CN116479164A - SNP locus, molecular marker, amplification primer and application of SNP locus and molecular marker related to soybean hundred-grain weight and size - Google Patents
SNP locus, molecular marker, amplification primer and application of SNP locus and molecular marker related to soybean hundred-grain weight and size Download PDFInfo
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Abstract
The invention belongs to the technical field of soybean molecular biology and molecular breeding, and particularly relates to SNP loci, molecular markers and amplification primers related to soybean hundred-grain weight and size and application thereof. The SNP locus is located in 6,606,131bp of soybean chromosome 4, the polymorphism is A or T, and the plant with the polymorphism of A has higher hundred grain weight and size than the plant with the polymorphism of T. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the nucleotide sequence of the molecular marker amplification primer is shown as SEQ ID NO.2-3, the contribution rate of the SNP locus to hundred-grain weight is 15.49-19.62%, and the additive effect is 3.56-4.41g. The SNP locus is used for developing a corresponding molecular marker, and the marker is used for selecting in northeast core germplasm resource sample groups, so that 75% of strains with 200bp of A of the molecular marker in 2020 and 2021 can be screened, the hundred grain weight is higher than 17.58g and 18.92g, the accuracy can reach 75%, the selection cost is greatly reduced, and the efficiency of improving the quality related to the grain size such as hundred grain weight is improved.
Description
Technical Field
The invention belongs to the technical field of soybean molecular biology and molecular breeding, and particularly relates to SNP loci, molecular markers and amplification primers related to soybean hundred-grain weight and size and application thereof.
Background
The soybean is the most important grain and oil crops in the world, and the cultivation of high-yield soybean varieties is the key of the development of soybean industry in China. The soybean yield is an extremely complex comprehensive character from the genetic aspect, consists of a plurality of specific quantitative characters, and has low efficiency and limited amplitude when the soybean unit yield is improved by conventional breeding means. However, with the development and perfection of molecular biology technology, the gene fine regulation technology can realize the polymerization and efficient utilization of excellent alleles, is the most critical technology for breakthrough large variety breeding, and is also a necessary means for improving the breeding capability of soybean varieties in the future. The development of excellent allele accumulation and its corresponding molecular markers is a prerequisite for achieving fine regulation and polymerization of genes.
The weight of the soybean hundred grains is an important index for determining the soybean yield; meanwhile, the grain size is also related to the germination vigor of the seeds; seed size is also an important factor affecting the end use of soybeans, small seeds are more suitable for natto and bean sprouts, while large seeds are more suitable for vegetable soybeans and tofu. Therefore, the molecular marker for the excellent allele of the soybean hundred-grain weight is discovered, so that not only can technical reserve be provided for the polymerization of the excellent allele of the high-yield soybean, but also clear guidance can be provided for the improvement of the soybean quality by a gene fine regulation technology.
In recent years, with the development of sequencing technology, researchers have had a more comprehensive understanding of soybean genomes. Whole Genome association analysis (Genome-Wide Association Studies, GWAS) is an advanced method of currently studying biological genomes by typing large-scale population DNA samples for genes related to biological phenotypes by whole Genome high-density genetic markers (such as SNPs or CNVs, etc.). Lam et al found 630 more than ten thousand SNPs by genomic resequencing 17 wild soybeans and 14 cultivated soybeans. At present, the association analysis is widely applied to plant researches such as soybean seed protein content, rice amino acid composition, triticale aluminum toxicity resistance and the like. In recent years, development of functional markers for a target trait using GWAS has become one of the hot spots in molecular biology research. The molecular marker assisted selection can obviously improve the genetic improvement process of soybean varieties.
To date, quantitative trait locus (Quantitative trait locus, QTL) localization studies have been performed on soybean hundred-grain weight and size-related genes by linkage analysis, and up to now, over 304 hundred-grain weight QTLs have been registered in the soybean genome database on all 20 chromosomes. However, most QTLs are micro-effective sites and are not validated, a large portion of which are repetitive locations. The genetic map is constructed by adopting relatively late SSR, AFLP, RFLP, RAPD and other low-density molecular markers, so that the locus is not accurate enough; and the molecular markers related to the current functional sites are mostly derived from recombinant inbred lines or single parent-mother constructed populations, and when the markers are applied to natural populations such as hybrid varieties, local varieties and the like, the molecular markers can not be used as molecular marker assisted breeding, and the genetic contribution rate of the sites in the natural populations can not be explained.
Therefore, it is necessary to find a marker which can more precisely locate regions and widely apply hybrid parents of different cultivated soybean groups to screen SNP (Single nucleotide polymorphism ) molecular markers related to soybean seed weight and size, and further to apply the marker to the breeding process of soybean yield and quality genetic improvement.
Disclosure of Invention
One of the purposes of the invention is to provide a SNP locus of soybean hundred grain weight and size, wherein the SNP locus is positioned at 6,606,131bp (the reference genome is G.max Wm82.A2. V1) of soybean chromosome 4, the polymorphism is A or T, the polymorphism of the SNP locus is that the plant of A has higher hundred grain weight and size than the plant of polymorphism T, and the locus is not reported in the past researches and related patents.
The second object of the present invention is to provide a molecular marker containing the SNP locus, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, the 200bp degenerate base W of the sequence is A or T, the region is not reported in previous researches and related patents, and the molecular marker belongs to co-dominant markers, is reliable and convenient to use, and provides great convenience for soybean high-yield and quality improvement breeding work.
The third object of the invention is to provide the molecular marked amplification primer, and the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 2-3.
The fourth object of the present invention is to provide the use of said SNP locus, said molecular marker or said amplification primer for screening or identifying high hundred grain weight and size soybean lines.
The fifth purpose of the invention is to provide the application of the SNP locus, the molecular marker or the amplification primer in soybean molecular breeding, transgenic soybean cultivation and soybean germplasm resource identification.
The invention aims at providing a method for identifying soybean hundred grain weight and grain size strains, which comprises the following steps:
and taking genomic DNA of soybean to be detected as a template, and performing PCR amplification by using the amplification primer, wherein if the 200bp of an amplification product is A, the soybean strain with high hundred-grain weight and size is obtained, and if the 200bp of the amplification product is T, the soybean strain with low hundred-grain weight and size is obtained.
The invention has the following beneficial effects:
the hundred-grain weight contribution rate of the SNP locus is 15.49-19.62%, and the additive effect is 3.56-4.41g. The SNP locus is used for developing corresponding molecular markers, and the markers are used for selecting northeast core germplasm resource sample groups, so that strain with 200bp A of the marker sequence of 75% in 2020 and 2021 can be screened, the hundred grain weight is higher than 17.58g and 18.92g, the accuracy can reach 75%, the selection cost is greatly reduced, and the efficiency of seed size related quality improvement breeding work such as hundred grain weight is improved.
Drawings
FIG. 1 is a graph showing the distribution of grain weight (100 SW), grain width (SW), grain thickness (ST) of 350 related groups of 2 years (E1: 2021, E2: 2020), grain weight of soybean seeds on the abscissa and the number of individual samples on the ordinate.
FIG. 2 is a population structure diagram of the related population obtained by the admix software based on SNP.
FIG. 3 is a Manhattan plot of MLM correlation analysis results of hundred grain weight, grain width, grain thickness of 350 natural populations for 2 years, with the ordinate being the negative logarithm of p-value (-log) 10 (p)) is chromosome, one point represents one SNP site; the dotted line is the negative logarithm of 1/SNP number, and the point above the dotted line indicates that the corresponding SNP marker is significantly correlated with hundred weight, where the arrow pointing to the point on the dotted line on chromosome 4 corresponds to a 6,606,131bp SNP.
FIG. 4 is a Box plot of phenotype differences of soybean hundred grain weight, grain width and grain thickness corresponding to molecular markers of soybean hundred grain weight, grain width and grain thickness of 350 natural populations for 2 years.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
In the invention, 3000 parts of soybean germplasm resources which are 40 ℃ N or less in China are collected and measured, and are collected and evaluated by soybean germplasm resource research team groups cultivated by soybean research institute of Jilin province agricultural sciences, and are stored in germplasm resource library of Jilin province agricultural sciences.
Example 1: construction and character determination of soybean seed hundred-grain weight associated population
In this example, 3000 parts of germplasm resources in a soybean germplasm resource pool are used, and the source of the germplasm resources covers most of the high latitude (north of 40 degrees N) soybean main production areas in China, including Heilongjiang province, jilin province, liaoning province, inner Mongolia, xinjiang province and the like. 3000 parts of resources are planted in the field, after the seeds are fully mature, the seeds are harvested, and 20 representative seeds are randomly selected for each variety to measure. Hundred-grain weight, grain length, width and height of 3000 parts of resources accord with normal distribution in a population, and the genetic diversity index is 3.90. 350 resources are extracted from the method, the hundred-grain weight genetic diversity index is still 3.90, and the 350 resources are used as the associated population. The operation steps are as follows:
group 10 and group 1 are classified according to group hundred particle weight average value (X) and standard deviation (delta)<X-2 delta, class 10 is not less than X+2 delta, and each class in the middle is different by 0.5 delta. Genetic diversity of each trait was evaluated using Shannon's informative index (H '), H ' = - Σp i lnP i ，P i The frequency of occurrence of the ith variation was represented, and the genetic diversity by calculating the hundred weight of 3000 resources was 3.90.
35 resources were randomly extracted from each cluster, the genetic diversity index H' of 350 resources was calculated, and when equal to 3.90, the 350 resources were determined to be the associated cluster, the hundred grain weight of the cluster was normally distributed (fig. 1).
Example 2: whole genome correlation analysis of soybean hundred grain weight and grain size
(1) The CTAB method is used for extracting 350 parts of resource single plant leaf DNA of related groups, the Thermo nanodrop 2000 is used for detecting the DNA concentration, and 1% agarose electrophoresis is used for detecting the purity and the integrity of the DNA, so that no degradation, no impurity pollution such as protein, polysaccharide and the like are required, and the concentration is more than 100 ng/uL.
(2) The whole genome re-sequencing technology of An Nuo Youda gene limited company is utilized to carry out whole genome re-sequencing on 350 resources, and the specific operation is as follows:
1) The enzyme digestion scheme is as follows: enzyme digestion prediction is carried out on the published soybean reference genome by utilizing enzyme digestion prediction software, enzyme digestion is carried out on each sample genome which is qualified in detection by using endonuclease RsaI and HaeIII, and SLAF fragments with genome fragments ranging from 364 bp to 414bp are selected.
(1) 2) sequencing flow: the SLAF Fragment obtained was subjected to 3' -end addition treatment with Klenow Fragment (3 '. Fwdarw.5 ' exo-) (NEB) and dATP at 37℃and to Dual-index sequencing adapter ligation, PCR amplification (PCR amplification primer: F: AATGATACGGCGACCACCGA; R: CAAGCAGAAGACGGCATACG), purification (Agencourt AMPure XP beads (Beckman Coulter, high Wycombe, UK)), mixing and cutting to select the Fragment of interest, and sequencing with IlluminaHiSeqTM after library quality inspection was passed. To evaluate the accuracy of the library building experiments, soybean ('Williams 82': g.max wm82.a2.v1) was used as a Control (Control) to perform the same process to participate in library building and sequencing, together with 112G reads data, with an average Q30 of 92.20% sequencing.
3) According to the positioning result of sequencing Reads on a reference genome, the GATK performs local heavy comparison (Local Realignment), GATK mutation detection, samtools mutation detection, and the steps of taking intersection mutation sites obtained by the two methods of GATK and samtools and the like so as to ensure the accuracy of SNP obtained by detection. The intersection of SNP markers obtained by the two methods is used as a final reliable SNP marker data set, and 3,306,713 group SNP are obtained in total.
(3) Phylogenetic tree is used to represent evolutionary relationships between species, and according to the relatedness between various organisms, various organisms are arranged on a branched tree-like chart, so that the evolutionary processes and the relatedness of the organisms are represented concisely. Based on SNP, a colony evolutionary tree of the sample is constructed by a MEGA5 software neighbor-joining algorithm.
(4) The genetic structure analysis of the population can provide the source of the blood system of the individual and the composition information thereof, and is an important genetic relationship analysis tool. Based on SNP, the population structure of samples was analyzed by an admixture software (FIG. 2), and clustering was performed assuming that the number of clusters (K value) of the samples was 1 to 16, respectively. And cross-verifying the clustering result, and determining the optimal clustering number as 8 according to the valley value of the cross-verifying error rate.
(5) Based on the SNP, principal component analysis (Principal components analysis, PCA) analysis was performed by TASSEL5 software to obtain a principal component cluster condition of the sample. Through PCA analysis, the relative approaching and the relative distant of the samples can be known, and the evolution analysis can be assisted.
(6) The correlation (relative kinshift) between two individuals in a natural population can be estimated using plink software. The genetic relationship itself is a relative value defining the genetic similarity between two specific materials and the genetic similarity between any material, and thus is defined as 0 directly when the genetic relationship value between two materials is less than 0 as a result.
(7) Based on association population SNP molecular marker data, genetic structure data, kinship matrix data and methionine content data, whole genome association analysis (Genome wide association study, GWAS) is performed by using a mixed linear model ((Mixed linear model, MLM) of GAPIT software, X is genotype, Y is phenotype, and finally each SNP locus can obtain an association result (figure 3) in terms of log 10 (p). Gtoreq.6.58 is a screening standard, SNP markers (A/T) which are remarkably related to hundred-grain weight, grain width and grain thickness are obtained at 6,606,131bp of chromosome 4, and detailed information is shown in Table 1.
TABLE 1 significant correlation of soybean seed weight hundred grain (100 SW) and grain width (SW), grain thickness (ST) SNP information
Example 3: application of significant correlation SNP markers of soybean hundred grain weight and grain size
SNP marker closely linked to soybean hundred grain weight is 6,606,131bp (A/T) of chromosome 4, named qSW4-1, which is a fragment obtained by PCR amplification with qSW4-1 primer using genomic DNA of the material to be identified as a template; the nucleotide sequence of qSW-1 is shown as SEQ ID NO.1, and the 200bp degenerate base W of the sequence is A or T.
Wherein, the amplification primer is:
qSW4-1-F：5’-TTGCGTACATGAGGACATGC-3’(SEQ ID NO.2)
qSW4-1-R：5’-TCTCAGTGTTGCCCACCTATT-3’(SEQ ID NO.3)
the specific steps of auxiliary judging the hundred grain weight and grain size of offspring of the variety by utilizing the SNP molecular marker are as follows:
(1) Extraction of genomic DNA of a material to be identified by CTAB method
1) Fresh leaves of soybean were taken, added with liquid nitrogen and ground into powder, and a proper amount was placed into a 1.5mL centrifuge tube.
2) 0.6mL of the preheated CTAB extract was added, mixed upside down several times, mixed in a water bath at 65℃for one hour, centrifuged at 12000rpm for 15min every 15min.
3) 0.6mL 24 was added: chloroform of 1 (V/V): the isoamyl alcohol solution is inverted and mixed for 5 to 10 times and centrifuged at 10000rpm for 15min.
4) The supernatant solution was transferred to another empty centrifuge tube using 24:1 (V/V) chloroform: the isoamyl alcohol solution was re-extracted once, then 50. Mu.L of RNase (10 mg/mL) was added and left at room temperature for 30min.
5) Adding isopropanol precooled at-20deg.C, centrifuging at 5000rpm for 10min at-20deg.C in a refrigerator for 30min, and removing supernatant.
6) The mixture was washed twice with 70% ethanol. And (3) drying, dissolving with sterilized water to obtain genome template DNA, and placing the genome template DNA into a refrigerator at 4 ℃ for later use.
7) The concentration of the DNA was detected with 0.8% agarose and diluted to the working concentration for PCR amplification.
2. And (3) carrying out PCR amplification by using SNP marker primers to obtain an amplification product.
1) PCR amplification system: the total volume was 20. Mu.L, including 10-50ng of genomic template DNA 3. Mu.L, 10. Mu. L Quick Taq HS DyeMix,10pmol of primers 2. Mu.L and ddH, respectively 2 O 3μL。
2) PCR amplification conditions: pre-denaturation at 94℃for 30s, annealing at 57℃for 30s, and extension at 72℃for 1min; cycling for 30 times; final extension at 72℃for 10min.
3. Judging hundred grain weight of seeds according to sequence comparison result
Sequencing analysis is carried out on the amplified product, wherein the average hundred particle weight of the strain subgroup with the 200bp A of the amplified product from the 5' end is obviously higher than that of the strain subgroup with the T at the position (figure 4). Wherein, the hundred grains of the strain with the 200bp A of 75% in 2020 and 2021 are higher than 17.58g and 18.92g. This shows that the marking is practically effective for assisting the selection.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. The SNP locus of the soybean hundred grain weight related to the size is characterized in that the SNP locus is positioned at 6,606,131bp of soybean chromosome 4, and the polymorphism is A or T.
2. The SNP locus of claim 1 wherein the plant having polymorphism a has a higher hundred grain weight and size than the plant having polymorphism T.
3. A molecular marker containing the SNP locus according to claim 1, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the 200bp degenerate base W of the sequence is A or T.
4. The molecular marker amplification primer of claim 3, wherein the nucleotide sequence of the amplification primer is shown in SEQ ID NO. 2-3.
5. Use of the SNP locus of claim 1, the molecular marker of claim 3 or the amplification primer of claim 4 for screening or identifying high hundred grain weight and size soybean variety resources.
6. Use of the SNP locus as defined in claim 1, the molecular marker as defined in claim 3 or the amplification primer as defined in claim 4 in soybean molecular breeding, cultivation of transgenic soybean, soybean germplasm resource identification.
7. A method for identifying different soybean hundred grain weight and grain size lines, comprising the steps of:
the genomic DNA of soybean to be detected is used as a template for PCR amplification, the amplification primer of claim 4 is used for amplifying the soybean strain with high hundred grain weight and size if the 200bp of the amplification product is A, and the soybean strain with low hundred grain weight and size if the 200bp of the amplification product is T.
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