CN116904636A - Molecular marker for detecting wheat stem WSC content QTL QWSC.caas-7DS and application - Google Patents
Molecular marker for detecting wheat stem WSC content QTL QWSC.caas-7DS and application Download PDFInfo
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Abstract
The invention discloses a Quantitative Trait Locus (QTL) for detecting WSC content of wheat stalksQWSC.caas‑7DSMolecular markers and application thereof, belonging to the technical field of biological breeding. Positioning a stem WSC content QTL on a 7D chromosome short arm by using a high-density genetic map of a Yangmai 16/Zhongmai 895DH population and 660K SNP thereofQWsc.caas‑7DSThe synergistic allele was from Zhongmai 895. Based on the research, the patent develops a novel drug for treating the cancerQWsc.caas‑7DSSNP markers within a regionAX‑109429088KASP-labeled primer set of (2), KASP-labeled primer set pair provided by the inventionQWsc.caas‑7DSSite(s)AX‑109429088The genotype of the sample is detected, the human error is low, the analysis flux is high, and the method is suitable for detecting a large number of samples. The KASP mark provided by the invention can be used for the QTL locus of WSC content of wheat stalksQWsc.caas‑7DSThe transformation of the excellent allele has important significance for accelerating the genetic improvement of the excellent allele by utilizing the QTL locus and improving the breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of crop molecular breeding, relates to a molecular marker for detecting a wheat stem water-soluble carbohydrate (WSC) content QTL QWSC.caas-7DS and application thereof, and in particular relates to a development and detection method of a KASP molecular marker for the wheat stem water-soluble carbohydrate content QTL QWSC.caas-7DS and application thereof.
Background
Wheat is an important grain crop, and improving the wheat yield has important significance for guaranteeing the grain safety of China and even the world. The filling period is a critical period in determining wheat yield, while the carbon source for grain filling is mainly derived from photosynthetic products made by flag leaves and water-soluble carbohydrates stored in stalks and leaf sheaths (Water soluble carbohydrate, WSC; ehdaie et al, 2006). Under normal moisture conditions, the contribution of stalk WSC to yield is 10% -20%, and under drought stress, the contribution to yield is as high as 30% -50% (Foulkes et al, 2002; rebetzke et al, 2008). In the evolution history of wheat varieties, the storage amount of the stem WSC is improved remarkably (Sadras and Lawson,2011; xiao et al, 2012), and the stem WSC has an important contribution to improving the grain weight and yield of the wheat varieties and improving the stress resistance and the stable yield of the wheat varieties. However, the stem materials of the current wheat variety still remain more in the middle and later stages of grouting, and a large genetic improvement space exists. Therefore, increasing the WSC content of stalks has become an important goal for wheat breeding and genetic improvement (Xue et al, 2008, drecer et al, 2009).
The study shows that the WSC content of the stem is highest from heading to 10 days after flowering, the stem is in a stable accumulation state about 20 days after flowering, then metabolic attenuation occurs, and the stem is at a lower level about 30 days after flowering. Therefore, it is of great importance to analyze the WSC content of wheat stalks 10 days after flowers (Xue et al, 2008; khoshor et al, 2014).
Single Nucleotide Polymorphisms (SNPs) are the most common form of genetic variation among plant individuals, and frequently occurring single nucleotide polymorphisms include base substitution, insertion and deletion, and are ideal molecular markers for genetic research of complex traits of plants.
Yangmai 16 and Zhongmai 895 are the main cultivars of winter wheat in the middle and downstream of Yangtze river and south China and Huang Huai winter wheat respectively. In production, the two varieties show the characteristics of quick grouting and high yield. (what is tiger, etc., 2014; liu Da is equivalent, 2017; guo Yongtao, etc., 2019). The genotype of a wheat-raising 16/middle wheat 895 Doubled Haploid (DH) population is analyzed by utilizing a wheat 660K SNP chip, a high-density linkage map is constructed, and a major QTL locus QWsc.caas-7DS and AX-109429088 which are 660K SNP markers in a QWsc.caas-7DS interval are positioned on a 7D chromosome short arm in combination with the measurement of the WSC content of a stalk 10 days after wheat flowers in the field (relevant research results are shown in detail in the paper of the doctor science).
The wheat 660K SNP chip itself contains 63 tens of thousands of probe sequences, and the genotype of a large number of different SNPs in a wheat material can be measured simultaneously by adopting the 660K SNP chip, but the genotype detection method is not suitable for the requirement of single SNP marker genotype detection. The competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) technology is a high-throughput, low-cost and low-error-rate SNP typing technology, which is widely applied to crop molecular marker-assisted breeding. Based on the detection, the patent converts 660K SNP marker AX-109429088 in the QWSC.caas-7DS interval into KASP marker, designs KASP marker primer group for detecting AX-109429088 genotype, and provides a good tool for breeding by utilizing stem WSC content QTL site QWSC.caas-7 DS.
Disclosure of Invention
The invention aims to provide a KASP marker for identifying the genotype of a tightly linked SNP marker AX-109429088 of a wheat stem WSC content QTL QWSC.caas-7DS with high throughput, low cost and high accuracy and application thereof.
In a first aspect, the present invention provides a KASP marker for detecting the genotype of SNP marker AX-109429088.
The SNP marker AX-109429088 is one of wheat 660K SNP chips (designed by Chinese national academy of agricultural sciences, produced by Affymetrix company), and the flanking sequence is SEQ ID NO:1, which is an antisense strand corresponding to the base sequence of positions 64443446 to 64443516 on the 7D chromosome in version 1.0 of the chinese spring genome of wheat material. SEQ ID NO:1 "[ A/G ]" at position 36 represents two polymorphic mononucleotide A or G of the SNP marker, i.e., the base at the position may be A or G in the actual wheat material. SEQ ID NO:1 is SEQ ID NO in the sequence table: 2, namely the sense strand corresponding to the base sequence from 64443446 to 64443516 on the 7D chromosome in the 1.0 version of the Chinese spring genome of the wheat material. SEQ ID NO:2 "[ T/C ]" at position 36 represents two polymorphic single nucleotides T or C of the SNP marker, which corresponds to base 64443481 of the 7D chromosome sense strand in version 1.0 of the Chinese spring reference genome of the wheat material, which may be T or C in the actual wheat material. The sequence information of the version 1.0 of the Chinese spring genome of the wheat material can be consulted or obtained from the website http:// 202.194.139.32.
The name of the KASP mark is AX-109429088-KASP, and at least comprises one primer group.
In a first aspect, the present invention provides a KASP marked primer set comprising:
1) A primer A;
primer A is (a 1) or (a 2) as follows:
(a1) SEQ ID NO:3, wherein the 1 st to 21 st positions of the sequence of the single-stranded DNA molecule are tag sequences A;
(a2) Setting SEQ ID NO:3 by substitution and/or deletion and/or addition of one or several nucleotides and which correspond to SEQ ID NO:3 a DNA molecule having the same function;
2) A primer B;
primer B is (B1) or (B2) as follows:
(b1) SEQ ID NO:4, wherein the 1 st to 21 st positions of the sequence of the single-stranded DNA molecule are tag sequences B;
(b2) Setting SEQ ID NO:4 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:4 a DNA molecule having the same function;
3) A primer C;
primer C is (C1) or (C2) as follows:
(c1) SEQ ID NO: 5;
(c2) Setting SEQ ID NO:5 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:5 DNA molecules having the same function.
Further, the a1 sequence of the primer A is a tag sequence A and SEQ ID NO in a sequence table from the 5 'end to the 3' end: 3, single-stranded DNA at positions 22-41;
the B1 sequence of the primer B is a tag sequence B and SEQ ID NO in a sequence table from the 5 'end to the 3' end: 4, single-stranded DNA at positions 22-41;
further, the nucleotide sequence of the tag sequence A is SEQ ID NO in the sequence table: 3, the nucleotide sequence of the tag sequence B is SEQ ID NO in the sequence table: 4 from position 1 to 21.
In a second aspect, the present invention provides a PCR reagent for identifying the genotype of the wheat SNP marker AX-109429088.
The PCR reagent for identifying the genotype of the wheat SNP marker AX-109429088 provided by the invention comprises the KASP marker primer set.
In some embodiments, the PCR reagents further comprise a PCR Mix;
the PCR Mix is as follows (d 1) or (d 2):
(d1) The kit comprises a fluorescent probe A, a fluorescent probe B and HiGeno DNA polymerase, wherein the 5' end of the fluorescent probe A is marked with a FAM fluorescent group, and the 3' end sequence is complementary with a tag sequence A at the 5' end of a primer A; the HEX fluorescent group is marked on the 5' end of the fluorescent probe B, and the 3' end sequence is complementary with the label sequence B on the 5' end of the primer B. The specific base bands in the middle of the sequences of the fluorescent probe A and the fluorescent probe B are provided with specific quenching groups, the sequences pass through hairpin structures, so that the fluorescent groups and the quenching groups are close to each other, and under the condition that PCR is not amplified, the quenching groups absorb the emitted light of the fluorescent groups, and no fluorescent signal is detected.
In the invention, the fluorescent Probe A, the fluorescent Probe B and the HiGeno DNA polymerase are existing technologies, and exist in HiGeno 2X Probe Mix A, which is a product of Beijing Jiacheng Biotechnology Co., ltd.
(d2) Comprising the fluorescent probe A, the fluorescent probe B, and the DNA polymerase described in (d 1) above, wherein the DNA polymerase is a DNA polymerase which is different from the HiGeno DNA polymerase described in (d 1) above and can be used for realizing a DNA polymerase chain reaction.
In a third aspect, the present invention provides a kit for identifying the genotype of SNP marker AX-109429088 in wheat.
The kit for identifying the genotype of the SNP marker AX-109429088 in wheat provided by the invention comprises the KASP marker primer set or the PCR reagent.
In a fourth aspect, the present invention provides the use of a KASP marker as described above or a primer set as described above or a PCR reagent as described above or a kit as described above.
The present invention provides the use of the above KASP markers or the above primer sets or the above PCR reagents or the above kits in any one of the following (1) to (8):
(1) Detecting or assisting in detecting genotype of tightly linked SNP marker AX-109429088 of QWsc.caas-7DS of QTL locus of WSC content of wheat stalk to be detected;
(2) Identifying or assisting in identifying the WSC content of the wheat stalks to be tested;
(3) Auxiliary screening or breeding of wheat single plants or strains or varieties with high WSC content;
(4) Auxiliary screening or breeding of wheat single plants or strains or varieties with low WSC content;
(5) Preparing a product for detecting or assisting in detecting the genotype of the SNP marker AX-109429088 of the wheat to be detected;
(6) Preparing a product for detecting or assisting in detecting the content QTL QWsc.caas-7DS of wheat stalk WSC to be detected;
(7) Preparing a product for screening or breeding single plants or strains or varieties of wheat with high WSC content;
(8) The method is used for preparing products for screening or breeding single plants or strains or varieties of wheat with low WSC content.
In a fifth aspect, the present invention provides a method for identifying the genotype of SNP marker AX-109429088 in wheat.
The method for identifying the genotype of the SNP marker AX-109429088 in the wheat comprises the following steps:
taking the genome DNA of the wheat to be detected as a template, and carrying out PCR amplification by adopting the KASP mark or the primer group and the PCR reagent or the kit to obtain a wheat amplification product to be detected;
carrying out fluorescent signal scanning and genotyping analysis on the wheat amplification product to be detected, and determining the genotype of the SNP marker AX-109429088 according to the genotyping result and the following principle:
if the fluorescence signal data of the amplified product of the wheat to be detected is subjected to typing and then gathered at a position near the upper side of the Y axis in a typing result coordinate system, the genotype of the SNP marker AX-109429088 in the wheat to be detected is TT;
if the fluorescence signal data of the amplified product of the wheat to be detected is clustered at a position near the right side of the X axis in the coordinate system after being typed, the genotype of the SNP marker AX-109429088 in the wheat to be detected is CC;
if the fluorescent signal data of the amplified product of the wheat to be detected is typed and then gathered at a position close to the middle position of a diagonal line in a coordinate system, the genotype of the SNP marker AX-109429088 in the wheat to be detected is a heterozygous genotype, which is represented by TC or CT.
In a sixth aspect, the present invention provides a method for identifying or aiding in the identification of the WSC content of a wheat straw under test.
The method for identifying or assisting in identifying the WSC content of the wheat stalks to be tested provided by the invention comprises the following steps:
using genome DNA of the wheat to be tested as a template, identifying the genotype of SNP marker AX-109429088 in the wheat to be tested by the method of the fifth aspect,
if the genotype of the SNP marker AX-109429088 in the material to be detected is TT, the wheat to be detected has relatively high content of the stalk WSC;
if the genotype of AX-109429088 in the wheat to be tested is CC, the wheat to be tested has a relatively low content of stalk WSC.
In a seventh aspect, the invention provides a method for assisting in screening or breeding a wheat single plant or strain or variety with high WSC content.
The method for assisting in screening or breeding the wheat single plant or strain or variety with high WSC content comprises the following steps:
the method of the fifth aspect is adopted to identify the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS in the wheat to be tested, the wheat material with the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS being TT or CT is selected in each generation of breeding, and finally, the wheat single plant or strain or variety with the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS being TT is bred.
In practical application, the wheat material with the genotype of the SNP marker AX-109429088 being TT can be used as a parent to be hybridized with the material with the genotype of the SNP marker AX-109429088 being TT or CT or CC, the method for detecting the genotype of the SNP marker AX-109429088 in wheat is adopted in the separation generation, and the single plant with the genotype of the marker AX-109429088 being TT or CT is selected until the stable single plant or strain or variety with the genotype of the tightly linked SNP marker AX-109429088 of the QTL locus QWsc.caas-7DS with the WSC content being TT is bred.
In an eighth aspect, the invention provides a method for assisting in screening or breeding a wheat single plant or strain or variety with low WSC content.
The method for assisting in screening or breeding the wheat single plant or strain or variety with low WSC content comprises the following steps:
the method of the fifth aspect is adopted to identify the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS in the wheat to be tested, the wheat material with the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS of CC or CT is selected in each generation of breeding, and finally, the wheat single plant or strain or variety with the genotype of the tightly linked SNP marker AX-109429088 of the stem WSC content QTL site QWsc.caas-7DS of CC is bred.
In practical application, the wheat material with the genotype of the SNP marker AX-109429088 of CC is used as a parent to be hybridized with the material with the genotype of the SNP marker AX-109429088 of CC or CT or TT, the method for detecting the genotype of the SNP marker AX-109429088 in the wheat to be detected is adopted in the separation generation, and the single plant with the genotype of the marker AX-109429088 of CC or CT is selected until the stable single plant or strain or variety with the genotype of the tightly linked SNP marker AX-109429088 of QWsc.caas-7DS of the QTL site of WSC content of CC is bred.
In the foregoing aspects, the higher WSC content of the stem in the present invention refers to the fact that when the effect of other gene loci affecting the WSC content on the genome of the wheat is equal, the gene loci correspond to SEQ ID NO in the sequence table on the 7D chromosome in the genome: 2, wherein the 36 th base of the gene fragment shown in the formula 2 is the homozygote of T, and the WSC content of the stem is higher than the WSC content of the stem 10 days after the flowers of the wheat to be detected, and the homozygote of which the base is C at the position. The fact that the WSC content of the stalk is low in the invention means that when the effects of other gene loci affecting the WSC content on the genome of the wheat are equal, the WSC content is equal to those of the gene loci corresponding to SEQ ID NO in a sequence table on a 7D chromosome in the genome: 2, wherein the 36 th base of the gene fragment shown in the formula 2 is C homozygote, and the WSC content of the stem is higher than that of the stem 10 days after flowers of the wheat to be detected, and the base of the stem is T homozygote. According to the invention, the WSC content of 174 families of a wheat raising 16/middle wheat 895DH group is measured in different environments, genotype analysis is carried out on the group by using a wheat 660K SNP chip, a high-density linkage map is constructed, a main effect QTL site for controlling the WSC content of wheat exists on a 7D chromosome short arm through QTL positioning, the site is marked as QWsc.caas-7DS, and the SNP marker AX-109429088 in the QTL interval is converted into a KASP marker AX-109429088-KASP which can be used for detecting or assisting in detecting and controlling the WSC content of the stalks with high flux, low cost and high accuracy. Experiments prove that: the KASP molecular marker AX-109429088-KASP can be used for molecular marker assisted selective breeding of wheat stem WSC content QTL QWsc.caas-7 DS. The invention provides a good tool for breeding and utilizing the QWsc.caas-7DS of the QTL locus of the WSC content of the stem, and has important practical significance for accelerating genetic improvement by utilizing the excellent allele of the QTL locus.
Drawings
FIG. 1 is a diagram of a genetic linkage around QWsc.caas-7DS of a 7D chromosome short arm QTL constructed using wheat 660K SNP chip markers. The numerical values in the figures represent the genetic position of the markers in cM; blue blocks represent genetic confidence intervals for the QTL QWsc.caas-7 DS.
FIG. 2 is a test result of high WSC content parent (middle wheat 895), low WSC content parent (Yangmai 16) and partial DH colony families using the KASP marker AX-109429088-KASP.
FIG. 3 shows the results of the detection of the middle wheat 895, yangmai 16, and another 161 wheat varieties using the KASP marker AX-109429088-KASP.
FIG. 4 is a test result of 10 days after flowers of two genotypic materials in 161 wheat varieties were detected using the KASP marker AX-109429088-KASP.
Detailed Description
The following examples illustrate the principles and features of the present invention, which are presented to facilitate a better understanding of the invention, but do not limit the scope of the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The primers used were all synthesized by the Optimazaceae biological gene company.
The wheat varieties used are all provided by the national center for wheat improvement.
In the following quantitative test, a wheat stalk WSC near infrared spectrum quantitative model and method constructed by Wang et al (2014) are adopted for measuring the content of the stalk WSC.
The quantitative tests in the following examples were all performed in triplicate, and the results averaged.
Example 1, acquisition of Tight linkage molecular marker AX-109429088 of wheat Gao Jing stalk WSC content QTL site QWsc.caas-7DS and KASP marker primer group thereof
Test material: middle wheat 895 and raised wheat 16 are respectively one of main-pushing varieties in Huang-Huai wheat regions and winter wheat regions in the middle and lower reaches of Yangtze river in China, and have the advantages of excellent comprehensive agronomic characters, quick grouting and high yield. The study uses Yangmai 16 as a female parent and Zhongmai 895 as a male parent to construct a Yangmai 16/Zhongmai 895DH population, which contains 174 families.
And (3) field test: DH colony is planted in a Henan Xinxiang test station in 2016-2017 and 2017-2018, a random block design is adopted, three repetitions are set, the DH colony is planted in a cell sowing mode, six rows of each cell are planted in a row length of 3m, and a row spacing is 25cm. The field management refers to the management of a local test station, and the control of powdery mildew, stripe rust, aphid and other diseases and insect pests is mainly carried out.
And (3) measuring the WSC content of the stems: recording flowering period of each test material, randomly cutting 20 main stems from each district 10 days after flowers, removing leaves and spikes, retaining stems, deactivating enzyme at 105 ℃ for 30min, and drying at 80 ℃. The stalk WSC content was determined by near infrared spectroscopy as constructed by Wang et al (2014). Each sample was subjected to 3 technical replicates and averaged for statistical analysis.
Genetic map construction: the related information of construction of Yangmai 16/Zhongmai 895DH population genetic map is described in detail in the university position treatise on construction of Yangmai 16/Zhongmai 895 double haploid population high-density genetic map and powdery mildew resistance QTL location published in 2019 by Xu Xiaoting.
Finding of QTL localization and linkage marker AX-109429088: and carrying out QTL positioning on WSC contents in different environments of 174 families of the DH group by using QTL Ici mapping V4.1 software and adopting a complete composite interval mapping method (ICIM). The LOD value is chosen to be 2.5 as the threshold. The positioning result shows that a main effect QTL locus (figure 1) for controlling the WSC content exists on a 7D chromosome short arm, wherein the two sides of the QTL interval of the positioning result of 2016-2017 are marked as AX-111666703 and AX-109429088, the LOD value is 2.68, and the explained phenotype variation rate is 4.31%; the two sides of the QTL interval of the 2017-2018 annual positioning result are marked as AX-109429088 and AX-109832413, the LOD value is 8.99, and the explained phenotype variation rate is 13.95%. A large number of sequence analyses, alignments and pre-experiments were performed, and the flanking sequences of SNP marker AX-109429088 were found to be specific on the 7D chromosome, so that they were converted into KASP markers for molecular marker assisted selection breeding.
Obtaining a primer set of KASP marker AX-109429088-KASP: SEQ ID NO:1 is a flanking sequence of SNP marker AX-109429088, which is an antisense strand corresponding to the 64443446 th to 64443516 th base sequence on the 7D chromosome in version 1.0 of the Chinese spring genome of wheat material. SEQ ID NO:1 "[ A/G ]" means two polymorphic single nucleotides A or G of the SNP marker, i.e., the base at the position in the actual variety is A or G. Using SEQ ID NO:1 at International wheat genome sequencing Consortium (IWSCC) website https:// heat-ugi. Versailles. Inra. Fr/BLAST search, 2 were obtained with SEQ ID NO:1, and the homologous sequences with the identity of more than 90% are SEQ ID NO in a sequence table respectively: 6, SEQ ID NO:7, which are located on the wheat 7D and 7B chromosomes, respectively. Wherein SEQ ID NO:6 is a nucleotide sequence of SEQ ID NO containing a marker AX-109429088 with a C base at the SNP site: 2.
for SEQ ID NO:6 and SEQ ID NO:7, performing multi-sequence alignment, and designing a set of KASP primer groups, which consists of a primer A, a primer B and a primer C according to the chromosome specificity and the general principle of KASP primer design. The 3' -end of the two upstream primers (primer A and primer B) is marked with allelic variation base C or T of AX-109429088, and the downstream primer (primer C) ensures the specificity of 7D chromosome amplified by PCR. The 5 'end of the upstream primer is connected with a fluorescent tag sequence, wherein the 5' end of the primer A is connected with a FAM fluorescent tag sequence: 5'-GAAGGTGACCAAGTTCATGCT-3', the 5' end of primer B is connected with HEX fluorescent tag sequence: 5'-GAAGGTCGGAGTCAACGGATT-3'.
The KASP marker AX-109429088-KASP developed based on the wheat stem WSC content QTL locus closely linked with SNP marker AX-109429088 and a primer group thereof consist of a primer A, a primer B and a primer C.
Primer A:5'-GAAGGTGACCAAGTTCATGCTCGAGCATTTCCGGTAATGGC-3' (SEQ ID NO:3, underlined sequence is FAM linker tag sequence A);
primer a may also be a primer sequence that encodes SEQ ID NO:3 by substitution and/or deletion and/or addition of one or several nucleotides and which correspond to SEQ ID NO:3 DNA molecules having the same function.
Primer B:5'-GAAGGTCGGAGTCAACGGATTCGAGCATTTCCGGTAATGGT-3' (SEQ ID NO:4, underlined sequence HEX linker tag sequence B);
primer B may also be a primer that encodes SEQ ID NO:4 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:4 a DNA molecule having the same function;
primer C:5'-GGGCAAGTATGTCACATCTTTATAT-3' (SEQ ID NO: 5), or alternatively SEQ ID NO:5 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:5 DNA molecules having the same function.
EXAMPLE 2 establishment of method for detecting SNP marker AX-109429088 genotype Using KASP marker
Detection of the Kasp marker AX-109429088-KASP the parent and part of the family of the Yangmai 16/Zhongmai 895DH population, in particular, comprises:
1. extracting genome DNA of each family of Yangmai 16/Zhongmai 895DH population by adopting a CTAB method, and diluting to obtain a template solution with the DNA concentration of about 30 ng/mu L. In addition, template DNA of Zhongmai 895 and Yangmai 16 were mixed in equal amounts to serve as a control template for heterozygous genotypes, and sterile ultrapure water was used as a blank control template.
2. And (2) taking the genomic DNA extracted in the step (1) as a template, and carrying out PCR amplification by adopting a KASP (KASP) mark primer group which is developed in the example (1) and used for detecting the genotype of a tightly linked marker AX-109429088 of wheat stem WSC content QTL locus QWSC.caas-7DS, so as to obtain an amplification product.
Preparing KASP marked primer working solution:
12. Mu.L (100. Mu.M) of the upstream primer (primer A and primer B) and 30. Mu.L (100. Mu.M) of the downstream primer (primer C) were each taken, and the mixture was supplemented to 100. Mu.L with sterile ultrapure water, and thoroughly mixed to prepare a KASP-labeled primer working solution for use.
PCR amplification reaction system: contains 2. Mu.L (about 30 ng/. Mu.L) of DNA template, 0.08. Mu. L, hiGeno2 of primer working solution, 2 XProbe Mix A (product of Beijing Jia Biotech Co., ltd., product No.: AQP-001L) and was supplemented to 5. Mu.L with sterile ultra-pure water.
The PCR reaction procedure was as follows: firstly, pre-denaturation at 95 ℃ for 15min; second, denaturation at 95℃for 20s, 65-58℃for 60s (0.7℃drop per cycle) for 10 cycles; thirdly, denaturation at 95 ℃ for 20s, annealing at 57 ℃ and extension for 60s, and 35 cycles; fourth, the temperature is kept at 37 ℃ for 60 seconds, and fluorescent signal scanning is carried out at 37 ℃. Experiments were performed with a control (NTC) without template DNA added to the reaction system, with 1 or more controls per plate.
3. Fluorescence scanning of PCR amplification products:
adopting a Roche LightCycler II instrument platform to scan fluorescent signals of the PCR amplification products, wherein the FAM excitation wavelength is 465nm, and the emission wavelength is 510nm; the HEX excitation wavelength was 533nm and the emission wavelength was 580nm.
4. Allelic typing:
analyzing the fluorescent signal scanning data by adopting genotyping software matched with a Roche LightCycler 480II instrument platform (the specific method refers to a software specification matched with the instrument), and determining the genotype of the SNP marker AX-109429088 of the wheat to be detected according to the analysis result as follows: the genotype of the sample shown blue in the coordinate system at a position near the right of the X-axis is the allele of the linked FAM fluorescent tag sequence (i.e., CC genotype); the genotype of a sample which is displayed as green at a position near the upper half part of the Y axis in a parting result coordinate system after parting of fluorescence signal data of a wheat amplification product to be detected is an allele type (namely TT genotype) connected with HEX fluorescence tag sequences; genotypes of samples that are clustered in the coordinate system near the middle of the diagonal and displayed as red are heterozygous genotypes (which can be represented by "CT" or "TC"); a sample that appears pink may not be able to determine its genotype due to poor quality of its genomic DNA, etc. The lower left hand corner of the coordinate system shows a black sample as a blank.
The KASP mark primer group is adopted to detect the parent and partial family genotype of Yangmai 16/Zhongmai 895DH population, the typing result is shown in figure 2, specifically: the fluorescence signal data of the wheat amplification product to be detected is subjected to typing and then gathered in a sample which is displayed as blue near the X axis, and the genotype is CC, wherein the sample comprises parent Yangmai 16; the fluorescence signal data of the amplified products are clustered near the Y axis to show green samples after typing, the genotype is TT, and the fluorescence signal data comprises parent middle wheat 895; the genotyping results of the samples of the Yangmai 16 and Zhongmai 895, which were equally mixed as the heterozygous genotype control templates, were collected in the coordinate system, and the genotype of the sample shown as red near the middle of the diagonal line was the heterozygous genotype, the genotype was CT (also indicated by "TC"), and the sample shown as black at the lower left corner was the blank control.
Example 3 use of the KASP marker AX-109429088-KASP to aid in the identification of wheat straw WSC content in breeding
1. Test material
The test materials include: 161 parts of wheat variety (Table 1), zhongmai 895, yangmai 16 and sterile ultra pure water. The DNA mixed samples of Zhongmai 895, yangmai 16 and Zhongmai 895 are used as a control group for detecting genotypes, water is used as a blank control, and 161 wheat varieties are used as a test group.
Measurement of WSC content of test group materials: 161 wheat varieties are planted in the Shandong Texas in 2016-2017 years, three repetitions are set by adopting a complete random block design, double rows of regions are set, the row length is 1.0m, the row width is 20cm, and the field management is carried out according to the management of a local test station. Recording the flowering period of each cell, randomly cutting 20 main stems from each cell 10 days after flowers, removing leaves and spikes, retaining stems, deactivating enzymes at 105 ℃ for 30min, and drying at 80 ℃. The stalk WSC content was determined by near infrared spectroscopy as constructed by Wang et al (2014). Each sample was subjected to 3 technical replicates and averaged for statistical analysis.
2. Test and control groups were tested with the KASP marker AX-109429088-KASP:
genomic DNA from each experimental material was extracted and the genotypes of AX-109429088 in the test and control materials were analyzed using the high-throughput molecular marker detection system described above, as described in example 2. The test results are shown in fig. 3: sterile ultrapure water is used as a blank control, and the parting result is gathered at the lower left corner of a fluorescent signal coordinate system and is displayed as black; yangmai 16 and 43 other varieties are typed, and the results are gathered near the position near the right side of the X axis and are displayed as blue, and the genotype is CC; the wheat 895 and the other 118 wheat varieties are clustered near the upper side near the Y axis, and the wheat varieties are green and have genotype of TT; the typing results of the DNA mixture sample of Yangmai 16 and Zhongmai 895 are gathered at the middle position of the diagonal line of the coordinate system, and are shown as red, and the genotype is "CT" (also indicated by "TC"). Specifically, the genotype results of 161 test wheat varieties using the KASP markers are shown in table 1, and the measurement results of the WSC content of the stalks 10 days after the flowers of 161 test wheat varieties are also shown in table 1. T-test is carried out on WSC contents of different genotype materials of 161 wheat varieties in a test group by adopting a PROC TTEST model in international SAS 9.2 statistical software, the results are shown in a table 2 and a figure 4, the variation range of the WSC contents of the varieties with genotype TT is 10.32-19.72%, and the average value is 15.56%; the variation range of the WSC content of the variety stem with the genotype of CC is 10.26-17.21%, and the average value is 13.87%. The T test result shows that under the P <0.001 level, the WSC content of the stem 10 days after the flowers of the variety with the genotype of TT is obviously higher than that of the variety with the genotype of CC. The results show that the KASP marker AX-109429088-KASP and the genotype detection system provided by the invention can be effectively used for molecular auxiliary selective breeding aiming at improving the WSC content of wheat stalks.
TABLE 1 detection of genotype results of 161 wheat varieties with KASP marker AX-109429088-KASP and WSC content of stalks 10 days after flowers of each variety
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Table 2 test of WSC content T of stalks 10 days after flower planting of 161 parts of wheat
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Claims (10)
- A kasp tagged primer set comprising:4) A primer A;primer A is (a 1) or (a 2) as follows:(a1) SEQ ID NO:3, wherein the 1 st to 21 st positions of the sequence of the single-stranded DNA molecule are tag sequences A;(a2) Setting SEQ ID NO:3 by substitution and/or deletion and/or addition of one or several nucleotides and which correspond to SEQ ID NO:3 a DNA molecule having the same function;5) A primer B;primer B is (B1) or (B2) as follows:(b1) SEQ ID NO:4, wherein the 1 st to 21 st positions of the sequence of the single-stranded DNA molecule are tag sequences B;(b2) Setting SEQ ID NO:4 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:4 a DNA molecule having the same function;6) A primer C;primer C is (C1) or (C2) as follows:(c1) SEQ ID NO: 5;(c2) Setting SEQ ID NO:5 via one or several nucleotide substitutions and/or deletions and/or additions and which correspond to SEQ ID NO:5 DNA molecules having the same function.
- 2. A PCR reagent comprising the KASP-labeled primer set according to claim 1.
- 3. The PCR reagent of claim 2, further comprising a PCR Mix; the PCR Mix is as follows (d 1) or (d 2):(d1) Comprises a fluorescent probe A, a fluorescent probe B and HiGeno DNA polymerase; wherein the 5 'end of the fluorescent probe A is marked with FAM fluorescent groups, and the 3' end sequence is complementary with the tag sequence A; the 5 'end of the fluorescent probe B is marked with HEX fluorescent groups, and the 3' end sequence is complementary with the tag sequence A;(d2) Comprising the fluorescent probe A, the fluorescent probe B, and the DNA polymerase described in (d 1) above, wherein the DNA polymerase is a DNA polymerase which is different from the HiGeno DNA polymerase described in (d 1) above and can be used for realizing a DNA polymerase chain reaction.
- 4. A kit comprising a KASP tagged primer set according to claim 1 and/or a PCR reagent according to any one of claims 2-3.
- 5. The use of a KASP-tagged primer set according to claim 1 or a PCR reagent according to any one of claims 2-3 or a kit according to claim 4, as follows (1) or (2) or (3) or (4) or (5) or (6) or (7) or (8):(1) Detecting or assisting in detecting genotype of tightly linked SNP marker AX-109429088 of QWsc.caas-7DS of QTL locus of WSC content of wheat stalk to be detected;(2) Identifying or assisting in identifying the WSC content of the wheat stalks to be tested;(3) Auxiliary screening or breeding of wheat single plants or strains or varieties with high WSC content;(4) Auxiliary screening or breeding of wheat single plants or strains or varieties with low WSC content;(5) Preparing a product for detecting or assisting in detecting the genotype of the SNP marker AX-109429088 of the wheat to be detected;(6) Preparing a product for detecting or assisting in detecting the content QTL QWsc.caas-7DS of wheat stalk WSC to be detected;(7) Preparing a product for screening or breeding single plants or strains or varieties of wheat with high WSC content;(8) The method is used for preparing products for screening or breeding single plants or strains or varieties of wheat with low WSC content.
- 6. A method for identifying the genotype of SNP marker AX-109429088 in wheat under test, comprising:taking the genome DNA of the wheat to be detected as a template, and adopting the KASP labeled primer group of claim 1 and/or the PCR reagent of any one of claims 2-3 and/or the kit of claim 4 to carry out PCR amplification to obtain the amplified product of the wheat to be detected;carrying out fluorescent signal scanning and genotyping analysis on the wheat amplification product to be detected, and determining the genotype of the SNP marker AX-109429088 according to the genotyping result and the following principle:if the fluorescence signal data of the amplified product of the wheat to be detected is subjected to typing and then gathered at a position near the upper side of the Y axis in a typing result coordinate system, the genotype of the SNP marker AX-109429088 in the wheat to be detected is TT;if the fluorescence signal data of the amplified product of the wheat to be detected is clustered at a position near the right side of the X axis in the coordinate system after being typed, the genotype of the SNP marker AX-109429088 in the wheat to be detected is CC;if the fluorescent signal data of the amplified product of the wheat to be detected is typed and then gathered at a position close to the middle position of a diagonal line in a coordinate system, the genotype of the SNP marker AX-109429088 in the wheat to be detected is a heterozygous genotype, which is represented by TC or CT.
- 7. A method for identifying or assisting in identifying the WSC content of wheat stalks to be tested, which is characterized by comprising the following steps:using genome DNA of the wheat to be detected as a template, and identifying the genotype of SNP marker AX-109429088 in the wheat to be detected by adopting the method of claim 6;if the genotype of the SNP marker AX-109429088 in the wheat to be detected is TT, the wheat to be detected has relatively high content of WSC in the stems;if the genotype of SNP marker AX-109429088 in the wheat to be tested is CC, the wheat to be tested has a relatively low content of stalk WSC.
- 8. A method for assisting in screening or breeding single plants or strains or varieties of wheat with high WSC content of stems, which is characterized by comprising the following steps:the method of claim 6 is adopted to identify the genotype of a tightly linked SNP marker AX-109429088 of a stem WSC content QTL site QWSc.caas-7DS in wheat to be detected, and the wheat material with the genotype of the SNP marker AX-109429088 being TT or CT is selected in each generation of breeding, and finally, a stable wheat single plant or strain or variety with the genotype of AX-109429088 being TT is bred.
- 9. A method for assisting in screening or breeding a wheat single plant or strain or variety with low WSC content, which is characterized by comprising the following steps:the method of claim 6 is adopted to identify the genotype of a tightly linked SNP marker AX-109429088 of a stem WSC content QTL site QWSc.caas-7DS in wheat to be detected, and the wheat material with the genotype of the SNP marker AX-109429088 of CC or CT is selected in each generation of breeding, and finally, a stable wheat single plant or strain or variety with the genotype of AX-109429088 of CC is bred.
- 10. The method according to claim 9, comprising: the wheat material with genotype of SNP marker AX-109429088 as CC is hybridized with the material with genotype of AX-109429088 as CC or CT or TT, the method of detecting or assisting in detecting the genotype of tightly linked SNP marker AX-109429088 of QTL locus QWSC.caas-7DS of the wheat stalk WSC content is adopted in the separation generation, and the single plant with genotype of marker AX-109429088 as CC or CT is selected until the stable wheat single plant or strain or variety with genotype of tightly linked SNP marker AX-109429088 of QWSC.caas-7DS of the WSC content as CC is bred.
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