CN111518933B - Wheat grain length related SNP marker and application thereof - Google Patents
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Abstract
The invention discloses a wheat grain length related SNP marker and application thereof, relating to the technical field of wheat molecular breeding, comprising an SNP marker positioned at 450729566bp of a wheat 7A chromosome, wherein the nucleotide of the SNP marker is G/C, and a method for judging the wheat grain length by utilizing the SNP marker, comprising the steps of extracting wheat DNA, utilizing a corresponding primer pair for amplification, carrying out enzyme digestion on an amplification product, and judging the type of the wheat grain length according to the enzyme digestion result; the invention provides a new detection means for wheat grain length selection, the detection method is simple and convenient, and the method is beneficial to improving the target and pertinence of molecular marker selection, thereby improving the efficiency of wheat high-yield molecular breeding.
Description
Technical Field
The invention relates to the technical field of wheat molecular breeding, in particular to a wheat grain length related SNP marker and application thereof.
Background
Wheat is one of the main food crops in China. With the increase of population and the reduction of cultivated land area, the improvement of yield is an important target of wheat breeding in China. Thousand kernel weight is one of three factors constituting wheat yield, and grain length, grain width and grain thickness are important factors determining the height of thousand kernel weight (Campbell et al, 1999; Chenjiahui et al, 2011). Research shows that thousand grain weight is in very obvious positive correlation with grain length and grain width (Li, et al, 2019). Therefore, the identification of major effective sites related to grain length and the development of molecular markers closely linked with the major effective sites are helpful for accelerating the molecular marker-assisted breeding process of new high-yield wheat varieties.
The data reports that wheat grain length-related QTLs are predominantly distributed throughout chromosomes 1B, 2A, 2B, 2D, 3A, 3B, 5A, 6B, 7A, 7B, and 7D (Campbell et al 1999; Tyagi et al 2015; Guo Li Jian et al 2017). Part of grain length related genes are also cloned in wheat, such as a glutamine synthetase gene TaGS1a for controlling grain weight related traits such as grain length-width ratio and the like is cloned on a wheat 6D chromosome by Guo et al (2013), and CAPS markers are developed; zhang et al (2014) clones a homologous gene TaGS-D1 of a grain weight gene OsGS3 for controlling rice grain length on a wheat 7D chromosome; dong et al (2014) has homologously cloned in wheat 7A chromosome a snake/GASA gene family member TaGASR7-a1 associated with thousand kernel weight and grain length; ma et al (2015) cloned a homolog gene TaGS5-3A of OsGS5 associated with cell differentiation in rice on wheat 3A chromosome, which was significantly associated with wheat grain length and grain weight; yang et al (2019) also isolated the homolog gene TaGL3-5A of rice GL3 gene in wheat by means of homologous cloning and developed a KASP marker that is significantly associated with thousand kernel weight and grain length of wheat.
However, the molecular genetic mechanism of grain length formation is still unclear, and new loci and new genes for controlling grain length are excavated, and molecular markers closely linked with the loci are developed, so that the method is beneficial to accelerating the wheat high-yield gene polymerization breeding process.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a wheat grain length related SNP marker and application thereof so as to identify more new grain length sites from different wheat materials;
another object of the present invention is to provide a method for identifying wheat grain length using the SNP marker, which solves the problem that wheat grain length cannot be identified rapidly.
The invention provides an SNP marker related to wheat grain length, wherein the SNP marker is positioned at 450729566bp of wheat 7A chromosome, and the nucleotide of the SNP marker is G/C.
The invention also provides a method for identifying the grain length of wheat, which comprises the following steps:
step 1, extracting wheat genome DNA to be detected
Step 2, carrying out PCR amplification on the wheat genome DNA obtained in the step 1 by using the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplification product;
step 3, carrying out enzyme digestion on the amplification product by using restriction enzyme RsaI to obtain an enzyme digestion product;
and 4, when the amplification product obtained in the step 2 can be enzyme-cut, the corresponding wheat is long-grain wheat, and if the amplification product obtained in the step 2 can not be enzyme-cut, the corresponding wheat is short-grain wheat.
Further, the PCR amplification system configuration of step 2: mu.L of 2.5mM dNTP, 0.25. mu.L of 10. mu.M primer, 1. mu.L of 10 XEasyTaq Buffer, 0.5U EasyTaq, 100ng of DNA template, and made up to 10. mu.L with double distilled water;
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 62 ℃ and 0.3 ℃ per cycle; extension at 72 ℃ for 30s for 40 cycles; extension at 72 ℃ for 8 min.
Further, in the step 3, the enzyme cutting system is as follows: mu.L of PCR product, 1. mu.L of 10 × CutSmart Buffer, 0.5U Rsa I, was made up to 10. mu.L with double distilled water.
Further, the wheat variety is specifically Jing 411 or Hongmangchun 21.
Compared with the prior art, the invention has the following advantages:
aiming at a new major locus Qgl, ahau-7A for controlling grain length, the invention discovers an SNP marker related to grain length and develops CAPS marker 7A-3738. Compared with SSR molecular markers, the CAPS marker has clear and distinguishable bands after electrophoresis detection, obvious band type difference among wheat varieties with different grain lengths, simple and convenient detection method, and is beneficial to improving the target property and pertinence of molecular marker selection, thereby improving the efficiency of high-yield molecular breeding of wheat.
Drawings
FIG. 1 is an agarose electrophoresis of the cleavage product, wherein; j is a Beijing 411 type strip; h is a Hongmanchun 21 type strip, and M is a strip of a 2K Marker;
FIG. 2 is a partial linkage map of a new major site Qgl. ahau-7A related to grain length in the Beijing 411/Hongmanchun 21 population.
Detailed Description
Examples
To verify the validity of the SNP markers proposed by the invention, the following verification experiments were carried out in this example:
(ii) determination of Grain Length (GL) phenotype
Respectively randomly selecting 300 wheat seeds of the wheat with the varieties of Jing 411 and Hongmangchun 21, respectively measuring grain length GL (mm) in 5-year harvest seasons of 2014, 2015, 2016, 2017 and 2018 by using a Wanshen SC-G type automatic seed test analyzer (provided by Hangzhou Wanshen detection science and technology Co., Ltd.), repeating the steps for three times, and taking the average value. Data consolidation is completed by Excel software, and correlation analysis between phenotype data and markers is completed by SPSS software. The grain length measurements of the parent Jing 411 and Hongmangchun 21 in 5 years are shown in Table 1.
Grain length determination (mm) in 5 years for RILs group parents Jing 411 and Hongmangchun 21
(II) extraction of wheat genome DNA
The test materials for DNA extraction are parent Jing 411 and Hongmanchun 21, 174 recombinant inbred line RIL (Jing 411/Hongmanchun 21) groups and 258 parts of Chinese wheat micro-core germplasm resources. The specific method comprises the following steps:
1. wheat single grains were ground, and 0.1g of 0.7ml of an extract (0.1M Tris-HCl (pH 8.5), 0.1M NaCl, 0.05M EDTA (pH 8.0), 2% SDS) was added thereto to lyse at a constant temperature of 60 ℃ for 45min with shaking.
2. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.
3. The supernatant was taken and added with equal volume of phenol: chloroform: isopentanol (25:24:1) was rotated upside down until the two phases did not separate quickly.
4. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.
5. The supernatant was taken and added with equal volume of phenol: chloroform: isopentanol (25:24:1) was rotated upside down several times.
6. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.
7. Adding equal volume of isopropanol into the supernatant, and standing in a refrigerator at-20 deg.C for 30 min.
8. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.
9. Washed twice with 70% ethanol.
10. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 min.
11. The precipitate is naturally dried in the air and stored at 4 ℃ for later use.
(III) design and sequencing of simplified genome digestion scheme
1. Selection of the enzyme digestion scheme: and finally selecting a wheat A genome as a reference genome for enzyme digestion prediction according to the information such as the size of the wheat genome, GC content and the like. And performing enzyme digestion prediction on the reference genome by enzyme digestion prediction software, and selecting the most appropriate enzyme digestion scheme.
2. The specific experimental process comprises the following steps: the genomic DNA of each sample that was found to be acceptable for the detection was digested with the restriction enzyme RsaI (New England Biolabs, NEB) according to the selected optimal digestion protocol. The resulting digested Fragment (SLAF tag) was treated with Klenow Fragment (3 ' → 5 ' exo-) (NEB) and dATP at 37 ℃ with A at the 3 ' end, ligated with Dual-index sequencing adapter, PCR amplified (PCR amplification primers: F, AATGATACGGCGACCACCGA; R, CAAGCAGAAGACGGCATACG), purified, mixed, cut with gel to select the desired Fragment, and the library was qualified and sequenced with IlluminaHiSeqTM 2500. To evaluate the accuracy of the digestion experiment, Nipponbare (Oryza sativa japonica) was used as a control for sequencing.
3. And identifying the original data obtained by sequencing by using the Dual-index to obtain the reads of each sample. The filtered adapters were evaluated for sequencing quality and data size of sequencing reads. And evaluating the enzyme cutting efficiency of the enzyme by the comparison efficiency of Control data, and further verifying the accuracy and the effectiveness of the experimental process. And (3) developing a large number of SLAF labels in parents and filial generations by adopting a method of comparing the reads with the genome, and screening the polymorphic SLAF labels. After the polymorphic SLAF label is subjected to genotype coding, the quality control is carried out according to the following standards:
(1) filtering reads with the sequencing depth of 10 multiplied by less than the parental stock;
(2) filtering tags with SNP number larger than 5;
(3) indicia with filtration integrity less than 70%;
(4) the label was severely biased (P <0.01) by filtration.
And constructing a high-density genetic map by using the controlled SLAF label through a HighMap mapping software, and using the map for QTL analysis after the map is qualified.
(IV) QTL analysis
The following three algorithms are used for QTL analysis:
1. an additive effect algorithm (ICIM-ADD) for drawing a complete composite interval in QTLICIMapping v4.1 software, LOD threshold values are calculated by 1000 times of random permutation tests, and the step interval is 1 cM.
2. A composite interval mapping method (MCIM) based on a mixed linear model in QTLnetwork v2.0 software sets a significance level (P) to be 0.05, and defaults to a step length of 1 cM.
3. QTL. gCImapping. GUI v1.1 software multi-site mixed model algorithm (gCIM), LOD value is set to 3.0, step 1 cM.
(V) wheat 660K SNP chip scanning
Constructing a high and low mixed pool based on the seed length phenotype values of the 174 families of the recombinant inbred line group, and scanning the high and low mixed pool by using a wheat 660K SNP chip (Beijing Boo classical Co.). The high and low mixing pools respectively comprise 1 long grain parent and 2 long grain mixing pools (5 extreme long grain families are selected for mixing in each long grain mixing pool) and 1 short grain parent and 2 short grain mixing pools (5 extreme short grain families are selected for mixing in each short grain mixing pool). The method comprises the steps of screening and extracting SNPs which are consistent in a pool and have genotype difference among pools in a 660K chip by utilizing Excel, carrying out enrichment analysis on the difference SNPs aiming at a major QTL (quantitative trait locus) section of a 7A chromosome, and selecting the difference SNPs positioned in a Qgl.
(six) development of CAPS marker 7A-3738 in QTL region
1. Differential SNP cleavage site analysis
The restriction enzyme site analysis of all SNPs in the target segment revealed that the G/C difference of the SNP AX-108743738 (450729566 bp on 7A chromosome) can be distinguished by the restriction endonuclease RsaI (New England Biolabs, NEB). The recognition sites for RsaI are: GT ^ AC.
2. Primer design
A sequence of SEQ ID NO.1 of 671bp in total length was obtained by BLAST on the wheat reference genome (IWGSC Refseq v2.0, website https:// urgi. versales. inra. fr/BLAST _ IWGSC/BLAST. php) with 35bp flanking sequences before and after AX-108743738 and extending 300bp before and after.
Then, using SEQ1 as a template, a pair of specific primers were designed using Primer Premier 5.0 software:
7A-3738-F:5'CTGCGATTTGGCGTCTT 3' SEQ ID NO.2
7A-3738-R:5'CATTCTGCTGCCCTGTG 3' SEQ ID NO.3
(VII) amplification, enzyme digestion and electrophoresis of target product
1. And (3) PCR amplification system configuration: mu.L of 2.5mM dNTP, 0.25. mu.L of 10. mu.M primer, 1. mu.L of 10 × EasyTaq Buffer, 0.5U EasyTaq, and 100ng of DNA template were made up to 10. mu.L with double distilled water.
2. PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 62 ℃ and 0.3 ℃ per cycle; extension at 72 ℃ for 30s for 40 cycles; extension at 72 ℃ for 8 min.
3. And (3) enzyme digestion system after amplification: mu.L of PCR product, 1. mu.L of 10 × CutSmart Buffer, 0.5U Rsa I, was made up to 10. mu.L with double distilled water.
4. And (3) enzyme digestion reaction program: 6 hours at 37 ℃.
After digestion, 3. mu.L of 6 XDNA Loading Buffer was added to the sample, and 5. mu.L of the digested product was fractionated by 2.5% agarose gel electrophoresis. The Jing 411 (long grain, G allelic type) band can be cut by enzyme, and the Hongmanchun 21 (short grain, C allelic type) band can not be cut (as shown in figure 1).
(eight) linkage analysis of CAPS markers in recombinant inbred populations
As shown in Table 2 and FIG. 2, CAPS markers 7A-3738 are encrypted to a high-density genetic map constructed by SLAF-seq, and the three algorithms of ICIM, gCIM and MCIM are adopted to carry out grain length QTL analysis, so that the 7A-3738 is closely linked with the 7A chromosome grain length-related major QTL Qgl.ahau-7A, and the average phenotype contribution rate is 11.93%.
New major site Qgl. ahau-7A information related to grain length detected in Jing 411/Hongmanchun 21 population
TABLE 2
Note: the likelihood of QTL presence for both ICIM and gCIM algorithms is represented by LOD values and the QTL significance for MCIM algorithms is represented by P values.
Verification of (nine) CAPS signatures in natural populations
The Mannich-Whitney test (U test) analysis showed that the difference between the GL phenotype values (2016GL and 2017GL) of grain length between wheat varieties carrying two allelic variant types (7A-3738a and 7A-3738b) of CAPS marker 7A-3738 in a natural population consisting of 258 wheat micro-core germplasm reaches a very significant level (Table 3)
258 parts of wheat micro-core germplasm are utilized to verify that CAPS markers 7A-3738 are extremely obviously related to grain length
TABLE 3
Note: indicates a very significant correlation between markers and traits at the 0.01 level; 7A-3738a is an allelic type consistent with Jing 411; 7A-3738b is an allelic type consistent with Miscanthus rubra spring 21.
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Claims (5)
1. An SNP marker associated with wheat grain length, wherein the SNP marker is located at 450729566bp of wheat 7A chromosome of reference genome IWGSC Refseq v2.0 version, and the nucleotide of the SNP marker is G/C.
2. A method for identifying wheat grain length, comprising the steps of:
step 1, extracting wheat genome DNA to be detected
Step 2, designing a PCR primer pair of SEQ ID NO.2 and SEQ ID NO.3 crossing the SNP marker of claim 1, and performing PCR amplification on the wheat genome DNA obtained in the step 1 by using the sequences of SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplification product;
step 3, carrying out enzyme digestion on the amplification product by using restriction enzyme RsaI to obtain an enzyme digestion product;
and 4, when the amplification product obtained in the step 2 can be enzyme-cut, the corresponding wheat is long-grain wheat, and if the amplification product obtained in the step 2 can not be enzyme-cut, the corresponding wheat is short-grain wheat.
3. The method of claim 2, wherein the PCR amplification system of step 2 is configured to: mu.L of 2.5mM dNTP, 0.25. mu.L of 10. mu.M primer, 1. mu.L of 10 XEasyTaq Buffer, 0.5U EasyTaq, 100ng of DNA template, and made up to 10. mu.L with double distilled water;
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 62 ℃ and 0.3 ℃ per cycle; extension at 72 ℃ for 30s for 40 cycles; extension at 72 ℃ for 8 min.
4. The method of claim 3, wherein the enzyme cutting system in the step 3 is as follows: mu.L of PCR product, 1. mu.L of 10 XCutSmart Buffer, 0.5U of RsaI, was made up to 10. mu.L with double distilled water.
5. The method according to any one of claims 2 to 4, wherein the wheat variety is in particular Jing 411 or Hongmangchun 21.
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