CN114045359A - KASP molecular marker related to wheat ear germination resistance and application thereof - Google Patents
KASP molecular marker related to wheat ear germination resistance and application thereof Download PDFInfo
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Abstract
The invention discloses an application of a KASP molecular marker related to wheat ear germination resistance. The invention provides an application of a composition for detecting the polymorphism or genotype (namely allele) of Sdr2A-Kasp locus in a wheat genome in preparing products for identifying or assisting in identifying the germination resistance of ears. The composition for detecting the Sdr2A-Kasp locus polymorphism and the genotype can be combined with other substances (such as substances for detecting the single nucleotide polymorphism or the genotype of other molecular markers related to the wheat pre-harvest sprouting) to prepare a high-throughput wheat pre-harvest sprouting resistant variety product.
Description
Technical Field
The invention relates to application of a KASP molecular marker, in particular to a KASP molecular marker related to wheat ear germination resistance and application thereof.
Background
Pre-harvest sprouting (PHS) refers to the sprouting of wheat over a spike that is exposed to the rain or in a humid environment prior to harvest. In particular, wheat white grain varieties are mainly used, the resistance to ear sprouting is weak, and once ear sprouting occurs, the loss caused by ear sprouting is large. The wheat field with the germination of the ears generally reduces the yield by 6-10 percent, and the activities of amylase, protease and lipase in the germinated wheat grains are greatly increased, so that the starch, protein and fat are degraded, the processing quality of the wheat is reduced, the quality of bread, noodles and steamed bread is seriously influenced, the volume of the bread processed by the wheat field is reduced, the interior of the wheat field is compact and not soft, and the toughness and elasticity of the noodles are reduced. Since ear sprouting also results in an increased level of asparagine in the flour, asparagine is readily converted to carcinogenic acrylamide at high temperatures when baking bread. Therefore, improving the germination resistance of wheat varieties is one of the important targets of wheat breeding.
The wheat head germination is a complex character controlled by multiple genes, morphological characteristics and physiological and biochemical characters (such as seed dormancy characteristics, seed coat color, alpha-amylase activity, glume inhibitors, ear structures and the like) of varieties can affect the wheat head germination to different degrees, wherein the seed dormancy characteristics are the most main factors influencing the resistance to the ear germination. Moreover, the wheat ear germination is greatly influenced by weather conditions, and the ear germination resistances of the same wheat variety in different years and different planting places are different, so that the identification in the field is difficult and the accuracy is low.
Molecular marker assisted selection (marker assisted selection MAS) is genotype-based selection, is not influenced by external environmental factors, and is increasingly widely applied to breeding practice. The common molecular marker types include RFLP, AFLP, SSR and the like, but the marker types cannot be co-separated with target genes. Functional markers (Functional markers) are developed based on gene characteristic sequences, are co-separated from target genes, greatly improve the accuracy of selection and have wide application prospects in MAS.
Moreover, the SSR markers or PCR markers are slow in detection speed, and breeding progeny materials are not easy to screen in a large scale. The KASP marker, namely competitive Allele Specific PCR (Kompetitive Allele Specific PCR), is a marker type developed in recent years, a primer is designed according to Specific SNP (single-base nucleotide polymorphism) or InDels (insertion/deletion) contained in an Allele, and a fluorescent group added at the tail end of the primer is used for detection, so that the genotyping work can be quickly and accurately completed, high-throughput analysis can be realized, the process of molecular marker-assisted selection is greatly accelerated, and the application prospect in crop breeding is wide. Therefore, the development of KASP markers for identifying wheat ear germination resistance has important theoretical significance and practical value.
Disclosure of Invention
The invention aims to solve the technical problem of how to identify the germination resistance of wheat ears in a high-throughput manner or in an auxiliary manner. In order to solve the technical problem, the invention provides any one application of A1-A3, A4 method as follows:
a1, application of a composition for detecting the polymorphism or genotype (namely allele) of Sdr2A-Kasp locus in wheat genome in identification or auxiliary identification of wheat ear germination resistance; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer which is P1 or P2:
p1, the PCR primer is a primer group consisting of single-stranded DNA with a nucleotide sequence of SEQ ID No.2, single-stranded DNA with a nucleotide sequence of 22-39 th sites of SEQ ID No.3 and single-stranded DNA with a nucleotide sequence of 22-40 th sites of SEQ ID No. 4;
p2, the PCR primer is a primer group consisting of single-stranded DNA with a nucleotide sequence of SEQ ID No.2, single-stranded DNA with a nucleotide sequence of SEQ ID No.3 and single-stranded DNA with a nucleotide sequence of SEQ ID No. 4.
A2, detecting the polymorphism or genotype (namely allele) of Sdr2A-Kasp locus in wheat genome, and applying the composition in preparing products for identifying or assisting in identifying wheat ear germination resistance; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer, and the PCR primer is the P1 or the P2.
A3, detecting the polymorphism or genotype (namely allele) of the Sdr2A-Kasp locus in the wheat genome, and applying the composition to wheat breeding or preparing a wheat breeding product; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer, and the PCR primer is the P1 or the P2.
The breeding aim comprises breeding the pre-harvest sprouting resistant wheat.
A4, identifying or assisting in identifying wheat ear germination resistance, including detecting the genotype of the wheat to be detected, and identifying or assisting in identifying the wheat ear germination resistance according to the genotype of the wheat to be detected; the genotype is the genotype of the Sdr2A-Kasp locus in the wheat genome; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the detection is carried out by using a PCR primer, and the PCR primer is the P1 or the P2.
Another technical problem to be solved by the present invention is how to perform wheat breeding.
In order to solve the technical problems, the invention provides the following technical scheme:
the method B1 and A4 are applied to wheat breeding.
The breeding aim comprises breeding the pre-harvest sprouting resistant wheat.
B2, a method for wheat breeding, comprising: detecting the polymorphism of Sdr2A-Kasp in a wheat genome A1, and selecting homozygous wheat with Sdr2A-Kasp locus G in the wheat genome as a parent to breed.
The breeding aim comprises breeding the pre-harvest sprouting resistant wheat.
Any of the following products 1) -3) containing a composition for detecting the polymorphism or genotype (i.e., allele) of the Sdr2A-Kasp locus in the wheat genome also belong to the protection scope of the invention:
1) products for detecting single nucleotide polymorphisms or genotypes associated with pre-harvest sprouting in wheat;
2) identifying or assisting in identifying the products with wheat ear germination resistance;
3) a product for wheat breeding.
In the application, the method and the product, the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1. The polymorphism or genotype (namely allele) for detecting the Sdr2A-Kasp locus in the wheat genome can be specifically the nucleotide type for detecting the Sdr2A-Kasp locus. The genotype of the Sdr2A-Kasp site in the wheat genome can be GG, AA or AG. The GG is homozygous with the Sdr2A-Kasp locus as G in the wheat genome, the AA is homozygous with the Sdr2A-Kasp locus as A in the wheat genome, and the AG is homozygous with the Sdr2A-Kasp locus as A and G in the wheat genome.
In the method A4, the identification or the auxiliary identification of the pre-harvest sprouting resistance of wheat according to the genotype of the wheat to be tested may be that the pre-harvest sprouting resistance of the wheat to be tested with the genotype of GG is higher than or is higher than the pre-harvest sprouting resistance of the wheat to be tested with the genotype of AA in a candidate manner.
In the above applications, methods and products, the wheat breeding is to cultivate pre-emergence resistant wheat or to breed pre-emergence resistant wheat.
In the above applications, methods and products, the composition for detecting the polymorphism or genotype (i.e., allele) of the Sdr2A-Kasp locus in the wheat genome may be a reagent and/or an apparatus required for determining the polymorphism or genotype of the Sdr2A-Kasp by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on one-step reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.
In the above applications, methods and products, the composition for detecting the polymorphism or genotype (i.e. allele) of the Sdr2A-Kasp locus in the wheat genome is 1), 2) or 3):
1) the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome contains a PCR primer for amplifying a wheat genome DNA segment including the Sdr2A-Kasp locus;
2) the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome is a PCR reagent containing the PCR primer;
3) a kit comprising 1) the PCR primer or 2) the PCR reagent.
In the above applications, methods and products, the PCR primers may be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radiolabels, e.g.32P; binding moieties such as biotin (biotin); haptens such as Digoxin (DIG); a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly (e.g., direct sequence read) without a label. The PCR primer can be a primer group consisting of single-stranded DNA with a nucleotide sequence of SEQ ID No.2, single-stranded DNA with a nucleotide sequence of SEQ ID No.3 and single-stranded DNA with a nucleotide sequence of SEQ ID No.4, wherein the SEQ ID No.3 consists of 39 nucleotides in the sequence table, and the 1 st to 21 st nucleotides are FAM linker sequences(as a marker), the 22 th to 39 th nucleotides are specific sequences; SEQ ID No.4 in the sequence table is composed of 40 nucleotides, the 1 st to 21 st nucleotides are HEX linker sequences (as markers), and the 22 nd to 40 th nucleotides are specific sequences.
In the above applications, methods and products, the product may be a reagent or a kit or a system, and the system may comprise a combination of reagents or kits, instruments and analytical software, such as a product consisting of PCR primers, PARMS master mix reagents, microplate reader and on-line software SNP decoder (http:// www.snpway.com/snpdecoder01/), a combination consisting of PCR primers, PARMS master mix reagents, on-line software SNP decoder and a fluorescence quantitative PCR instrument. The product can comprise the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome.
The invention discloses a novel KASP marker for detecting wheat ear germination resistance. The specific primer group provided by the invention consists of single-stranded DNA shown in SEQ ID No.2, single-stranded DNA shown in SEQ ID No.3 and single-stranded DNA shown in SEQ ID No.4, wherein the single-stranded DNA shown in SEQ ID No.3 and the single-stranded DNA shown in SEQ ID No.4 are provided with fluorescent labeling joints. In one embodiment of the invention, the primer group with the fluorescent labeled joint is used for amplifying wheat genome DNA of two groups of multiple samples including Sdr2A-Kasp sites, a high-throughput genotyping detector PHERAStar SNP is used for carrying out fluorescence signal processing, the nucleotide types of the Sdr2A-Kasp sites are determined, and the germination index of each sample to be detected is determined. Experiments prove that in the group 1 population consisting of 83 wheat varieties and the group 2 population consisting of 61 wheat varieties, the germination index of the homozygous wheat variety with Sdr2A-Kasp locus G is obviously lower than that of the homozygous wheat variety with Sdr2A-Kasp locus A, and the ear germination resistance of the homozygous wheat variety with Sdr2A-Kasp G is obviously higher than that of the homozygous wheat variety with SNP locus Sdr2A-Kasp A. The Sdr2A-Kasp is an SNP molecular marker related to the wheat ear germination resistance, and the specific primer group provided by the invention can be used for identifying or assisting in identifying the wheat ear germination resistance, screening wheat ear germination resistance varieties, assisting in breeding by the wheat molecular marker, and breeding and cultivating wheat with resistance to ear germination. The polymorphism of Sdr2A-Kasp is directly expressed in a DNA form and can be detected in each tissue and each development stage of wheat, thereby being beneficial to conveniently and rapidly predicting the germination resistance of wheat ears. In practical application, in order to improve the accuracy, the composition for detecting the Sdr2A-Kasp locus polymorphism and the genotype can be combined with other substances (such as substances for detecting other single nucleotide polymorphisms or genotypes related to the wheat pre-harvest sprouting) to prepare a product for identifying the wheat pre-harvest sprouting resistant variety. The specific primer group is applied to the wheat pre-harvest sprouting resistance molecular marker auxiliary selection, and a wheat variety (germplasm) with higher pre-harvest sprouting resistance can be quickly screened out, so that the breeding process of a new pre-harvest sprouting resistance wheat variety is accelerated. The invention has important theoretical significance and economic value for utilizing the molecular marker to assist in selecting the wheat variety resistant to pre-harvest sprouting.
Drawings
FIG. 1 shows the specific binding site of Sdr2A-Kasp labeled primer pair and the sequence shown in SEQ ID No.1 in example 1 of the present invention.
FIG. 2 is a diagram of the genotyping results of group 1 wheat varieties detected by Sdr2A-Kasp marker in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
All primer synthesis in this example was performed by Shanghai Biotechnology engineering services, Inc.
Example 1
In the early stage, the inventor clones a TaSdr-A1 gene located on a wheat 2A chromosome and develops a functional marker Sdr2A for identifying wheat ear germination resistance, but the marker is CAPS (enzyme digestion amplification polymorphic sequence), PCR and restriction endonuclease digestion are required, the operation steps are complicated, and high-throughput detection is not easy to realize. Therefore, the invention develops the KASP marker Sdr2A-Kasp of the gene according to the SNP sites contained in the TaSdr-A1 gene, and greatly accelerates the detection speed of wheat ear germination resistance.
1. Application of KASP marker Sdr2A-Kasp to detect common wheat variety TaSdr-A1 allele type
A SNP site exists in a common wheat variety TaSdr-A1, which is the 643 th nucleotide of SEQ ID No.1, the nucleotide type is G or A, and the letter R represents. One allele of TaSdr-A1 is GG (i.e. pure combination of G at 643 st nucleotide of SEQ ID No. 1), and wheat of the type is taken as TaSdr-A1a (also called G base type); the other allele of TaSdr-A1 is AA (i.e. homozygous for A at 643 of SEQ ID No. 1), and wheat of this type is taken as TaSdr-A1b (also called A base type).
The method for detecting the allele type of each common wheat variety TaSdr-A1 in each group by using KASP marker Sdr2A-Kasp in batches comprises the following two steps: PCR amplification and genotyping.
(1) PCR amplification
Extracting common wheat genome DNA by CTAB method, adding 400. mu.l TE to dissolve. The DNA is subjected to quality detection by 1% agarose gel electrophoresis, and the extracted DNA is required to have no obvious impurity, clear band and no degradation. PCR amplification is carried out by using the wheat genome DNA as a template.
Preparing KASP labeled primer working solution: designing KASP primers according to SNP at 643 site of wheat grain dormancy TaSdr-A1 gene, wherein polymorphism of the SNP site is A/G base difference, the sequences of the primers are shown in Table 1, and a schematic diagram of specific binding site of the primers and the sequence shown in SEQ ID No.1 is shown in FIG. 1.
TABLE 1 KASP marker primer sequences for identifying common wheat TaSdr-A1 gene allelic variation
Amplifying a fragment of which the 643 th nucleotide of the SEQ ID No.1 is G by the single-stranded DNA molecules shown in the SEQ ID No.2 and the SEQ ID No.3, and reading a fluorescent signal of a fluorescent group combined with the FAM sequence by using an enzyme-labeling instrument or a fluorescent quantitative PCR instrument;
the single-stranded DNA molecules shown in SEQ ID No.2 and SEQ ID No.4 amplify a fragment of which the 643 st nucleotide of SEQ ID No.1 is A, and a fluorescence signal of a fluorescent group combined with the HEX sequence can be read by an enzyme-labeling instrument or a fluorescent quantitative PCR instrument.
Mu.l of the forward primer (100. mu.M), 12. mu.l of the reverse primer-1 (100. mu.M) and 12. mu.l of the reverse primer-2 (100. mu.M) were taken and supplemented with sterile ultrapure water to 100. mu.l to prepare a KASP-labeled primer working solution, which was stored at-20 ℃ for further use.
The PCR amplification system is as follows: template DNA 2.2. mu.l (concentration 20-50 ng/. mu.l), primer working solution 0.056. mu.l, 50mM MgCl2(LGC Co., Lot No.:10364672) 0.08. mu.l, KASP 2 × Master Mix (LGC Co., LotNo.10371613) 2.5. mu.l, and the reaction system was supplemented with sterile ultrapure water to 5. mu.l.
The PCR reaction program is: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 deg.C for 20s, and renaturation for 20s (the first renaturation temperature is 65 deg.C, and the temperature is reduced by 1 deg.C per cycle) for 10 cycles; denaturation at 95 deg.C for 10s, and renaturation at 57 deg.C for 1min for 30 cycles; storing at 10 deg.C.
(2) Genotyping
KASP SNP detection is carried out by using a 96-well plate or a 384-well plate, and after the PCR reaction is finished, fluorescence data reading is carried out on the reaction product by using a high-throughput genotyping detector PHERAStar SNP scanner (LGC company). The fluorescence excitation wavelength of FAM is 485nm, and the emission wavelength is 520 nm; the HEX fluorescence excitation wavelength is 535nm, and the emission wavelength is 556 nm. The fluorescence scanning result is graphically displayed by using Klustercaller software, and the GG genotype (TaSdr-A1a) has FAM fluorescence and is distributed near the x axis; the AA genotype (TaSdr-A1b) has HEX fluorescence and is distributed near the y axis; the heterozygous type is distributed around the diagonal and the sample with no detected signal is distributed around the origin.
2. Application of KASP marker Sdr2A-Kasp to detection of common wheat variety pre-harvest sprouting resistance
Experiments are carried out on two groups of common wheat varieties, The first group of common wheat includes 83 winter wheat varieties, The second group of common wheat includes 61 varieties, all varieties are known varieties and are respectively described in non-patent documents, namely, The set of common wheat varieties TaSdr-A1a associated with present-modified wheat varieties in Chinese patent documents, closed of set of common genes (Tar) associated with present-modified wheat varieties and horizontal modification of a functional wheat, and The second group of common wheat varieties includes 83 winter wheat varieties, 61 varieties of common wheat varieties, all varieties of which are known varieties, namely, wheat varieties and horizontal varieties, and three varieties of common varieties of varieties, wheat varieties and horizontal modification of a functional wheat varieties, and combinations of varieties of white varieties and horizontal varieties of wheat varieties, wheat varieties and horizontal varieties of wheat varieties of varieties, and varieties of trees, and varieties of trees, such as well-1, the wheat research center has preservation in the institute of grain and oil crops of the academy of agriculture and forestry of Hebei province, and the public can obtain the wheat research center from the institute of grain and oil crops of the academy of agriculture and forestry of Hebei province, so that the application experiment can be repeated and the wheat research center cannot be used for other purposes.
(1) And detecting the wheat variety of the 1 st group by utilizing Sdr2A-Kasp markers. The wheat variety of the group 1 is 83 parts in total, the wheat variety is planted in Beijing, and the genotype detection result is shown in figure 2.
After wheat harvest, Germination indexes (Germination Index) of grains are detected. The germination index detection method comprises the following steps: placing two layers of filter paper in a culture dish, soaking the filter paper by using sterile water, then placing the seeds on the filter paper downwards in the abdominal furrow, culturing at room temperature, investigating the number of germinated seeds every day, investigating continuously for 7 days, and calculating the germination index of the seeds. And (4) characterizing the germination resistance of the ear through the germination index of the seeds.
In the formula, n1、n2、…、n7The number of germinated seeds on day 1d, 2d, … d and 7d are shown in sequence. total grains represent the total number of kernels investigated. Three replicates were run and the results averaged and are shown in table 2. 83 parts of Chinese winter wheat variety: 19 varieties are TaSdr-A1a (GG) genotypes, and the average value of the germination indexes is 19.6; 64 varieties are TaSdr-A1b (AA) genotypes, and the average value of the germination indexes is 39.3; the germination index of the wheat variety carrying the allele TaSdr-A1a is lower than that of the wheat variety carrying the allele TaSdr-A1b, and the two have significant difference (P)<0.01), the results are shown in Table 3.
TABLE 2 detection results and germination index of group 1 wheat variety Sdr2A-Kasp marker
TABLE 3 statistical results of the relationship between TaSdr-A1 gene allelic variation type and grain germination index of group 2 common wheat
Description of the drawings: the different letters following the germination index represent that the difference reaches a very significant level (P < 0.01).
(2) And detecting the wheat variety of the 2 nd group by using Sdr2A-Kasp markers. The wheat variety of the group 2 is 61 parts in total and planted in Henan Anyang. And (3) detecting the Germination Index (Germination Index) of the grains after wheat is harvested, wherein the Germination Index measuring method is the same as the Germination Index measuring method in the step (1). Three replicates were run and the results averaged and are shown in table 4. 61 parts of Chinese winter wheat variety: 10 varieties are TaSdr-A1a genotypes, and the average value of the germination indexes is 16.4; 51 varieties are TaSdr-A1b genotypes, and the average value of the germination indexes is 41.2; the germination index of the wheat variety carrying the allele TaSdr-A1a was lower than that of the wheat variety carrying the allele TaSdr-A1b, with a significant difference (P <0.01), and the results are shown in Table 5.
TABLE 4 detection results and germination index of group 2 wheat variety Sdr2A-Kasp
TABLE 5 statistical analysis results of the relationship between TaSdr-A1 gene allelic variation type and grain germination index of group 2 common wheat
Description of the drawings: the different letters following the germination index represent that the difference reaches a very significant level (P < 0.01).
In conclusion, the germination index of the wheat variety with the Sdr2A-Kasp genotype GG (TaSdr-A1a, also called as G base type) is obviously lower than that of Sdr2A-Kasp genotype AA (TaSdr-A1b, also called as A base type), which indicates that the ear germination resistance of wheat with the SNP site Sdr2A-Kasp genotype TaSdr-A1a is obviously higher than that of wheat with the SNP site Sdr2A-Kasp genotype TaSdr-A1 b.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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ccggcgaccg cgagtaccgg cgggaatctg acgatgctct cgaccatggt ggcgggcgat 420
gaggaggagg aggaggagga gagggatatc cccgtggagc gcgacctgct gcggaagctg 480
ctggagccca aggtgatctc gccgcgggcg atgcgccccg tgggatccac catccacgtc 540
gaatccatcg tccacggcgc cgtcgacgcg gccagcagca cggccgcctc gaagacggcg 600
gaggaggtgg aggcggaggt ggagaccgac gcgctgccgg cgrtcgtcac ggactcgagc 660
aaccgcgtcc ggctggtgaa cgacgcgtac aaggagatgg tgggcgcgcc cgagtgcctg 720
tggctcggcg cggtggccgc gtcgaggagg atcagcgggg aggtggcgct ggtggtggcc 780
gagcaggcga cgctgccgga gtccccaggg gggttctcgt gcacggcgaa gatcgagtgg 840
gagtgcggcg gcggcgagcg ggcttccatc catgcagcgt gcgacgtcag ccggctgcag 900
tgcgagtaca ggcactacct cttcgcctgg aggtttcgcg ccgccgatgc atcatcgccc 960
gccgacagcc accgcgccgg cggcgaagca tga 993
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatccaccat ccacgtcga 19
<210> 3
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaaggtgacc aagttcatgc tgctcgagtc cgtgacgac 39
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaaggtcgga gtcaacggat ttgctcgagt ccgtgacgat 40
Claims (10)
1. The application of the composition for detecting the polymorphism or genotype of Sdr2A-Kasp locus in wheat genome in identification or auxiliary identification of wheat ear germination resistance; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer which is P1 or P2:
p1, the PCR primer is a primer group consisting of single-stranded DNA with a nucleotide sequence of SEQ ID No.2, single-stranded DNA with a nucleotide sequence of 22-39 th sites of SEQ ID No.3 and single-stranded DNA with a nucleotide sequence of 22-40 th sites of SEQ ID No. 4;
p2, the PCR primer is a primer group consisting of single-stranded DNA with a nucleotide sequence of SEQ ID No.2, single-stranded DNA with a nucleotide sequence of SEQ ID No.3 and single-stranded DNA with a nucleotide sequence of SEQ ID No. 4.
2. The application of the composition for detecting the polymorphism or genotype of Sdr2A-Kasp locus in wheat genome in preparing products for identifying or assisting in identifying wheat ear germination resistance; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer, wherein the PCR primer is the P1 of claim 1 or the P2 of claim 1.
3. The application of a composition for detecting the polymorphism or genotype of an Sdr2A-Kasp locus in a wheat genome in wheat breeding or in preparing a wheat breeding product, wherein the Sdr2A-Kasp locus is an SNP locus in the wheat genome, the nucleotide type of the SNP locus is G or A, and the SNP locus is the 643 th nucleotide of SEQ ID No. 1; the composition comprises the PCR primer, wherein the PCR primer is the P1 of claim 1 or the P2 of claim 1.
4. The method for identifying or assisting in identifying the wheat ear germination resistance comprises the steps of detecting the genotype of wheat to be detected, and identifying or assisting in identifying the wheat ear germination resistance according to the genotype of the wheat to be detected; the genotype is the genotype of the Sdr2A-Kasp locus in the wheat genome; the Sdr2A-Kasp site is an SNP site in a wheat genome, the nucleotide type is G or A, and the SNP site is the 643 th nucleotide of SEQ ID No. 1; the detection is performed by using PCR primer, the PCR primer is the P1 of claim 1 or the P2 of claim 1.
5. Use of the method of claim 4 in wheat breeding.
6. A method of breeding wheat comprising: detecting the polymorphism of the Sdr2A-Kasp locus in the wheat genome as claimed in claim 1, and selecting homozygous wheat with the Sdr2A-Kasp locus G in the wheat genome as a parent to breed.
7. The product containing the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome is any one of C1) -C3):
C1) products for detecting single nucleotide polymorphisms or genotypes associated with pre-harvest sprouting in wheat;
C2) identifying or assisting in identifying the products with wheat ear germination resistance;
C3) a product for wheat breeding.
8. The use according to any one of claims 1-3 and 5, the method of claim 4 or 6, or the product of claim 7, wherein: the wheat breeding is to cultivate the wheat with the spike germination resistance or breed the wheat with the spike germination resistance.
9. The use according to any one of claims 1-3, 5 and 8, the method of claim 4, 6 or 8 or the product of claim 7 or 8, wherein: the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome is D1), D2) or D3):
D1) the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome contains a PCR primer for amplifying a wheat genome DNA segment including the Sdr2A-Kasp locus;
D2) the composition for detecting the polymorphism or genotype of the Sdr2A-Kasp locus in the wheat genome is a PCR reagent containing the PCR primer;
D3) a kit containing the PCR primer described in D1) or the PCR reagent described in D2).
10. The use, method or product according to claim 9, wherein: the PCR primer is a primer group consisting of a single-stranded DNA shown by SEQ ID No.1 in a sequence table, a single-stranded DNA shown by SEQ ID No.2 in the sequence table and a single-stranded DNA shown by SEQ ID No.3 in the sequence table.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111298290.9A CN114045359A (en) | 2021-11-04 | 2021-11-04 | KASP molecular marker related to wheat ear germination resistance and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111298290.9A CN114045359A (en) | 2021-11-04 | 2021-11-04 | KASP molecular marker related to wheat ear germination resistance and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861667A (en) * | 2016-04-20 | 2016-08-17 | 中国农业科学院作物科学研究所 | Molecular marker and specific primer for identifying wheat grain germination traits and application thereof |
| CN107217098A (en) * | 2017-06-23 | 2017-09-29 | 四川农业大学 | The KASP molecular labeling related to wheat anti growing out character and its application |
| CN113528700A (en) * | 2021-07-22 | 2021-10-22 | 湖北省农业科学院粮食作物研究所 | KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application |
-
2021
- 2021-11-04 CN CN202111298290.9A patent/CN114045359A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861667A (en) * | 2016-04-20 | 2016-08-17 | 中国农业科学院作物科学研究所 | Molecular marker and specific primer for identifying wheat grain germination traits and application thereof |
| CN107217098A (en) * | 2017-06-23 | 2017-09-29 | 四川农业大学 | The KASP molecular labeling related to wheat anti growing out character and its application |
| CN113528700A (en) * | 2021-07-22 | 2021-10-22 | 湖北省农业科学院粮食作物研究所 | KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application |
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| JACQUELINE BATLEY: "《Methods in Molecular Biology》", 31 December 2015, 施普林格科学出版社 * |
| 邹景伟等: "120 份小麦品种(系)重要性状功能基因的KASP检测", 《分子植物育种》 * |
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