CN116790794A - Kasp markers of anti-melanosis QTL QBP.caas-2AL and QBP.caas-5BL - Google Patents
Kasp markers of anti-melanosis QTL QBP.caas-2AL and QBP.caas-5BL Download PDFInfo
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Abstract
The application discloses a KASP mark of anti-melanosis QTL QBP.caas-2AL and QBP.caas-5 BL. The application provides application of single nucleotide polymorphism of SNP locus AX-179560327 and/or single nucleotide polymorphism of SNP locus AX-179388377 on wheat genome as markers in identification or auxiliary identification of black embryo disease resistance of wheat. According to the application, KASP primers are developed according to two markers, and molecular marker assisted screening can be performed on the black embryo genes. The application has important significance for cultivating the wheat variety with the enhanced melanosis antibody.
Description
Technical Field
The application relates to the technical field of biology, in particular to a KASP marker of anti-melanosis QTL QBP.caas-2AL and QBP.caas-5 BL.
Background
Wheat black embryo disease is a seed disease, and has a tendency to be significantly aggravated in recent years due to global climate change and improvement of soil conditions. The main wheat producing areas such as Yangtze river basin, huang-Huai river basin and the like in China are seriously ill. The main symptoms of the melanosis are that the embryo part of the seed grains presents obvious black brown spots, and the appearance, the nutrition quality and the processing quality of the seed grains are seriously affected. New wheat national standards (GBl 351-1999) clearly list black embryo kernels as imperfect kernels. According to the standard of wheat commodity grain acquisition in China, the imperfect grains are degraded when exceeding 6 percent. At present, no conclusion is drawn on the pathogenesis of the black embryo disease, including the theory of mycology and the theory of enzymatic browning, and the severity of the occurrence of the disease is also influenced by a plurality of environmental factors.
The breeding and utilizing of disease-resistant varieties is an effective means for reducing the damage of black embryo diseases, and the resistance of the black embryo diseases is a complex quantitative trait controlled by multiple genes. The method is an effective method for reducing the damage of the black embryo disease by excavating the black embryo disease resistance locus, developing a molecular marker closely linked with the black embryo disease resistance locus and cultivating the resistance variety by utilizing a molecular marker-assisted selective breeding strategy. With the development of wheat 9K, 50K, 90K and 660K chips and the second generation sequencing technology, SNP markers are widely applied. However, SNP marker information is mainly derived from chips or resequencing, and is too costly to use on a large scale. The SNP marker is converted into the KASP marker with relatively low detection cost, and has important significance for wheat molecular marker assisted selective breeding (MAS). Currently, hundreds of KASP markers have been developed and validated that correlate with wheat disease resistance, processing quality, and nutritional quality.
In recent years, with the completion of sequencing of Chinese spring wheat and the progress of new generation sequencing and chip technology, genetic analysis research on complex properties of wheat is rapidly increasing, but so far, genetic research on wheat black embryo disease is less. Some anti-blackembryo related molecular markers have been identified and developed at present, but no report of direct application to blackembryo disease-resistant breeding has been seen. Therefore, developing a black embryo disease resistance KASP marker useful in breeding is important for accelerating the disease-resistant breeding process of wheat black embryos.
Disclosure of Invention
The application aims to provide KASP markers of anti-melanosis QTL QBP.caas-2AL and QBP.caas-5 BL.
In a first aspect, the application claims the use of a single nucleotide polymorphism of SNP site AX-179560327 and/or a single nucleotide polymorphism of SNP site AX-179388377 on the wheat genome as a marker in any of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) And preparing a product for screening out the wheat single plants with relatively weak resistance to the black embryo diseases.
The SNP site AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP site is A or C. The SNP site AX-179560327 corresponds to 753.6Mb (https:// ugi. Versailles. Inra. Fr/blast_iwgsc /) (hereinafter the same) of wheat reference genome Chinese Spring RefSeq v 1.0.0.
The SNP site AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on the wheat 5B chromosome, and the nucleotide at the SNP site is G or A. The SNP site AX-179388377 corresponds to 673.0Mb (https:// ugi. Versailles. Inra. Fr/blast_iwgsc /) (hereinafter the same) of wheat reference genome Chinese Spring RefSeq v 1.0.0.
In a second aspect, the application claims the use of a substance for detecting a single nucleotide polymorphism at SNP site AX-179560327 on the wheat genome and/or a substance for detecting a single nucleotide polymorphism at SNP site AX-179388377 on the wheat genome, in any of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) And preparing a product for screening out the wheat single plants with relatively weak resistance to the black embryo diseases.
The SNP site AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP site is A or C.
The SNP site AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on the wheat 5B chromosome, and the nucleotide at the SNP site is G or A.
Wherein the substance for detecting a single nucleotide polymorphism of said SNP site AX-179560327 on the wheat genome may be KASP primer A as described in the third aspect hereinafter or reagent A as described in the fourth aspect hereinafter or a kit containing said reagent A.
Wherein the substance for detecting the single nucleotide polymorphism of the SNP site AX-179388377 on the wheat genome is KASP primer B described in the third aspect below or reagent B described in the fourth aspect below or a kit containing the reagent B.
In a third aspect, the application claims a kit of primers for use in or in assisting in the identification of wheat's resistance to black-embryo disease.
The kit primer for identifying or assisting in identifying the black embryo disease resistance of the wheat can be any one of the following primers:
(B1) KASP primer a; the KASP primer A consists of a primer 1, a primer 2 and a primer 3; the primer 1 is single-stranded DNA with a fluorescent tag sequence A and 22-41 positions of SEQ ID No.1 from the 5 'end to the 3' end; the primer 2 is single-stranded DNA with a fluorescent tag sequence B and 22-41 positions of SEQ ID No.2 from the 5 'end to the 3' end; the primer 3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 3.
(B2) KASP primer b; the KASP primer B consists of a primer 4, a primer 5 and a primer 6; the primer 4 is single-stranded DNA with a fluorescent tag sequence C and 22-39 positions of SEQ ID No.4 from the 5 'end to the 3' end; the primer 5 is single-stranded DNA with a fluorescent tag sequence D and 22-39 positions of SEQ ID No.5 from the 5 'end to the 3' end; the primer 6 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 6.
(B3) Consists of the KASP primer A in (B1) and the KASP primer B in (B2).
Wherein, the tag sequence A is a fluorescent tag sequence FAM, and the nucleotide sequence of the tag sequence A can be the 1 st-21 st position of SEQ ID No. 1; the tag sequence B is a fluorescent tag sequence HEX, and the nucleotide sequence of the tag sequence B can be 1 st to 21 st positions of SEQ ID No. 2; the tag sequence C is a fluorescent tag sequence FAM, and the nucleotide sequence of the tag sequence C can be 1 st to 21 st positions of SEQ ID No. 4; the tag sequence D is a fluorescent tag sequence HEX, and the nucleotide sequence of the tag sequence D can be 1 st to 21 st positions of SEQ ID No. 5.
Further, the primer 1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer 2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2.
Further, the primer 4 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 4; the primer 5 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 5.
In a fourth aspect, the application claims a reagent or kit for identifying or aiding in the identification of wheat's resistance to black-embryo disease.
The kit claimed in the application contains the reagent; the reagent may be any of the following:
(C1) A reagent a comprising a KASP primer a as described in the third aspect above;
(C2) Reagent B containing KASP primer B described in the fourth aspect;
(C3) Consists of the reagent A described in (C1) and the reagent B described in (C2).
In a fifth aspect, the application claims specific DNA molecules.
The specific DNA molecule claimed by the application is shown as SEQ ID No.7 or SEQ ID No. 8.
Wherein M at position 36 of SEQ ID No.7 is A or C; r at position 36 of SEQ ID No.8 is G or A.
In a sixth aspect, the application claims the use of a primer set as described in the third aspect hereinbefore or a reagent or kit as described in the fourth aspect hereinbefore or a specific DNA molecule as described in the fifth aspect hereinbefore for any of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) Preparing a product for screening out wheat single plants with relatively weak resistance to the black embryo disease;
(A9) Wheat breeding.
In (A9), the purpose of the breeding is to obtain a wheat variety with enhanced resistance to black-embryo disease.
In a seventh aspect, the application claims any of the following methods:
method I: a method for comparing the resistance of wheat to be tested to black embryo disease can comprise the following steps (D1) or (D2):
(D1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and determining the resistance strength of the wheat to be detected to the black embryo disease according to the genotype of the wheat to be detected as follows: the resistance of the wheat to be tested of genotype C to the black-embryo disease is stronger than or the candidate is stronger than the resistance of the wheat to be tested of genotype A to the black-embryo disease;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the genotype C is homozygote of C at the SNP locus AX-179560327 on the wheat genome;
the A genotype is homozygous type A with the nucleotide at the SNP locus AX-179560327 on the wheat genome.
(D2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and determining the resistance strength of the wheat to be detected to the black embryo disease according to the genotype of the wheat to be detected as follows: the resistance of the A genotype to the black-embryo disease of the wheat to be tested is stronger than or the candidate is stronger than the resistance of the G genotype to the black-embryo disease of the wheat to be tested;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the A genotype is homozygous A at the SNP locus AX-179388377 on the wheat genome;
the G genotype is homozygous G at the SNP site AX-179388377 on the wheat genome.
Method II: a method for breeding a wheat single plant or strain or variety with relatively high resistance to black-embryo disease can comprise the following steps of (E1) or (E2):
(E1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, selecting the wheat to be detected with the C-C genotype as a parent for breeding, and selecting the wheat with the C-C genotype in each breeding generation to finally obtain a wheat single plant or strain or variety with relatively strong black embryo resistance;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the genotype C is homozygous C at the SNP site AX-179560327 on the wheat genome.
(E2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, selecting the wheat to be detected with A genotype as a parent for breeding, and selecting the wheat with A genotype in each breeding generation to finally obtain a wheat single plant or strain or variety with relatively strong black embryo resistance;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the A genotype is homozygous type A with the nucleotide at the SNP locus AX-179388377 on the wheat genome.
Method III: a method for selecting a wheat individual having relatively weak resistance to blackembryo disease, comprising the steps of (F1) or (F2):
(F1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and eliminating the single plant of the wheat to be detected with the A genotype;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the A genotype is homozygous type A with the nucleotide at the SNP locus AX-179560327 on the wheat genome.
(F2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and eliminating the single plant of the wheat to be detected with G genotype;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the G genotype is homozygous G at the SNP site AX-179388377 on the wheat genome.
In the above method, the detection of the nucleotide at the SNP site AX-179560327 on the genome of the wheat to be tested may be accomplished by direct sequencing, or may be performed according to a method comprising the steps of: performing PCR amplification of the genomic DNA of the test wheat using the reagent A described in the fourth aspect, performing fluorescent signal scanning on the amplified product, and determining the genotype of the SNP site AX-179560327 in the genome of the test wheat as follows: if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence A, the wheat to be detected is A: A genotype; and if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence B, the wheat to be detected is of the C:C genotype.
In the above method, the detection of the nucleotide at the SNP site AX-179388377 on the genome of the wheat to be tested may be accomplished by direct sequencing, or may be performed according to a method comprising the steps of: performing PCR amplification on the genomic DNA of the wheat to be tested by using the reagent B described in the fourth aspect, performing fluorescent signal scanning on the amplified product, and determining the genotype of the SNP site AX-179388377 in the genome of the wheat to be tested according to the following steps: if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence C, the wheat to be detected is A: A genotype; and if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence D, the wheat to be detected is G:G genotype.
In a specific embodiment of the application, the resistance to the black embryo disease is reflected by the black embryo rate of the seeds. The high black embryo rate of the seeds indicates weak resistance to the black embryo disease, and the low black embryo rate of the seeds indicates strong resistance to the black embryo disease.
In the present application, the wheat may be selected from the filial generation of middle wheat 578 and ataxia wheat 22 or 165 parts of wheat material in table 2.
The application determines two wheat anti-black-embryo genes QTL and linkage molecular markers thereof, wherein the QTL is named as QBP.caas-2AL (AX-110623096-AX-179560327, 753.2-753.6 Mb) and QBP.caas-5BL (673.0-679.7 Mb, AX-110623096-AX-179560327) respectively, and under 6 detected environmental conditions, 3.61-13.49% and 3.58-9.13% of phenotype variation can be explained. And KASP primers are developed according to the corresponding markers, and molecular marker assisted screening can be performed on the black embryo genes. The application has important significance for cultivating the wheat variety with the enhanced melanosis antibody.
Drawings
FIG. 1 is a graph of QBP.caas-2AL and QBP.caas-5BL for the middle wheat 578 XJi wheat 22RIL population.
FIG. 2 shows the genotyping results of Kasp_2A_BP and Kasp_5B_BP on 165 wheat varieties. Black circles are blank control and green circles are heterozygous.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1, discovery of 2 Blackdisease resistance QTLs of the middle wheat 578 XJi wheat 22RIL population, and acquisition and use of KASP markers thereof
1. Phenotype data acquisition
Using Zhongmai 578 as female parent and Jimai 22 as male parent, adopting single grain transmission method to construct F containing 262 families 5 RIL population. Zhongmai 578/Jimai 22RIL colony planted in Henan New countryside (34 DEG 53'N,113 DEG 23' E) and commercial hills (33 DEG 43'N,114 DEG 49' E) in 2020-2021 years adopts complete random block design, three times of repetition and single wayA row area, wherein the row length is 1.0m, the row width is 0.2m, and 30 seeds are uniformly sown in each row; 2021-2022 was planted in Henan New countryside and Shandong Texas (37℃45'N,116℃37' E), using Latin square design, three replicates, 6 row zones, 3.0m row length, 0.2m row width, 270 seeds per square meter mechanical drill. In the whole wheat growing process, the field management is carried out according to a local conventional cultivation technology. After normal wheat harvesting and threshing, randomly selecting 200 seeds from each repeated single plant line, counting the number of black embryo seeds by adopting a unified standard (black brown spots are visible on the embryo parts of the seeds), and calculating the black embryo rate.
Extracting 262 family young leaf genome DNA by adopting a CTAB method, measuring the DNA concentration by using a Nanodrop2000c spectrophotometer, regulating the DNA sample to a standard concentration of 50 ng/. Mu.l, detecting the DNA quality by using 0.8% agarose gel, and carrying out SNP typing on the DNA with qualified quality. SNP analysis was performed using a 50K SNP chip completed by the cooperation of the institute of crop science and Affymetrix Axiom, national academy of agricultural sciences.
2. Linkage map construction
RILs and both parents were genotyped using a wheat 50KSNP chip from CapitalBio Corporation (http:// www.capitalbio.com), containing a total of 55,224 SNPs. Genotype data filtering criteria was screening for non-polymorphic markers, families with a marker deletion rate greater than 20% and markers with a minimum allele frequency of less than 30%, the remaining 9354 high quality polymorphic markers were used for subsequent analysis. The filtered polymorphic markers were treated with BIN function in Icimapping v4.2 (http:// www.isbreeding.net/; meng et al 2015) to separate the same genotyping markers into one BIN, creating 34 linkage clusters, including 1501 BIN markers altogether. Genetic linkage maps were mapped using JoinMap v4.0 and MapChart v2.32 software (https:// www.wur.nl/en/show/Mapchart.htm; voorriss, 2002).
3. QTL analysis
And detecting the QTL by using a complete interval mapping method of Icimapping v4.2, and selecting an LOD threshold value to be 2.8. The chromosomes 2A and 5B were mapped to 2 more stable QTLs designated QBP.caas-2AL and QBP.caas-5BL (FIG. 1). The QBP.caas-2AL flank is marked as AX-110623096-AX-179560327, and the physical interval is 753.2-753.6Mb. Under different environmental conditions, 3.61-13.49% of phenotypic variation can be explained (see Table 1, FIG. 1); the flanking marker AX-179560327 (SNP site) was transformed into Kasp_2A_BP, and the genotype of 165 wheat varieties was examined. The QBP.caas-5BL flank is marked as AX-179388377-AX-110910964, and the physical interval is 673.0-679.7Mb. Under different environmental conditions, 3.58-9.13% of phenotypic variation can be explained (see Table 1, FIG. 1); the flanking marker AX-179388377 (SNP site) was transformed into Kasp_5B_BP, and the genotype of 165 wheat varieties was detected.
TABLE 1 detection of QBP.caas-2AL and QBP.caas-5BL by composite interval mapping method
Note that: BLUE represents the best linear unbiased estimate, similar to the mean of 4 environments, and can be used as an environment for QTL positioning.
4. Design and utilization of KASP primer
1. Design of KASP primer
Corresponding to the Kasp_2A_BP marker, the SNP site AX-179560327 is located at physical position 753.6Mb (https:// urgi.versailles. Inra. Fr/blast_iwgsc /) of wheat reference genome (2A chromosome) Chinese Spring RefSeq v 1.0.0. The SNP variation between the middle wheat 578 and the Jimai 22 for the marker AX-179560327 (sense strand) closely linked to QBP.caas-2AL and its surrounding nucleotides are shown in SEQ ID No.7 (M stands for A or C).
Designing a KASP marker primer sequence according to the sense strand of SNP locus AX-179560327, as follows:
upstream primer F1:5' -GAAGGTGACCAAGTTCATGCTCCTCGACAAGTTTCATGTTCATGT-3' (SEQ ID No.1, underlined is the specific fluorescent tag sequence FAM);
the upstream primer F2:5' -GAAGGTCGGAGTCAACGGATTCCTCGACAAGTTTCATGTTCATGG-3' (SEQ ID No.2, underlined is the specific fluorescent tag sequence HEX);
the downstream primer R:5'-GCACGCTCTTCAATCTGGGA-3' (SEQ ID No. 3).
The last base at the 3' -end of the two upstream primers corresponds to SNP site AX-179560327 (sense strand). SNP site AX-179560327 is A or C (represented by M in SEQ ID No. 7) at position 36 of wheat 2A chromosome corresponding to SEQ ID No.7 (sense strand).
The upstream primer F1 is used for amplifying the condition that the nucleotide at the SNP site AX-179560327 (sense strand) on the wheat 2A chromosome is A, and the upstream primer F2 is used for amplifying the condition that the nucleotide at the SNP site AX-179560327 (sense strand) on the wheat 2A chromosome is C; the downstream primer R is a universal primer.
The single-stranded DNA molecule shown in SEQ ID No.1 and the single-stranded DNA molecule shown in SEQ ID No.3 amplify a fragment with genotype A of A homozygous at nucleotide at SNP site AX-179560327 (sense strand) on wheat 2A chromosome.
The single-stranded DNA molecule shown in SEQ ID No.2 and the single-stranded DNA molecule shown in SEQ ID No.3 amplify a fragment with genotype C of C homozygote of the nucleotide at SNP site AX-179560327 (sense strand) on wheat 2A chromosome.
The single-stranded DNA molecule shown in SEQ ID No.1, the single-stranded DNA molecule shown in SEQ ID No.2 and the single-stranded DNA molecule shown in SEQ ID No.3 amplify a fragment with the genotype of A: C heterozygote of the nucleotide at SNP site AX-179560327 (sense strand) on wheat 2A chromosome.
Corresponding to the Kasp_5B_BP marker, the SNP site AX-179388377 is located at physical position 673.0Mb (https:// urgi.versailles. Inra. Fr/blast_iwgsc /) of wheat reference genome (5B chromosome) Chinese Spring RefSeq v 1.0.0. The SNP variation between the middle wheat 578 and the Jimai 22 and the surrounding nucleotide thereof for the marker AX-179388377 (sense strand) closely linked to QBP.caas-5BL are shown in SEQ ID No.8 (R stands for A or G).
Designing a KASP marker primer sequence according to the antisense strand of SNP locus AX-179388377, as follows:
upstream primer F1':5' -GAAGGTGACCAAGTTCATGCTGACGCAAACGCAAACGGT-3' (SEQ ID No.4, underlined is the specific fluorescent tag sequence FAM);
upstream primer F2':5' -GAAGGTCGGAGTCAACGGATTGACGCAAACGCAAACGGC-3' (SEQ ID No.5, underlined)An heterofluorescent tag sequence HEX);
downstream primer R':5'-GCTCACTAGAAGCTGACGCA-3' (SEQ ID No. 6).
The last base at the 3' -end of the two upstream primers corresponds to SNP site AX-179388377 (antisense strand). SNP site AX-179388377 is A or G (represented by R in SEQ ID No. 8) at position 36 of wheat 5B chromosome corresponding to SEQ ID No.8 (sense strand).
The upstream primer F1 'is used for amplifying the condition that the nucleotide at the SNP site AX-179388377 (antisense strand) on the wheat 5B chromosome is T (corresponding to the sense strand, the nucleotide at the SNP site is A), and the upstream primer F2' is used for amplifying the condition that the nucleotide at the SNP site AX-179388377 (antisense strand) on the wheat 5B chromosome is C (corresponding to the sense strand, the nucleotide at the SNP site is G); the downstream primer R' is a universal primer.
The single-stranded DNA molecule shown in SEQ ID No.4 and the single-stranded DNA molecule shown in SEQ ID No.6 amplify a fragment in which the nucleotide at the SNP site AX-179388377 (antisense strand) on the wheat 5B chromosome is homozygous for T (corresponding to the sense strand, the genotype of the SNP site is homozygous for A: A).
The single-stranded DNA molecule shown in SEQ ID No.5 and the single-stranded DNA molecule shown in SEQ ID No.6 amplify a fragment in which the nucleotide at the SNP site AX-179388377 (antisense strand) on the wheat 5B chromosome is C-homozygous (corresponding to the sense strand, the genotype of the SNP site is G: G-homozygous).
The single-stranded DNA molecule shown in SEQ ID No.4, the single-stranded DNA molecule shown in SEQ ID No.5 and the single-stranded DNA molecule shown in SEQ ID No.6 amplify fragments with the nucleotides of T and C heterozygous (corresponding to the sense strand, the genotype of the SNP locus is A: G heterozygous) at the SNP locus AX-179388377 (antisense strand) on the wheat 5B chromosome.
2. Establishment of KASP detection method
Principle of KASP: two forward competitive primers (primer 5 'end has base sequence complementary pairing with fluorophore HEX and FAM, other sequences only have difference at SNP of 3' end) and one reverse common primer; the PCR reaction system contains a fluorescent group and a quenching group modified universal sequence (Master Mix is provided by LGC company), two forward primers can emit light with two different colors, if the locus is homozygous, a single fluorescence is emitted, and if the locus is heterozygous, two kinds of fluorescence are emitted simultaneously.
The KASP marker PCR amplification system was as follows: 2.0. Mu.l of KASP 2 XMaster Mix (LGC, cat# 13448166), 0.048. Mu.l of KASP primer (3 primers mixed at a total concentration of 50. Mu.M, with a molar ratio of two upstream primers to one downstream primer of 2:2:5), 1.952. Mu.l of template DNA (50 ng/. Mu.l). Amplification was performed using a 384-well PCR apparatus (BIO-RAD, S1000TMthermal Cycler) as follows: 94 ℃ for 15min;94 ℃ for 20s, 63-55 ℃ for 1min (1 ℃ drop per cycle), 10 cycles; 94℃for 20s, 55℃for 60s,32 cycles. The final fluorescence data were read from the PCR amplified products in an auto-focusing fluorescent multifunctional microplate reader (PHERAstarplus SNP, BMG LABECH) and the data were then imported into Klumtercalaller v3.4 (LGC, hoddesdon, UK) for genotyping.
For Kasp_2A_BP marker (SNP site AX-179560327): if the fluorescence signal data of the amplified product is analyzed by a genotyping software KlumterCaller to be close to the Y axis (FAM fluorescence signal), the genotype of the locus (sense strand) is represented as AA homozygosity; if the fluorescence signal data of the amplified product is analyzed by a genotyping software KlumterCaller to be close to the X axis (HEX fluorescence signal), the genotype of the locus (sense strand) is represented as CC homozygosity; if the fluorescence signal data of the amplified product is located in the middle of the X-axis and the Y-axis (FAM and HEX signals are simultaneously present) by genotyping software KiusterCaller analysis, the genotype representing the site (sense strand) is AC heterozygous.
For Kasp_5B_BP marker (SNP site AX-179388377): if the fluorescence signal data of the amplified product is analyzed by a genotyping software KlumterCaller to be close to the Y axis (FAM signal), the genotype of the locus (sense strand) is represented as AA homozygosity; if the fluorescence signal data of the amplified product is analyzed by a genotyping software KlumterCaller to be close to the X axis (HEX signal), the genotype of the locus (sense strand) is represented as GG homozygosity; if the fluorescence signal data of the amplified product is located in the middle of the X axis and the Y axis (FAM and HEX signals are simultaneously present) by genotyping software KlumterCaller analysis, the genotype representing the site (sense strand) is AG heterozygous.
3. Kasp detection
The experimental materials were 165 parts of Huang-Huai wheat variety, see in particular Table 2.
165 parts of Huang Huai wheat region varieties are planted in Henan Anyang (35 DEG 12'N,113 DEG 37' E) (2012-2013, 2013-2014 and 2014-2015 years) and Anhui/28717S (35 DEG 12'N,113 DEG 37' E) (2012-2013 and 2013-2014 years). All experiments were performed using a random block design, 3 replicates, 3 rows of blocks, 2m rows long, 25cm row spacing, 50 grains/row. And performing field management according to a local conventional management rule, and performing blackembryo disease resistance identification. After normal wheat harvesting and threshing, randomly selecting 200 seeds from each repeated single plant line, counting the number of black embryo seeds by adopting a unified standard (black brown spots are visible on the embryo parts of the seeds), and calculating the black embryo rate.
And (3) respectively extracting genome DNA of all experimental materials, taking the genome DNA as a template, and detecting by using the KASP primer designed in the step (2), wherein the specific operation is shown in the step (2).
The results are shown in Table 2 and FIG. 2.
165 wheat varieties were tested using Kasp_2A_BP markers, and the results showed that 56 varieties were medium wheat 578 genotype (AA), 85 varieties were Jimai 22 (CC) genotype, and the average black embryo rate of CC homozygous varieties was reduced by 16.73% compared with AA homozygous varieties, with a significant difference at the 0.05 level (Table 3).
165 wheat varieties were detected using Kasp_5B_BP markers, and the results showed that 47 varieties were medium wheat 578 genotype (GG), 95 varieties were Jimai 22 genotype (AA), and that the average black embryo rate of the AA homozygous varieties was 18.15% lower than that of the GG homozygous varieties, with a significant difference at the 0.05 level (Table 3).
Table 2, genotype test results of 165 wheat varieties
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Note that: in the table, HK1 is named HK1/6/NVSR3/5/BEZ/TVR/5/CFN/BEZ// SU92/CI13645/3NAI60 in detail.
Kasp_2A_BP is a KASP marker for verifying the 2A chromosome QTL, AA and CC represent the genotypes of Zhongmai 578 and Jimai 22 respectively, and NN represents the deletion;
Kasp_5B_BP is a KASP marker used to verify the 5B chromosome QTL, GG and AA represent the middle wheat 578 and the Jimai 22 genotypes, respectively, and NN represents the deletion.
TABLE 3 Natural population black embryo Effect of QBP.caas-2A and QBP.caas-5B 165 parts
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. The use of a single nucleotide polymorphism of SNP site AX-179560327 and/or a single nucleotide polymorphism of SNP site AX-179388377 on the wheat genome as a marker in any one of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) Preparing a product for screening out wheat single plants with relatively weak resistance to the black embryo disease;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the SNP site AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on the wheat 5B chromosome, and the nucleotide at the SNP site is G or A.
2. Use of a substance for detecting a single nucleotide polymorphism of SNP site AX-179560327 on a wheat genome and/or a substance for detecting a single nucleotide polymorphism of SNP site AX-179388377 on a wheat genome, in any of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) Preparing a product for screening out wheat single plants with relatively weak resistance to the black embryo disease;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the SNP site AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on the wheat 5B chromosome, and the nucleotide at the SNP site is G or A.
3. Use according to claim 1 or 2, characterized in that: the substance for detecting the single nucleotide polymorphism of the SNP site AX-179560327 on wheat genome is KASP primer A as described in claim 4 or 5 or reagent A as described in claim 6 or 7 or a kit containing the reagent A;
the substance for detecting the single nucleotide polymorphism of the SNP site AX-179388377 on the wheat genome is the KASP primer B as set forth in claim 4 or 5 or the reagent B as set forth in claim 6 or 7 or a kit containing the reagent B.
4. A set of primers for use in or in assisting in the identification of wheat's resistance to black-embryo disease, characterized in that: the set of primers is any one of the following:
(B1) KASP primer a; the KASP primer A consists of a primer 1, a primer 2 and a primer 3; the primer 1 is single-stranded DNA with a fluorescent tag sequence A and 22-41 positions of SEQ ID No.1 from the 5 'end to the 3' end; the primer 2 is single-stranded DNA with a fluorescent tag sequence B and 22-41 positions of SEQ ID No.2 from the 5 'end to the 3' end; the primer 3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 3;
(B2) KASP primer b; the KASP primer B consists of a primer 4, a primer 5 and a primer 6; the primer 4 is single-stranded DNA with a fluorescent tag sequence C and 22-39 positions of SEQ ID No.4 from the 5 'end to the 3' end; the primer 5 is single-stranded DNA with a fluorescent tag sequence D and 22-39 positions of SEQ ID No.5 from the 5 'end to the 3' end; the primer 6 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 6;
(B3) Consists of the KASP primer A in (B1) and the KASP primer B in (B2).
5. The set of primers of claim 4, wherein: the fluorescent tag sequence A is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence A is the 1 st-21 st position of SEQ ID No. 1; the fluorescent tag sequence B is a fluorescent tag sequence HEX, and the nucleotide sequence of the fluorescent tag sequence B is the 1 st-21 st position of SEQ ID No. 2; the fluorescent tag sequence C is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence C is the 1 st-21 st position of SEQ ID No. 4; the tag sequence D is a fluorescent tag sequence HEX, and the nucleotide sequence of the tag sequence D is the 1 st-21 st position of SEQ ID No. 5;
further, the primer 1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer 2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2; and/or
Further, the primer 4 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 4; the primer 5 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 5.
6. A reagent or kit for identifying or aiding in the identification of black-embryo resistance in wheat, characterized in that: the kit contains the reagent; the reagent is any one of the following:
(C1) A reagent a comprising a KASP primer a according to claim 4 or 5;
(C2) A reagent b comprising the KASP primer b of claim 4 or 5;
(C3) Consists of the reagent A described in (C1) and the reagent B described in (C2).
7. Specific DNA molecules are shown as SEQ ID No.7 or SEQ ID No. 8.
8. Use of the set of primers of claim 4 or 5 or the reagent or kit of claim 6 or the specific DNA molecule of claim 7 in any of the following:
(A1) Identifying or aiding in the identification of wheat black-embryo resistance;
(A2) Preparing a product for identifying or aiding in the identification of blackembryo disease resistance of wheat;
(A3) Comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A4) Preparing a product for comparing the resistance strength of the wheat to be tested to the black embryo disease;
(A5) Selecting a single plant or strain or variety of wheat with relatively strong black embryo disease resistance;
(A6) Preparing a product for breeding a wheat single plant or strain or variety with relatively strong resistance to black embryo diseases;
(A7) Screening out wheat single plants with relatively weak resistance to the black embryo diseases;
(A8) Preparing a product for screening out wheat single plants with relatively weak resistance to the black embryo disease;
(A9) Wheat breeding.
9. The method comprises the following steps:
method I: a method for comparing the resistance of wheat to be tested to black embryo disease comprises the following steps of (D1) or (D2):
(D1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and determining the resistance strength of the wheat to be detected to the black embryo disease according to the genotype of the wheat to be detected as follows: the resistance of the wheat to be tested of genotype C to the black-embryo disease is stronger than or the candidate is stronger than the resistance of the wheat to be tested of genotype A to the black-embryo disease;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the genotype C is homozygote of C at the SNP locus AX-179560327 on the wheat genome;
the A genotype is homozygous A at the SNP locus AX-179560327 on the wheat genome;
(D2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and determining the resistance strength of the wheat to be detected to the black embryo disease according to the genotype of the wheat to be detected as follows: the resistance of the A genotype to the black-embryo disease of the wheat to be tested is stronger than or the candidate is stronger than the resistance of the G genotype to the black-embryo disease of the wheat to be tested;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the A genotype is homozygous A at the SNP locus AX-179388377 on the wheat genome;
the G genotype is homozygote of G at the SNP locus AX-179388377 on the wheat genome;
method II: a method for breeding a wheat single plant or strain or variety with relatively high resistance to black-embryo disease, comprising the steps of (E1) or (E2):
(E1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, selecting the wheat to be detected with the C-C genotype as a parent for breeding, and selecting the wheat with the C-C genotype in each breeding generation to finally obtain a wheat single plant or strain or variety with relatively strong black embryo resistance;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the genotype C is homozygote of C at the SNP locus AX-179560327 on the wheat genome;
(E2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, selecting the wheat to be detected with A genotype as a parent for breeding, and selecting the wheat with A genotype in each breeding generation to finally obtain a wheat single plant or strain or variety with relatively strong black embryo resistance;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the A genotype is homozygous A at the SNP locus AX-179388377 on the wheat genome;
method III: a method for selecting a wheat individual having relatively weak resistance to blackembryo disease, comprising the steps of (F1) or (F2):
(F1) Detecting the nucleotide at SNP locus AX-179560327 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and eliminating the single plant of the wheat to be detected with the A genotype;
the SNP locus AX-179560327 is positioned at the 36 th position of SEQ ID No.7 on a wheat 2A chromosome, and the nucleotide at the SNP locus is A or C;
the A genotype is homozygous A at the SNP locus AX-179560327 on the wheat genome;
(F2) Detecting the nucleotide at SNP locus AX-179388377 on the genome of the wheat to be detected, determining the genotype of the wheat to be detected, and eliminating the single plant of the wheat to be detected with G genotype;
the SNP locus AX-179388377 is positioned at the 36 th position of SEQ ID No.8 on a wheat 5B chromosome, and the nucleotide at the SNP locus is G or A;
the G genotype is homozygous G at the SNP site AX-179388377 on the wheat genome.
10. The method according to claim 9, wherein: detecting the nucleotide at the SNP locus AX-179560327 on the genome of the wheat to be detected is carried out according to the method comprising the following steps: PCR amplification of the genomic DNA of the test wheat using the reagent A of claim 6, fluorescent signal scanning of the amplified product, and then determining the genotype of the SNP site AX-179560327 in the genome of the test wheat as follows: if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence A, the wheat to be detected is A: A genotype; if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence B, the wheat to be detected is of a C:C genotype;
and/or
Detecting the nucleotide at the SNP locus AX-179388377 on the genome of the wheat to be detected is carried out according to the method comprising the following steps: PCR amplification of the genomic DNA of the test wheat using the reagent B of claim 6, fluorescent signal scanning of the amplified product, and then determining the genotype of the SNP site AX-179388377 in the genome of the test wheat as follows: if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence C, the wheat to be detected is A: A genotype; and if the fluorescent signal of the amplified product of the wheat to be detected is the signal corresponding to the fluorescent tag sequence D, the wheat to be detected is G:G genotype.
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