application of the SNP marker in wheat powdery mildew resistant breeding is also the protection scope of the invention.
In a second aspect of the present invention, there is provided a gene for detecting and resisting powdery mildew developed based on KASP technologyPm5ePrimers for closely linked SNPs; the gene for resisting powdery mildewPm5eThe closely linked SNPs were AX-95000860 and AX-94638908, respectively; the physical position of AX-95000860 is 708115366, and the base at the SNP site is C or T; the physical position of the molecular marker AX-94638908 is 721220492, and the base at the SNP site is C or T;
wherein, the primer sequences for detecting AX-95000860 are respectively shown in SEQ ID NO. 1-3; the sequences of the primers for detecting AX-94638908 are shown in SEQ ID NO.4-6 respectively.
In a third aspect of the present invention, there is provided a detection reagent or a kit comprising the above primer.
The invention also provides application of the primer, the detection reagent or the kit in the preparation of wheat powdery mildew resistant materials.
The invention also provides application of the primer, the detection reagent or the kit in molecular marker assisted wheat powdery mildew resistance selective breeding.
The invention also provides application of the primer, the detection reagent or the kit in breeding wheat resources with powdery mildew resistance.
In a fourth aspect of the invention, a gene for detecting and resisting powdery mildew is providedPm5eA method of closely linked SNPs comprising the steps of:
(1) extracting DNA of a wheat sample to be detected;
(2) taking 3 mul of template DNA with the concentration of 20 ng/mul, 0.0825 mul of mixed primer shown by SEQ ID NO.1-3 or mixed primer shown by SEQ ID NO.4-6 and 2 XMaster Mix 3 mul for PCR amplification;
(3) and analyzing the genotype of the PCR amplification product by using a fluorescence detector.
Preferably, in step (2), the PCR amplification conditions are:
1) pre-denaturation at 94 ℃ for 5 min;
2) denaturation at 94 ℃ for 20 s;
3) annealing at 65 ℃ for 30s, and circulating the steps 2) to 3) for 10 times, wherein the annealing temperature is reduced by 0.8 ℃ in each circulation;
4) denaturation at 94 ℃ for 20 s;
5) annealing at 57 ℃ for 30s, and circulating the steps 4) -5) for 38 times;
6) storing at 4 ℃.
The invention has the beneficial effects that:
the invention provides two wheat powdery mildew resistance genes with important utilization value in breedingPm5eThe SNP markers (marker AX-95000860 and marker AX-94638908) of (1), and the KASP detection marker is developed based on these two newly found SNP markers, and can be used for rapid, efficient and high-throughput detectionPm5eDistribution in wheat varieties and distribution in segregating populations is favorable toPm5eFine localization and cloning, and can be paired in breeding populationsPm5ePerforming marker assisted selection usingPm5eImproving the powdery mildew resistance of wheat, reducing the influence on the wheat yield and improving the powdery mildew resistance breeding effect of wheat
Drawings
FIG. 1: AX-95000860 labeled KASP typing results, the dots near the horizontal axis represent the C (powdery mildew resistance) genotype, the dots near the vertical axis represent the T (powdery mildew resistance) genotype, and the dots at the diagonal line represent the heterozygote; x represents a well to which no DNA was added, ■ represents ddH2O blank control.
FIG. 2: AX-94638908 labeled KASP typing results, the dots near the horizontal axis represent the C (powdery mildew resistance) genotype, the dots near the vertical axis represent the T (powdery mildew resistance) genotype, and the dots near the diagonal line represent the heterozygote type; x represents a well to which no DNA was added, ■ represents ddH2O blank control.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, SNP related to wheat powdery mildew resistance is screened out and developed into KASP detection marker capable of accurately detecting SNP sites, so that the important significance is provided for the selection of wheat powdery mildew resistant materials, the improvement of wheat yield and quality and the like. To developPm5eThe invention utilizes 35K gene chip technology, screens out probes with polymorphic SNP loci between a disease resistance pool and a disease susceptibility pool by constructing the disease resistance pool and the disease susceptibility pool, is used for searching a wheat genome sequence database, finds out the probes which are compared to 7BL chromosome loci, and has 88 probes in total. According to the results of the studies published by the predecessors,Pm5ethe gene was located between two EST sequences CJ729392 and CJ584170, which are 23.5cM apart genetically. Using the comparison result to select the specific probe between the two EST sequences, and further comparing and analyzing the probe containing SNP specific site between the two EST sequences to convert SNP into KASP marker containing powdery mildew resistant genePm5eThe rejuvenation 30 of the strain is hybridized with 214F obtained by high-susceptibility powdery mildew material Chancellor2:3Genotyping in generation group, and finding two heels by linkage analysisPm5eKASP marker with a closer gene linkage, useful forPm5eHigh-throughput detection and marker-assisted breeding.
The invention provides two wheat powdery mildew resistance genes with important utilization value in breedingPm5eThe molecular markers of (a) are respectively: molecular marker AX-95000860 and molecular marker AX-94638908; the two molecular markers (namely SNP) are both positioned on wheat 7BL chromosome, wherein the physical position of the molecular marker AX-95000860 SNP is 708115366, and the base at the SNP site is C or T; the physical position of the molecular marker AX-94638908 SNP is 721220492, and the base at the SNP site is C or T. (SNP location was determined from the currently published wheat gene sequence database).
SNP marker AX-95000860 and marker AX-94638908 are the first markers of the inventionIt was found and proposed that these two SNP markers andPm5ethe two SNP markers are closely linked and applied toPm5eThe gene transfer has important practical significance for accelerating the breeding of powdery mildew resistant wheat varieties and improving the breeding efficiency.
In order to detect the specificity of the two key SNPs in the Chinese wheat powdery mildew group, the method is favorable for resisting powdery mildew genesPm5eThe invention discloses a primer for detecting SNP closely linked with powdery mildew resistance gene Pm5e based on KASP technology, wherein the primer sequence is as follows:
AX-95000860-KASP-FAM_C-R：5＇CAGGATTGGACTCGGCTGGAAAC3＇，SEQ ID NO.1；
AX-95000860-KASP-HEX_T-S:5＇CAGGATTGGACTCGGCTGGAAAT3＇，SEQ ID NO.2；
AX-95000860-KASP-R:5＇ATGTCAGGTCACCACGATGC3＇，SEQ ID NO.3。
AX-94638908-KASP-FAM_C-R：5＇ATGATAACATGCTGCGCATGAC3＇，SEQ ID NO.4；
AX-94638908-KASP- HEX_T-S:5＇ATGATAACATGCTGCGCATGAT3＇，SEQ ID NO.5；
AX-94638908-KASP-R:5＇TACACAAACTAGGTGGAGGTACAAC3＇，SEQ ID NO.6。
after obtaining the above primers, the inventors further provide a specific detection method as follows:
1. extracting the whole genome DNA of the wheat to be detected by using a CTAB method;
2. 214F from 30 rejuvenates hybridized with Chancellor2Performing phenotype identification on powdery mildew of the population, and selecting high-resistance and high-sensitivity F2Respectively mixing 15 single plants into an anti-pool and a sensing pool, and finding out SNP (single nucleotide polymorphism) sites near a target gene by using a 35K gene chip;
3. these SNP sites were converted into KASP detection markers using primer5.0, and the genomic DNA extracted in the previous step was subjected to PCR amplification on a Q-Cycler 96 PCR instrument (Hain Life science UK. UK) and SNP detection on an ABI Quant study 12K Flex real-time fluorescent quantitative PCR system (Life Technologies Corporation USA);
the criterion for the decision is determined by the color of the dots and the position of the cluster on the horizontal or vertical axis: the signal is blue, is gathered near the horizontal axis and is gathered together with the powdery mildew resistance control, and the powdery mildew resistance genotype is judged; the signal is red, and is gathered together with the longitudinal axis and the susceptible powdery mildew control to judge the susceptible powdery mildew genotype; the signal is green, and is not aggregated with two coordinate axes and resistance and susceptibility controls, and the heterozygous genotype is judged near a y = x straight line;
4. analyzing the above obtained andPm5edistribution of SNP markers closely linked to genes in wheat population.
The specific method comprises the following steps:
extracting the whole genome DNA of the wheat to be detected by using a CTAB method:
(a) placing young and tender wheat leaves in a 96-hole deep-hole plate, freezing by using liquid nitrogen, and grinding into powder on a tissue grinder; (b) adding 800 μ L CTAB buffer solution preheated to 65 ℃ into each hole in a 96-hole deep-hole plate, placing in a 65 ℃ water bath for 90min, and slightly shaking once every 10min to fully crack DNA; (c) adding 800 μ L chloroform isoamyl alcohol mixed solution with volume ratio of 24:1, and shaking gently for 10 min; (d) centrifuging at 12000r at 4 deg.C for 10min, collecting 600 μ L supernatant, and placing in a new clean 96-well deep-well plate (with the numbers corresponding to each other); (e) add 600. mu.L of pre-cooled isopropanol and 60. mu.L of 3M sodium acetate (pH = 5.2) and shake gently to see white DNA floc generation, place in-20 deg.C refrigerator for 60min to increase DNA yield. (f) Centrifuging at 4000r for 10min, pouring out supernatant, washing the precipitate with precooled 70% ethanol for 2-3 times, and air-drying until no alcohol smell exists; (g) adding 200 mul of ultrapure water to dissolve the DNA, and storing in a refrigerator at-20 ℃ for later use after the DNA is fully dissolved in the water.
AndPm5edevelopment of gene-linked KASP detection markers:
at present, 6 andPm5eclosely linked markers, comprising 4 SSR markers (Xgwm 783, Xgwm1267, Xwmc364 and Xbarc 065) and 2 EST markers (CJ 729392 and CJ 584170). Disease-resistant parent rejuvenation 30 containsPm5eThe gene, and the other parent, Chancellor, appeared to be highly susceptible to powdery mildew. Creating F with these two anti-sense materials2Separating the population of the plants,the total number of the strains is 214, and the phenotype identification of powdery mildew is carried out at the seedling stage and the adult stage. Respectively selecting high-resistance and high-sensitivity F according to the identification result2Each individual plant is 15 plants, and an anti-infection gene pool are constructed. And (3) identifying the polymorphic SNP sites of the anti-influenza gene pool by using a 35K gene chip technology, wherein 88 SNP sites are identified on 7 BL. The probe sequence containing the SNP site and two EST mark sequences are compared on a reference genome of Chinese spring by using a URGI website, 27 probes positioned between the two EST marks are screened, and a primer5.0 is used for designing a KASP primer. At 214F2Genotyping in segregating populations, finding two pairsPm5eThe SNP marker linked with the gene is further developed into a KASP detection marker.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The methods adopted by the invention are all the prior art in the field, and details are not shown.
Example 1: wheat genome DNA extraction
1) Collection of DNA-extracted leaves
In the three-leaf stage of the wheat, selecting young and tender wheat leaves, putting the young and tender wheat leaves into a corresponding 96-hole deep-hole plate according to the number, and operating on an ice box to prevent DNA degradation;
2) extracting wheat genome DNA by using a CTAB method; storing at-20 deg.C for use.
The steps are as follows:
(a) placing young and tender wheat leaves in a 96-hole deep-hole plate, freezing by using liquid nitrogen, and grinding into powder on a tissue grinder; (b) adding 600 μ L CTAB extract preheated to 65 deg.C into each well of 96-well deep-well plate, placing in 65 deg.C water bath for 60min, and gently shaking once every 10min to fully crack DNA; (c) adding 600 μ L of chloroform isoamyl alcohol mixed solution with volume ratio of 24:1, and shaking gently for 10 min; (d) centrifuging at 12000r at 4 deg.C for 10min, collecting 500 μ L supernatant, and placing in a new clean 96-well deep-well plate (with the numbers corresponding to each other); (e) add 500. mu.L of pre-cooled isopropanol and 50. mu.L of 3M sodium acetate (pH = 5.2) and shake gently to see white DNA floc generation, place in-20 deg.C refrigerator and freeze for 20min to increase DNA yield. (f) Centrifuging at 4000r for 10min, pouring out supernatant, washing the precipitate with precooled 70% ethanol for 2-3 times, and air-drying until no alcohol smell exists; (g) adding 200 mul of ultrapure water to dissolve the DNA, and storing in a refrigerator at-20 ℃ for later use after the DNA is fully dissolved in the water.
The preparation method of the solution used in the above step is as follows:
(a) preparation of CTAB extracting solution
Deionized water was added to a volume of 1000 ml.
（b）3M NaAc (pH=5.2)
Weighing 40.8g of sodium acetate trihydrate, pouring the sodium acetate trihydrate into a beaker, adding water to 80ml, adjusting the pH value to 5.2 by using glacial acetic acid, and fixing the volume to 100 ml.
（c）1M Tris-HCl (pH=8.0)
Dissolving 121.1g Tris (or 157.6g Tris HCl) in 800ml water, cooling to room temperature, adding concentrated hydrochloric acid (or NaOH) to adjust pH to 8.0 (about 4.2 ml), and diluting to 1000 ml.
（d）0.5M EDTA·2Na (pH =8.0)
186.1g of EDTA-2 Na was added to 800ml of water, a pH meter was placed in the solution, the pH was adjusted with NaOH (about 20g of NaOH pellets were required), and the EDTA-2 Na was dissolved by stirring with NaOH, and the solution was clarified by adjusting the pH to about 7.8, and the volume was adjusted to 1000 ml.
(e) Chloroform/isoamyl alcohol (24: 1)
Chloroform according to volume ratio: isoamyl alcohol =24: 1.
(f) 1000ml of 70% alcohol
300ml of deionized water was added to 700ml of absolute ethanol.
Example 2: rejuvenation in 30Pm5eDevelopment of closely linked markers in genes
At present, 6 andPm5eclosely linked markers, comprising 4 SSR markers (Xgwm 783, Xgwm1267, Xwmc364 and Xbarc 065) and 2 EST markers (CJ 729392 and CJ 584170). Because of the low specificity of SSR markers, the KASP detection marker developed in this study was done on the basis of two EST markers. The disease-resistant material rejuvenation 30 containsPm5eThe gene, Chancellor, appears to be highly susceptible to powdery mildew. Creation of F Using these two resistant parents2The colony is separated, 214 strains are totally obtained, and the phenotype identification of powdery mildew is carried out at the seedling stage and the adult stage. Respectively selecting high-resistance and high-sensitivity F according to the identification result2Each individual plant is 15 plants, and an anti-infection gene pool are constructed. And (3) identifying the SNP sites of the anti-influenza gene pool by using a 35K gene chip technology, wherein 88 SNP sites are identified on 7 BL. The probe sequence containing the SNP locus and two EST mark sequences are compared on a reference genome of Chinese spring by using a URGI website, 27 probes positioned between the two EST marks are screened, and a primer5.0 is designed into a KASP detection mark. 16 markers with hairpin structure, primer dimer formed between primers, and Tm value over 3 deg.C between upstream and downstream primers are designed by removing at F2Genotyping is carried out in a segregation population, 3 KASP detection markers can well distinguish the genotypes of all individuals, and only two KASP detection markers and the KASP detection markers disclosed in the patent are discovered through linkage analysisPm5eThe genes are closely linked.
Example 3: primer dilution was mixed with KASP assay primers:
after three primers of AX-95000860 were diluted to 100. mu.M with Tris HCl, the ratio by volume of AX-95000860-KASP-FAM _ C-R: AX-95000860-KASP-HEX _ T-S: AX-95000860-KASP-R: tris HCl = 6: 6: 15: 23, subpackaging and storing at-20 ℃ as KASP assay primers.
The method for diluting and mixing the primer AX-94638908 was the same as that for AX-95000860.
Example 4: PCR amplification system and procedure
Preparation of PCR System
The system was prepared on ice according to the following table, 3. mu.L (about 20 ng/. mu.L) of template DNA, 2 × Master mix 3. mu.L (LGC Group UK), and 0.0825. mu.L of KASP assay primer (primers supplied by Saimer Feishell technology (China) Co., Ltd.).
| Medicine and food additive | Volume of |
| Template DNA | 3 μ L (about 20ng/μ L) |
| 2x Master Mix | 3μL |
| KASP assay primer | 0.0825μL |
| Sum of | 6.0825μL |
The PCR procedure was as follows:
1) pre-denaturation at 94 ℃ for 5 min;
2) denaturation at 94 ℃ for 20 s;
3) annealing at 65 ℃ for 30s (0.8 ℃ per cycle), and cycling through steps 2) -3) 10 times;
4) denaturation at 94 ℃ for 20 s;
5) annealing at 57 ℃ for 30s, and circulating the steps 4) -5) for 38 times;
6) storing at 4 ℃.
Example 5: analysis of results
The amplified PCR system was subjected to SNP distribution results collection using an ABI Quant Studio 12K Flex real-time fluorescent quantitative PCR System, and the distribution results of AX-95000860 are shown in FIG. 1, in which the horizontal axis represents powdery mildew resistance genotype and the vertical axis represents powdery mildew resistance genotypeThe axis is the genotype of the susceptible powdery mildew, 30 rejuvenescence is hybridized with chancellor F2After 214 materials of the generation group are detected, 48 powdery mildew resistant genotype materials (the genotype is the same as that of powdery mildew resistant rejuvenation 30 of the invention) and 51 powdery mildew susceptible genotype materials (the genotype is the same as that of powdery mildew resistant Chanchalor of the invention) are obtained, 112 heterozygous genotype materials (the heterozygous genotype is different from that of powdery mildew resistant and susceptible control parents of the invention) are obtained, and 3 materials have no genotype detected due to the DNA quality.
The distribution results of AX-94638908 are shown in FIG. 2, and the detection and analysis of 214 populations of rejuvenation 30 and chancellor hybrid F2 generations similarly resulted in 56 powdery mildew resistant genotype materials, 47 powdery mildew resistant genotype materials, 111 heterozygous genotype materials, and 2 materials with no genotype detected due to DNA quality. And transforming the phenotype into the genotype according to the result and combining the identification result of the powdery mildew phenotype, drawing a linkage map, and further researching to accelerate the fine positioning and cloning of the gene.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> high-flux detection marker of wheat powdery mildew resistance gene Pm5e and application thereof in breeding
<130> 2018
<160> 6
<170> PatentIn version 3.5
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Claims (8)
1. The application of SNP markers related to wheat powdery mildew resistance in wheat powdery mildew resistance breeding; the SNP markers are respectively a marker AX-95000860 and a marker AX-94638908; the marker AX-95000860 and the marker AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of the marker AX-95000860 is 708115366, and the basic group at the SNP site is C or T; the physical position of the marker AX-94638908 is 721220492, and the base at the SNP site is C or T;
the physical position of the SNP marker is determined based on the Chinese spring reference genome with the version number of IWGSC reference v1.0 on the URGI website;
the C genotype of the marker AX-95000860 is the powdery mildew resistance genotype, and the T genotype is the powdery mildew susceptibility genotype; the genotype C of AX-94638908 is powdery mildew resistant genotype, and the genotype T is powdery mildew susceptible genotype.
2. KaSP technology development-based powdery mildew resistance gene for detectionPm5ePrimers for SNPs closely linked to each other, characterized in that the SNP is a gene resistant to powdery mildewPm5eThe closely linked SNPs were AX-95000860 and AX-94638908, respectively; AX-95000860 and AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of AX-95000860 is 708115366, and the base at SNP site is C or T; the physical position of AX-94638908 is 721220492, and the base at the SNP site is C or T; the physical position of the SNP is determined based on the Chinese spring reference genome with the version number of IWGSC reference v1.0 on the URGI website;
wherein the sequence of the primer for detecting AX-95000860 is shown in SEQ ID NO. 1-3; the sequence of the primer for detecting AX-94638908 is shown in SEQ ID NO. 4-6.
3. A test kit comprising the primer of claim 2.
4. The application of the primer of claim 2 or the detection kit of claim 3 in the preparation of wheat powdery mildew resistant materials, wherein the primer or the detection kit is used for detecting powdery mildew resistant genesPm5eClosely linked SNPs, which are a marker AX-95000860 and a marker AX-94638908, respectively, wherein the C genotype of the marker AX-95000860 is the powdery mildew resistance genotype, and the T genotype is the powdery mildew susceptibility genotype; the C genotype of AX-94638908 is powdery mildew resistant genotype, and the T genotype is powdery mildew susceptible genotype;
the marker AX-95000860 and the marker AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of AX-95000860 is 708115366, and the base at SNP site is C or T; the physical position of AX-94638908 is 721220492, and the base at the SNP site is C or T; the physical location of the SNP marker was determined based on the Chinese spring reference genome, version number IWGSC reference v1.0, on the URGI website.
5. Use of the primer according to claim 2 or the detection kit according to claim 3 in molecular marker assisted selective breeding of wheat for powdery mildew resistance, wherein the primer or the detection kit is used for detecting powdery mildew resistance genePm5eClosely linked SNPs, which are a marker AX-95000860 and a marker AX-94638908, respectively, wherein the C genotype of the marker AX-95000860 is the powdery mildew resistance genotype, and the T genotype is the powdery mildew susceptibility genotype; the C genotype of AX-94638908 is powdery mildew resistant genotype, and the T genotype is powdery mildew susceptible genotype;
the marker AX-95000860 and the marker AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of AX-95000860 is 708115366, and the base at SNP site is C or T; the physical position of AX-94638908 is 721220492, and the base at the SNP site is C or T; the physical location of the SNP marker was determined based on the Chinese spring reference genome, version number IWGSC reference v1.0, on the URGI website.
6. Use of the primer according to claim 2 or the detection kit according to claim 3 for breeding wheat resources with powdery mildew resistance, wherein the primer or the detection kit is used for detecting and resisting powdery mildew genePm5eClosely linked SNPs, which are a marker AX-95000860 and a marker AX-94638908, respectively, wherein the C genotype of the marker AX-95000860 is the powdery mildew resistance genotype, and the T genotype is the powdery mildew susceptibility genotype; the C genotype of AX-94638908 is powdery mildew resistant genotype, and the T genotype is powdery mildew susceptible genotype;
the marker AX-95000860 and the marker AX-94638908 are both located on wheat 7BL chromosome, wherein the physical position of AX-95000860 is 708115366, and the base at SNP site is C or T; the physical position of AX-94638908 is 721220492, and the base at the SNP site is C or T; the physical location of the SNP marker was determined based on the Chinese spring reference genome, version number IWGSC reference v1.0, on the URGI website.
7. Gene for detecting and resisting powdery mildewPm5eThe method for detecting closely linked SNPs is characterized by comprising the following steps:
(1) extracting DNA of a wheat sample to be detected;
(2) taking 3 mul of template DNA with the concentration of 20 ng/mul, 0.0825 mul of mixed primer shown by SEQ ID NO.1-3 or mixed primer shown by SEQ ID NO.4-6 and 2 XMaster Mix 3 mul for PCR amplification;
(3) and detecting the fluorescent signal of the PCR amplification product by using a fluorescence detector to perform genotyping.
8. The method of claim 7, wherein in step (2), the PCR amplification conditions are:
1) pre-denaturation at 94 ℃ for 5 min;
2) denaturation at 94 ℃ for 20 s;
3) annealing at 65 ℃ for 30s, and circulating the steps 2) to 3) for 10 times, wherein the annealing temperature is reduced by 0.8 ℃ in each circulation;
4) denaturation at 94 ℃ for 20 s;
5) annealing at 57 ℃ for 30s, and circulating the steps 4) -5) for 38 times;
6) storing at 4 ℃.
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| CN109295249B (en) * | 2018-10-28 | 2021-08-03 | 山东农业大学 | Wheat powdery mildew adult-plant-stage resistance QTL and molecular marker thereof |
| CN109468409B (en) * | 2019-01-04 | 2022-04-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | High-throughput detection marker for wheat powdery mildew resistance gene Pm2b and application thereof |
| CN111763249B (en) * | 2019-03-08 | 2021-11-16 | 中国科学院遗传与发育生物学研究所 | Plant powdery mildew resistance related protein Pm5e, and coding gene and application thereof |
| CN111719008B (en) * | 2019-03-19 | 2021-11-09 | 中国科学院遗传与发育生物学研究所 | SNP coseparated with wheat powdery mildew disease-resistant gene Pm5e and application thereof |
| CN113234852B (en) * | 2021-06-30 | 2022-11-04 | 四川省农业科学院作物研究所 | Molecular marker and primer group for identifying wheat powdery mildew resistance and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001098479A2 (en) * | 2000-06-20 | 2001-12-27 | Plant Bioscience Limited | Plant resistance gene |
| CN101659988A (en) * | 2008-08-29 | 2010-03-03 | 李祥 | SSR markers of powdery mildew resistance gene in wheat Fu Zhuang 30 and method for acquiring same |
| RU2535985C1 (en) * | 2013-08-02 | 2014-12-20 | Федеральное государственное бюджетное учреждение науки Институт цитологии и генетики Сибирского отделения Российской академии наук (ИЦиг СО РАН) | Method of creation of lines of spring soft wheat with elongated period of earing and complex resistance to fungal diseases |
-
2018
- 2018-04-19 CN CN201810353673.3A patent/CN108300799B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001098479A2 (en) * | 2000-06-20 | 2001-12-27 | Plant Bioscience Limited | Plant resistance gene |
| CN101659988A (en) * | 2008-08-29 | 2010-03-03 | 李祥 | SSR markers of powdery mildew resistance gene in wheat Fu Zhuang 30 and method for acquiring same |
| RU2535985C1 (en) * | 2013-08-02 | 2014-12-20 | Федеральное государственное бюджетное учреждение науки Институт цитологии и генетики Сибирского отделения Российской академии наук (ИЦиг СО РАН) | Method of creation of lines of spring soft wheat with elongated period of earing and complex resistance to fungal diseases |
Non-Patent Citations (8)
| Title |
|---|
| 5份小麦农家品种的抗白粉病基因分析及定位;徐晓丹;《中国博士学位论文全文数据库农业科技辑》;20170715(第7期);D046-11 * |
| Genetic mapping of three alleles at the Pm3 locus conferring powdery mildew resistance in common wheat (Triticum aestivum L.);Xiu-Qiang Huang等;《Genome》;20041231;第47卷(第6期);第1130-1136页 * |
| Identification of Pm58 from Aegilops tauschii;Andrew T Wiersma等;《Theor Appl Genet》;20170731;第130卷(第6期);第1123-1133页 * |
| Identification of single nucleotide polymorphism (SNP) markers closely linked with powdery mildew resistance gene Pm5e in wheat;Yuan XiuFang等;《Journal of Plant Breeding and Crop Science》;20200331;第12卷(第1期);第1-7页 * |
| Identification of the resistance gene to powdery mildew in Chinese wheat landrace Baiyouyantiao;Xu Xiao dan等;《JOURNAL OF INTEGRATIVE AGRICULTURE》;20180131;第17卷(第1期);第37-45页 * |
| Microsatellite mapping of the powdery mildew resistance gene Pm5e in common wheat (Triticum aestivum L.);X Q Huang等;《Theor Appl Genet》;20030331;第106卷(第5期);第858-865页 * |
| 利用SNP 芯片和BSA 分析规模化定位小麦抗白粉病基因;吴秋红等;《作物学报》;20180131;第44卷(第1期);第1-14页 * |
| 小麦抗白粉病基因Pm5e的SSR标记研究;朱玉丽等;《分子植物育种》;20081231;第6卷(第6期);第1080-1084页 * |
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