CN109706263B - SNP molecular marker linked with wheat stripe rust resistance gene QYr. sicau-1B-1 and application - Google Patents
SNP molecular marker linked with wheat stripe rust resistance gene QYr. sicau-1B-1 and application Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular breeding, and provides an SNP molecular marker linked with a wheat stripe rust resistance gene QYr.sicau-1B.1 and application thereof, wherein the SNP molecular marker is KASP-Yr and can be obtained by amplifying a primer with a nucleotide sequence shown as SEQ ID NO. 1-3. Detection and analysis show that the molecular marker can accurately track the wheat stripe rust resistance QTL QYr. sicau-1B.1, and predict the stripe rust resistance of wheat, thereby facilitating molecular design breeding. The invention also discloses a method for identifying the molecular marker of the wheat stripe rust resistance QTL QYr.sicau-1B.1, and the method provided by the invention can enhance the accuracy of stripe rust resistance prediction so as to quickly screen out a wheat variety or a wheat strain with increased stripe rust resistance for breeding and greatly accelerate the breeding process of a wheat high-yield variety.
Description
Technical Field
The invention belongs to the field of molecular biology and crop genetic breeding, and particularly relates to a molecular marker related to wheat stripe rust resistance, in particular to a wheat stripe rust resistance gene QYr.sicau-1B-1 linked SNP molecular marker, a primer pair for amplifying the molecular marker and application of the molecular marker.
Background
Wheat is an important grain crop in China, the perennial planting area is more than 2000 million hectares, and the wheat occupies about 27 percent of the grain crop area; the total yield is more than 1 hundred million tons, and accounts for about 22 percent of the yield of the food crops. Wheat stripe rust is an important disease in wheat production in China, when the wheat stripe rust is invaded by stripe rust, yellow necrotic spots appear on wheat leaves, yellow summer spores are generated, and the spores are turned into black at the later stage; the rust stripe causes the leaf to be green and unable to carry out photosynthesis, thereby reducing yield, and even causing no grain harvest in serious cases. Many researches at home and abroad are dedicated to the research of new resistant wheat varieties, and the excavation of the resistant genes plays an important role in breeding and popularizing disease-resistant varieties.
The wheat strain '20828' shows near-immune resistance to the epidemic physiological races of the stripe rust in China for more than 10 years in the southwest ecological region, has the characteristics of more small spikes, proper plant height, compact plant type and the like, and is a preferred parent for breeding.
Single Nucleotide Polymorphism (SNP) refers to a DNA sequence Polymorphism caused by a change such as a transition, a transversion, an insertion, or a deletion at a specific Nucleotide position in DNA in a genome. The technology is that known sequence information is utilized to compare and search SNP sites, and then specific primers are designed by utilizing the discovered variation sites to carry out PCR amplification on genome DNA or cDNA, so as to obtain specific polymorphic products based on the SNP sites, and finally, the polymorphism of the products is analyzed by utilizing the electrophoresis technology. The SNP markers have the advantages of large quantity and wide distribution; uneven distribution among individual genes and the entire genome; SNP allele frequencies are easily estimated.
KASP is a novel genotyping technology with low cost and high throughput characteristics by competitive Allele Specific PCR (KASP) developed by LGC (Laboratory of the Goverment Chemist) (http:// www.lgcgenomics.com), carries out accurate double-Allele genotyping on SNP and In Del sites by Specific matching of terminal bases of primers, and is widely applied to molecular marker-assisted selection of crops such as rice, wheat, soybean and the like.
Previously, researchers performed QTL mapping on the stripe rust resistance trait, and found that the QTL related to the stripe rust resistance trait exists widely in wheat and is distributed in each chromosome, wherein the largest number of stripe rust resistance QTL are mapped on the 1B chromosome. However, due to the high variation of the small species of pathogenic bacteria, a large number of genes for resisting stripe rust have or are losing resistance, so that many researches at home and abroad are dedicated to the research on new resistant wheat varieties, and the disease-resistant genes and the genetic characteristics of the wheat resistance need to be known, so that the wheat resistance is more reasonably and effectively applied, and more efficient resistant varieties are developed.
Disclosure of Invention
The invention aims to provide a molecular marker closely linked with wheat stripe rust resistance QTL QYr. sicau-1 B.1.
The invention also aims to provide the molecular-labeled fluorescent quantitative PCR primer.
The third purpose of the invention is to provide the application of the molecular marker tightly linked with the wheat stripe rust resistance QTL QYr.
The purpose of the invention is realized by the following technical scheme:
based on the purposes, the applicant utilizes the stripe rust resistant wheat strain '20828' as a female parent and takes the wheat strain 'CN 16' as a male parent for hybridization to obtain a hybrid F1，F1Selfing the single plant to obtain F2At F2Using single seed propagation up to F8And thirdly, obtaining a recombinant inbred line containing 199 lines to form a genetic mapping population. For phenotype identification of stripe rust of a recombinant inbred line group, parents '20828', 'CN 16' and DNA of a recombinant inbred line group plant are extracted, and a wheat 55K SNP chip is used for positioning a wheat stripe rust resistance QTL. The wheat 55K SNP chip is an economic medium-density SNP chip developed on the basis of a wheat 660K SNP chip. The chip contains about 55,000 wheat SNP markers which are uniformly distributed on 21 chromosomes, wherein each chromosome averagely contains 2,600 markers, the average genetic distance between the markers is about 0.1cM, and the average physical distance is less than 300Kb, so that the chip is suitable for general germplasm resource diversity analysis, genetic mapping and new gene discovery, comparative genome analysis, and variety registration and identification (fingerprint analysis).
According to 55K SNP chip data, a genetic map is constructed by using JoinMap4.0. Combining with stripe rust phenotype data of a population, detecting the QTL by using a complete Interval Mapping-ADD (ICIM-ADD) in QTL IciMapping 4.0 and using BLUP (best linear unbiased prediction) values of 2016-, stably expressed wheat stripe rust resistance QTL QYr. sicau-1B.1 is positioned in the 1.3cM interval on the long arm of the 1B chromosome, physical mapping of flanking markers and screening of genes located within the interval at intervals of 1Mbp, co-screening to obtain 61 genes, cloning of these genes at the parents '20828' and 'CN 16', for obtaining the polymorphic sites and developing the molecular markers, 30 KASP primers (table 1) are designed totally, and finally the marker KASP-Yr is tightly linked with the stripe rust resistant QTL QYr.sicau-1 B.1.
TABLE 110 pairs of KASP primer sequences
The wheat stripe rust resistant QTL QYr. sicau-1B.1 comes from a female parent '20828', the QTL is positioned on the long arm of a wheat chromosome 1B, and the physical position of a genome version of RefSeqv1.0 is 474 Mb. The invention provides application of the wheat stripe rust resistance QTL QYr. sicau-1B.1 in regulation and control of wheat stripe rust resistance.
Further, the invention provides a molecular marker KASP-Yr of wheat stripe rust resistance QTL QYr.sicau-1B.1, which is tightly linked with wheat stripe rust resistance QTL QYr.sicau-1 B.1. The molecular marker and wheat stripe rust resistant QTL QYR.sicau-1B.1 are co-located on the long arm of wheat 1B chromosome, and the genetic distance between the molecular marker and QYR.sicau-1B.1 is less than 1.3 cM. The polymorphism of the SNP molecular marker is A/G. The nucleotide sequence containing the SNP molecular marker is shown as SEQ ID NO. 31.
The molecular marker KASP-Yr of the wheat stripe rust resistant QTL QYr. sicau-1B.1 is obtained by PCR amplification of a primer pair with a nucleotide sequence shown as SEQ ID NO. 1-3. The 3 primers comprise 2 upstream primers and 1 universal downstream primer, and the 5' ends of the 2 upstream primers are respectively modified with different fluorescent groups; the nucleotide sequences of the 3 primers are respectively shown as SEQ ID NO.1, 2 and 3.
The average LOD value of this SNP molecular marker was 21.39, accounting for about 25.52% of phenotypic variation.
The invention provides a specific primer combination for detecting a wheat stripe rust resistance QTL QYr. sicau-1B.1 linked SNP molecular marker, which contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-3.
The kit containing the specific primer combination belongs to the protection scope of the invention.
The invention provides the application of the molecular marker KASP-Yr, the specific primer combination and a kit containing the specific primer combination in the improvement of wheat germplasm resources or the creation of wheat stripe rust resistant materials or the application of wheat molecular assisted breeding.
The invention provides the application of the molecular marker KASP-Yr, the specific primer combination and a kit containing the specific primer combination in identifying wheat with stripe rust resistance or cultivating wheat with stripe rust resistance.
The invention provides the application of the molecular marker KASP-Yr, the specific primer combination and a kit containing the specific primer combination in identifying the wheat stripe rust resistant gene QYr.
The application of the invention specifically comprises the steps of taking genome DNA of a plant sample to be detected as a template, carrying out fluorescent quantitative PCR amplification by using fluorescent quantitative PCR primers, carrying out genotyping by using amplification results, and identifying a plant capable of reading fluorescence modified by the primers SEQ ID NO.2 as a plant containing wheat stripe rust resistant QTL QYr.sicau-1 B.1.
The invention provides a molecular marking method for identifying wheat stripe rust resistance QTL QYr.sicau-1B.1, which takes DNA of a material to be identified as a template, uses three specific primer pairs with primer sequences respectively shown as SEQ ID NO. 1-3 to carry out PCR amplification and reads a fluorescence value, and if the fluorescence modified by SEQ ID NO.2 appears, the wheat with stripe rust resistance QTL QYr.sicau-1B.1 can be judged.
Specifically, the application includes the following steps:
1) extracting the genome DNA of a plant to be detected;
2) taking the genome DNA of a plant to be detected as a template, and performing PCR amplification reaction and reading a fluorescence value in a CFX96Real-Time System by using a primer of an amplification molecular marker KASP-Yr;
3) and detecting the fluorescence of the PCR amplification product, and if HEX fluorescence can be read, determining that the plant to be detected is the wheat resource with the stripe rust resistance.
The amplification system of the PCR amplification is as follows: 5 μ L Master Mix, three primers SEQ ID No: 1. 2 and 3 at a concentration of 10 ng/. mu.L, 120. mu.L, and 300. mu.L, respectively, and ddH2O 460. mu.L were added and mixed to be used as mixed primers, 1.4. mu.L of the mixed primers, 5ng of the template DNA, and double distilled water were added to a total amount of 10. mu.L, and at least 3 independent blanks were added in place of the DNA template in the double distilled water.
The procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles; after completion, fluorescence readings were taken.
The invention discloses a molecular marker KASP-Yr located on a wheat 1B chromosome and linked with wheat stripe rust resistance, wherein the molecular marker is a flanking marker of a stripe rust resistance QTL QYr.sicau-1B.1 on a long arm of the wheat 1B chromosome, and the linkage degree is high. The marker can be used for detecting the stripe rust resistance QTL on the wheat 1B chromosome, and rapidly screening plants with the locus, thereby facilitating the molecular assisted breeding of high-resistance wheat. The molecular marker KASP-Yr provided by the invention is closely linked with the stripe rust resistance QTL QYr. sicau-1B.1 on the wheat 1B, and can be used for positioning the stripe rust resistance character of the wheat, so that diseased plants are eliminated in the breeding process, the breeding work efficiency is improved, and a foundation is provided for the research of stripe rust resistance genes of the wheat.
The invention discloses a stripe rust resistance QTL QYr.sicau-1B.1 from wheat 20828' for the first time, which is positioned on the long arm of a wheat 1B chromosome and can obviously improve the stripe rust resistance of the wheat. The QTL has higher utilization value in wheat disease resistance (regulation and control of stripe rust resistance) breeding.
The invention discloses a molecular marker KASP-Yr for accurately detecting stripe rust resistance QTL QYr.sicau-1B.1 of wheat '20828' based on a fluorescent quantitative PCR platform for the first time, and the molecular marker KASP-Yr is a codominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification.
The molecular marker KASP-Yr disclosed by the invention is remarkably related to stripe rust resistant QTL QYr. sicau-1B.1, presents coseparation marker characteristics, has high accuracy for molecular marker-assisted selection, improves the selection and identification efficiency of wheat stripe rust resistant varieties suitable for different environments, and has high success rate.
Drawings
FIG. 1 shows the location of wheat stripe rust resistance QTL QYr. sicau-1B.1 on chromosome 1B in example 1 of the present invention.
FIG. 2 shows the fluorescence readings of the molecular marker KASP-Yr assay of the recombinant inbred line strain of '20828' × 'CN 16' in example 1; wherein, HEX (blue, '20828') fluorescence is a stripe rust resistant strain, FAM (orange, 'CN 16') fluorescence is an infectious strain; green fluorescence is a heterozygous strain; black fluorescence is blank.
FIG. 3 shows the fluorescence reading results of the detection of molecular marker KASP-Yr of the plant of the recombinant inbred line strain of '20828' x common wheat line 'SY 95-71' in example 2 of the present invention; wherein, HEX (blue, '20828') fluorescence is a stripe rust resistant strain, FAM (orange, 'SY 95-71') fluorescence is an infectious strain; green fluorescence is a heterozygous strain; black fluorescence is blank.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The wheat germplasm resources used in the embodiment of the invention are from a germplasm resource library of the wheat research institute of Sichuan university of agriculture.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 obtaining of wheat stripe rust resistance Gene QYr. sicau-1B-1 and its molecular marker KASP-Yr
(1) Hybridizing the wheat strain '20828' as female parent and the wheat strain 'CN 16' as male parent to obtain hybrid F1，F1Selfing the single plant to obtain F2At F2Using single ear propagation up to F8And thirdly, obtaining a recombinant inbred line containing 199 lines to form a genetic mapping population.
(2) Phenotype identification of stripe rust of a recombinant inbred line population, after inducing materials SY95-71 to fully develop diseases, recording disease resistance reaction of the tested materials in the field, judging each strain in the population according to 1-9 level classification standards for the reaction type, and recording the judgment result as morphological marking data of a stripe rust resistance gene QYr. The position thus mapped on the linkage map is the position of the QYr. sicau-1B.1 gene.
(3)55K SNP chip analysis
a) DNA extraction: the DNA of parent '20828', 'CN 16' and the plant of the recombinant inbred line population is extracted by a CTAB method.
b) The extracted DNA is subjected to quality detection by using an ultramicro spectrophotometer, and is sent to a company for genotype analysis after being qualified, and the genotype analysis of the parents and mapping group in the research is completed by a 55K SNP chip developed by combining Beijing Boao crystal and Kanji Kangchi Biotechnology Limited company (http:// www.capitalbiotech.com) and Jia Sungqi topic, and the chip can be sold in the market.
c) Constructing a linkage map: according to 55K SNP chip data, a genetic map is constructed by using JoinMap4.0. Combining with stripe rust phenotype data of a population, using an integral complex Interval Mapping-ADD, ICIM-ADD in QTL IciMapping 4.0, setting a threshold LOD to be more than or equal to 2.5, using BLUP (best linear unbiased prediction) values of 4 ecological points and 4 ecological point stripe rust in three years of 2016 + 2018 to detect the QTL, locating the wheat stripe rust resistance QTL QYR.sicau-1B.1, and calculating the position of the QYR.sicau-1B.1 and the genetic distance between molecular markers.
d) Densification of genetic map and acquisition of closely linked molecular markers: in order to compact a genetic map and obtain molecular markers closely linked with the stripe rust resistant QTL QYr.sicau-1B.1, the flanking markers are physically positioned and genes positioned in the interval are screened by utilizing a 55K SNP chip data positioning result, the genes are cloned at parents '20828' and 'CN 16', polymorphic sites are obtained and molecular markers are developed, a DNMAN is utilized to design KASP primers (30 pieces and 10 pairs of KASP primers are designed together) (table 1), and finally the markers KASP-Yr and the stripe rust resistant QTL QYr.sicau-1B.1 are closely linked, the average LOD value of the SNP molecular markers is 21.39, and about 25.52% of phenotypic variation is explained.
e) And (6) carrying out analysis. 1 molecular marker is finally obtained from 10 pairs of KASP primers and is closely linked with the stripe rust resistant QTL QYr. The results are shown in FIGS. 1 and 2.
Example 2 application of molecular marker KASP-Yr in selection control of stripe rust resistant QTL QYr
(1) A common wheat line '20828' resistant to stripe rust is used as a female parent, a common wheat line 'SY 95-71' susceptible to stripe rust is used as a male parent to construct a recombinant inbred line, and 163 lines are randomly selected from the progeny lines.
(2) The obtained 163 strains were subjected to KASP-Yr marker detection by the specific method: extracting DNA of 163 strains; taking the DNA as a template, taking a specific primer pair of a molecular marker KASP-Yr as a primer to carry out PCR amplification and carry out fluorescence reading, wherein the labeled KASP-Yr primer is as follows:
primer on FAM tag: (FAM tag sequence underlined)
5’-GAAGGTGACCAAGTTCATGCTCTTGTGTGTGACTGTACCATA -3’(SEQ ID No.1)
Primers on HEX tag: (lower slide part HEX tag sequence)
5’-GAAGGTCGGAGTCAACGGATTCTTGTGTGTGACTGTACCAT G-3’(SEQ ID No.2)
A universal downstream primer:
5’-CTGATACAGATAGGTAGCAT-3’(SEQ ID No.3)
the amplification system of the PCR amplification is as follows: 5 μ L Master Mix, three primers SEQ ID No: 1. 2 and 3 at a concentration of 10 ng/. mu.L, 120. mu.L, and 300. mu.L, respectively, and ddH2O 460. mu.L were added and mixed to be used as mixed primers, 1.4. mu.L of the mixed primers, 5ng of the template DNA, and double distilled water were added to a total amount of 10. mu.L, and at least 3 independent blanks were added in place of the DNA template in the double distilled water.
The procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles; after completion, fluorescence readings were taken.
As a result of the fluorescence reading (see FIG. 3), the genotype of the plant in which HEX (blue) fluorescence consistent with that of '20828' was detected was designated as A, and the genotype of the plant exhibiting FAM (orange) fluorescence in the same manner as that of 'SY 95-71' was designated as B, and the plant was designated as a susceptible strain. The BLUP values for the genotypes and 4 ecotype point rust of the respective lines are shown in Table 2. The average disease index of plants of the same type as '20828' containing the stripe rust resistant QTL QYr. sicau-1B.1 in the marker KASP-Yr was 37.56, which is very significantly lower than the disease index of plants of the type 'SY 95-71' (average 65.35). The actual result is consistent with the expected result, which shows that the stripe rust resistant QTL QYr.sicau-1B.1 of the invention has the effect of obviously improving the stripe rust resistance; meanwhile, the molecular marker KASP-Yr can be used for tracing and identifying the stripe rust resistant QTL QYr.
TABLE 2 '20828' × 'SY 95-71' recombinant inbred line KASP-Yr genotype and phenotype correspondence results
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (5)
1. The specific primer combination of the SNP molecular marker for detecting the wheat stripe rust resistance is characterized by comprising a primer with a nucleotide sequence shown as SEQ ID NO. 1-3.
2. A kit comprising the specific primer combination of claim 1.
3. Use of the specific primer combination of claim 1 or the kit of claim 2 for identifying stripe rust resistant wheat.
4. The application of claim 3, wherein the genome DNA of a plant sample to be detected is used as a template, fluorescent quantitative PCR amplification is carried out by using fluorescent quantitative PCR primers shown in SEQ ID No. 1-3, genotyping is carried out by using an amplification result, and the plant which can read the fluorescence modified by the primer SEQ ID No.2 is identified as the wheat resource with the stripe rust resistance.
5. The use of claim 4, wherein the amplification reaction system of the fluorescence quantitative PCR: 5 μ L Master Mix, 3 primers SEQ ID NO.1, 2 and 3 at 10ng/μ LAdding 120 mu L, 120 mu L and 300 mu L respectively and adding ddH2Mixing O460 muL and then using the mixture as a mixed primer, adding 1.4 muL of the mixed primer, 5ng of template DNA and double distilled water to the total amount of 10 muL, and simultaneously adding at least 3 independent blanks for replacing the DNA template with the double distilled water;
fluorescent quantitative PCR procedure: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s and renaturation/elongation at 61 ℃ for 60s for 10 cycles; denaturation at 94 ℃ for 20s and renaturation/elongation at 55 ℃ for 60s for 26 cycles;
genotyping the results obtained; and detecting the fluorescence of the PCR amplification product, wherein if the fluorescence modified by the primer SEQ ID NO.2 can be read, the plant to be detected is the wheat resource with the stripe rust resistance.
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