CN113870946A

CN113870946A - Method for screening SNP (single nucleotide polymorphism) loci of rice and method for identifying rice varieties

Info

Publication number: CN113870946A
Application number: CN202111290339.6A
Authority: CN
Inventors: 张群宇; 陈杰湖; 周峰; 王曼; 刘耀光; 王红梅; 袁健铭
Original assignee: Guangzhou Scgene Technology Co ltd; South China Agricultural University
Current assignee: Guangzhou Scgene Technology Co ltd; South China Agricultural University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2021-12-31

Abstract

The invention discloses a method for screening a rice SNP locus and a method for identifying a rice variety, which relate to the technical field of biological molecules.

Description

Method for screening SNP (single nucleotide polymorphism) loci of rice and method for identifying rice varieties

Technical Field

The invention relates to the technical field of biological molecules, in particular to a method for screening rice SNP loci and a method for identifying rice varieties.

Background

Indica rice and japonica rice are two most deeply and mainly used directions for the differentiation of common cultivated rice. In general, indica rice is different from japonica rice in morphology and geographical distribution. The south indica and north japonica rice layout is formed in the process of planting the long rice. However, with the acceleration of modern breeding process, the discovery of affinity genes and interspecies heterosis are continuously utilized, so that the boundary between indica and japonica becomes more and more fuzzy.

And new requirements are put forward for indica-japonica rice identification under new situations. At present, the purchase price of rice is different between indica rice and japonica rice, and the indica rice and the japonica rice are generally distinguished by observing grain shapes in the market, but the method is not accurate, and is particularly important for classifying more and more indica rice and japonica rice filial generations.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for screening rice SNP loci and a method for identifying rice varieties.

The invention is realized by the following steps:

in a first aspect, an embodiment of the present invention provides a method for screening a rice SNP site, including: obtaining SNP candidate loci of rice based on the information of N rice sample genomes, wherein N is more than or equal to 2; the N rice samples are samples obtained after non-core group samples are removed; and screening the SNP candidate sites to obtain target sites for identifying rice varieties.

In a second aspect, the embodiment of the present invention provides a method for identifying a rice variety, which includes detecting a target site of a rice sample to be detected, where the target site is obtained by screening according to the method for screening a rice SNP site described in the previous embodiment.

In a third aspect, the embodiments of the present invention provide a kit for identifying a rice variety, comprising: reagents for detecting the genotype of the target site; the target site is obtained by screening the rice SNP site screening method as described in the previous embodiment.

In a fourth aspect, the embodiments of the present invention provide an application of a reagent for detecting the genotype of a target site in the preparation of a kit for distinguishing or identifying rice varieties, wherein the target site is obtained by screening according to the method for screening a rice SNP site described in the previous embodiments.

The invention has the following beneficial effects:

according to the method, before the SNP loci for identifying the rice varieties are screened, rice samples for obtaining the SNP loci are filtered in advance, non-core group samples are filtered, and then the SNP loci are screened and detected, so that the pertinence and the effectiveness of the SNP loci for identifying and analyzing specific rice varieties can be remarkably improved, and a way is provided for the effective identification of the specific rice varieties and the construction of high-quality heterogeneous gene maps of the rice.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of a sample screening rule at the chromosome level;

FIG. 2 is a profile of each chromosome of the rice sample before screening in example 1 along the PC1 axis;

FIG. 3 is a profile of each chromosome of the rice sample after screening in example 1 along the PC1 axis;

FIG. 4 is a graph showing the distribution of the whole genome levels of rice samples on the PC1, PC2 and PC3 axes before and after screening in example 1;

FIG. 5 is a graph showing the difference in Hp distribution curves before and after screening of rice samples in test example 1;

FIG. 6 is a graph showing the difference in the number of SNPs and the number of genes obtained before and after screening in test example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The invention provides a method for screening rice SNP loci, which comprises the following steps: obtaining SNP candidate loci of rice based on the information of genomes of N rice samples, wherein N is more than or equal to 2, and the N rice samples are samples from which non-core group samples are removed; and screening the SNP candidate sites to obtain target sites for identifying rice varieties.

The "data of N cases of rice chromosomes and/or genomes" herein can be obtained by the existing gene database or by the way of self-detection, and the method for obtaining the SNP sites based on the sample genome information can be obtained based on the existing technology, and is not described again.

The breeding of new species is carried out in a hybrid rice breeding mode in rice breeding, and due to differences of breeding targets, breeding algebra and the like, a part of samples in the bred rice variety have more heterozygous genotypes, and the heterozygous genotypes are defined as non-core population samples including non-core population indica rice and non-core population japonica rice.

At present, SNP candidate sites obtained based on a conventional screening method cannot effectively reflect the specificity of a specific rice variety, and the sites obtained by screening cannot effectively identify and research the SNP candidate sites. Therefore, it is necessary to finely screen rice samples, and remove the samples of hybrid rice to obtain the core sites of specific rice varieties.

In a preferred embodiment, N.gtoreq.10; n is more than or equal to 50; n is more than or equal to 100. The larger the sample size, the more convincing the result.

In a preferred embodiment, the step of removing the non-core population sample comprises: performing PCA analysis on each chromosome of N rice samples respectively, dividing the N samples into 2 groups, taking the midpoint of two groups in a PC1 axis as a center (baseline), and marking the chromosomes corresponding to 1% -10% of the samples closest to the center in a PCA distribution diagram as a mixed type. The rice has 12 chromosomes, and after PCA analysis is respectively carried out on each chromosome, when the number of marked chromosomes of 1 sample is more than or equal to 2 times, the sample is filtered and removed and is not used for subsequent SNP locus screening.

In a preferred embodiment, the SNP candidate site is a Non-Synonymous mutated SNP site (Non-Synonymous SNP). The SNP includes intergenic region SNP, gene non-coding region SNP and gene coding region SNP, the gene coding region SNP includes non-synonymous mutation and synonymous mutation, and the synonymous mutation is used for the mutation which does not cause the coding amino acid change, and the non-synonymous mutation is used for the mutation which causes the amino acid change. Such SNPs are more representative, and the change of SNP results in the change of codon-encoded amino acid, thereby resulting in the change of protein sequence and three-dimensional structure, and having direct influence on gene function.

In a preferred embodiment, the step of screening for the SNP candidate site comprises: and calculating the heterozygosity fraction Hp of the SNP candidate sites in the population, and taking the sites smaller than a set threshold value of Hp as initial target sites.

The heterozygosity score Hp is a parameter for evaluating gene polymorphism and studying the ensemble genetics, and is calculated by the following formula:

H_p＝2＊Σn_MAJΣn_MIN/(Σn_NAI+Σn_MIN)²；

wherein, Σ n_MAJThe sum of the maximum genotype frequencies in the observation window, the maximum genotype frequency is the ratio of the maximum genotype to the population; sigma-n_MINIs the sum of the minimum genotype frequencies in the observation window; the observation window may be selected to be a region of a specific length on the genome, such as 100-150 kb. In the embodiment of the invention, the observation window is 1bp, namely the position of the SNP locus.

Hp is in the range of 0 to 0.5, and when Hp is equal to 0.5, it represents Σ n_MAJAnd Σ n_MINEqual indicates that the probability of two genotypes appearing in the population is the same, whereas when the Hp value is equal to 0, it indicates that there is only one genotype (homozygous).

Preferably, the set threshold value of Hp is 0.001-0.05; more preferably, the set Hp threshold is 0.001-0.005, and compared with other Hp values, the SNP sites obtained by screening based on the set Hp threshold can more stably and accurately identify specific rice varieties, so that a way is provided for effectively identifying the specific rice varieties and constructing excellent rice seed gene maps.

In a preferred embodiment, the step of screening the SNP candidate sites after obtaining the initial target site further comprises: and marking the gene with the SNP sites in the coding region as the initial target site as a target gene, and acquiring the SNP site in the coding region of the target gene as a final target site. The advantages of such an arrangement are: the method can screen out more representative sites and genes in different rice varieties, the genes containing target sites and common SNP sites still have less representativeness to the identification of the rice varieties, the SNP may not be enough to cause the change of gene functions, and the genes of which the SNP is the initial target site in a coding region are marked, so that the initial target sites in all the target genes are obtained as the final target sites, and the effectiveness and pertinence of the finally obtained SNP sites can be obviously improved. In a preferred embodiment, the rice is selected from at least one of indica and japonica rice.

The embodiment of the invention also provides a method for identifying rice varieties, which comprises the step of detecting the genotype of the target site of a rice sample to be detected, wherein the target site is obtained by screening according to the screening method for the rice SNP site described in the embodiment.

In a preferred embodiment, the rice variety includes at least one of indica and japonica rice.

The embodiment of the invention also provides a kit for identifying rice varieties, which comprises: reagents for detecting the genotype of the target site; the target site is obtained by screening according to the screening method of the rice SNP site described in any embodiment.

In alternative embodiments, the type of reagent may be at least one of a primer pair, a probe, and a gene chip.

In addition, the embodiment of the invention also provides application of a reagent for detecting the genotype of the target site in preparing a kit for distinguishing or identifying rice varieties, wherein the target site is obtained by screening according to the screening method of the rice SNP site in any embodiment.

In a preferred embodiment, the rice is selected from at least one of indica and japonica rice.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

A method for screening rice core SNP sites is provided, which comprises the following steps.

1. Data acquisition

Selecting rice genome DNA sequencing data disclosed by NCBI (NCBI public sequencing data database, website: https:// www.ncbi.nlm.nih.gov/sra /), wherein 44697 parts of sequencing data included in an Illumina sequencing platform account for 99.62% of the total data volume.

2. SNP detection

The SNP detection adopts bwa (version is not limited, http:// bio-bw. sourceform. net /) alignment software to align the double-end sequences, the alignment selects aln strategy or mem strategy, and the parameters select default parameters. Aligned sequences were sorted using samtools (version unlimited, http:// samtools. sourceform. net /), and SNP detection was performed using GATK (version unlimited, https:// software. branched infection. org/GATK /), with 3.07M SNP sites detected in total.

3. PCA analysis and sample screening

PCA Analysis the detected SNP sites were analyzed using PLINK (version v1.9 or higher, http:// www.cog-genomics. org/PLINK/1.9/) and Genome-wide Complex transaction Analysis (GCTA) (version not limited, http:// cNsgenomics. com/software/GCTA /). After analysis filtering of SNP sites with MAF <0.05(MAF is the minimum allele frequency), PCA analysis was performed on 12 rice chromosomes, respectively. At the chromosome level, the rice is divided into 2 groups according to the PCA analysis result on the PC1 coordinate axis, and the 2 groups are indica rice group and japonica rice group respectively by combining the rice sample information observation. According to the sample distribution condition on the PC1 coordinate axis, part of rice varieties are scattered and distributed between indica rice and japonica rice populations, and the part of rice varieties are considered as mixed types (hybrid varieties, non-core population indica rice or non-core population rice) by combining with the analysis of an evolutionary tree. In order to reduce the interference of mixed rice varieties on subsequent analysis, a python program is adopted to filter the varieties, and the filtering/screening rule can refer to fig. 1, wherein in the PCA distribution of indica rice and japonica rice, the midpoint of the PC1 axis is the center (dotted line) and 10% of samples closest to the center position are defined as non-indica rice and/or non-japonica rice core population samples of the chromosome. If a sample with more than 2 chromosomes is labeled as a non-core population type, the rice sample is filtered and not used for subsequent analysis. By the sample filtering strategy, 1205 varieties of core indica rice populations and 655 varieties of core japonica rice sample populations are selected.

In this example, the profile of each chromosome in the rice sample before screening on the PC1 axis is shown in FIG. 2, and the profile of each chromosome in the rice sample after screening on the PC1 axis is shown in FIG. 3. The profiles of the whole genome levels of the rice samples before and after screening on the PC1, PC2 and PC3 axes are shown in FIG. 3.

4. Hp and Fst analysis

Hp represents a porous heterojunction score: h_p＝2＊Σn_MAJ∑n_MIN/(∑n_MAJ+Σn_MIN)²(ii) a And analyzing and calculating by adopting a python program according to the statistical method of Hp, and respectively calculating the Hp values of the indica rice population and the japonica rice population.

Fst (fixed coefficient) adopts an R language software package hierfstat (version: 0.04-22, https:// cran.r-project. org/web/packages/hierfstat/index. html), the range of Fst value is 0-1, 0 represents that the genotype is not fixed at all, the base proportion in different groups is the same (Hp corresponding to each group is equal to 0.5), and the Fst value equal to 1 represents that the genotypes between the two groups are different (Hp corresponding to each group is equal to 0 and the genotypes of the two groups are different).

5. Labeling target sites and target genes

Screening of encoding genes the Hp values of non-synonymous mutant SNP sites in the coding region of each gene were counted according to the gene annotation information of Nipponbare (MSU 7.0). If all non-synonymous mutation SNP loci of the gene have Hp values less than 0.001(Hp setting threshold value) and Fst values more than or equal to 0.95 in indica rice population or japonica rice population, the gene is judged to be simultaneously selected (marked as target gene) in indica rice and japonica rice; if all the SNP sites of the gene with changed amino acids are only in japonica rice with Hp value less than 0.001 and Fst value less than 0.95, judging the japonica rice as a candidate gene; if all the SNP sites of the gene in which the amino acids are changed have Hp values of less than 0.001 and Fst values of less than 0.95 only in indica rice, the SNP site (target site) is judged to be the indica rice candidate SNP site.

In total, 2459 indica rice and japonica rice selected SNP loci (in 1226 genes), 1788 japonica rice selected SNP loci (in 1389 genes) and 828 indica rice selected SNP loci (in 828 genes) are screened, and 5636 SNP loci are screened, which correspond to 3224 coding genes.

6. Gene chip specific primer design

According to the flanking sequence of the SNP locus, a specific short DNA sequence is designed for each SNP locus according to the base complementary pairing principle. The method comprises the following steps: primer3 is adopted to design Primer sequences on two sides of the SNP site, the distance between one Primer sequence and the SNP site is required to be smaller than 150bp, the distance between the SNP site and the 3' end of the Primer is required to be larger than 10bp, and the amplified sequences can be detected by 150bp read-length high-throughput sequencing without being interfered by the Primer sequences. The amplification length is not more than 500 bp.

After the design of the primers is finished, adopting blastn (version unlimited) comparison software to compare the primer sequence pair to a reference genome, under the condition that the similarity is more than 95 percent and the last 3 bases at the 3' end are completely matched, at least one of the upstream primer and the downstream primer is specifically combined to the SNP target region, and if the similarity is not more than 95 percent, redesigning the primers.

Example 2

A method for screening a rice core SNP site is provided, which is substantially the same as in example 1, except that: step 3 is omitted: PCA analysis and sample screening.

Example 3

A method for screening a rice core SNP site is provided, which is substantially the same as in example 1, except that: hp set threshold < 0.05.

Example 4

A method for screening a rice core SNP site is provided, which is substantially the same as in example 1, except that: hp is set to a threshold value < 0.01.

Example 5

A method for screening a rice core SNP site is provided, which is substantially the same as in example 1, except that: hp is set to a threshold value < 0.1.

Example 6

A method for screening a rice core SNP site is provided, which is substantially the same as in example 1, except that: in step 5, all SNP sites (synonymy mutated and non-synonymy mutated) of the coding region of the gene are analyzed and screened for Hp values, and sites meeting a set threshold are used as final selected sites/target sites.

Experimental example 1

Experiments were performed based on the screening methods for core SNP sites provided in example 1 (post-screening) and example 2 (pre-screening or non-screening). And comparing the Hp value distribution curves of the numbers of the SNP sites of the indica type rice and the japonica type rice in the example 2 and the example 1, in the Hp distribution curves before screening the samples, the maximum value of the Hp distribution curves of the indica type rice and the japonica type rice is not equal to 0, and is mainly caused by the interference of the non-core population samples, so that the maximum value is shifted to the right, as shown in fig. 5, and the results show that the curves before screening and after screening are obviously changed from the curves before screening.

The differences in the number of selected SNP sites and the corresponding genes detected are shown in FIG. 6. The result shows that before screening, only a few genes are displayed to be simultaneously selected by indica rice and japonica rice, and a small part of the genes are selected by indica rice or japonica rice. After the sample is screened, the number of the selected genes of both indica and japonica rice or the selected genes of indica and japonica rice is greatly improved, and the main reason for the improvement is to remove the interference of non-core indica and japonica rice samples to obtain the result.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for screening rice SNP sites is characterized by comprising the following steps:

obtaining SNP candidate loci of rice based on the information of N rice sample genomes, wherein N is more than or equal to 2; the N rice samples are samples obtained after non-core group samples are removed;

and screening the SNP candidate sites to obtain target sites for identifying rice varieties.

2. The method for screening SNP loci of rice as claimed in claim 1, wherein the step of removing the non-core population sample comprises:

carrying out PCA analysis on each chromosome of the rice sample respectively, dividing the rice sample into 2 groups, taking the midpoint of two groups in a PC1 axis as a center, and marking the chromosome corresponding to 1% -10% of the sample closest to the center in a PCA distribution diagram as a mixed type;

when the number of marked chromosomes of 1 sample is more than or equal to 2 times, the rice sample is removed.

3. The method of screening SNP sites in rice according to claim 2, wherein the SNP candidate sites are nonsynonymous mutated SNP sites.

4. The method for screening SNP loci in rice according to claim 1, wherein the step of screening SNP candidate loci comprises: calculating heterozygosity fraction Hp of the SNP candidate sites in the rice population, and taking the sites meeting the set threshold value smaller than Hp as initial target sites;

preferably, the calculation formula of Hp is as follows:

H_p＝2＊Σn_MAJΣn_MIN/(Σn_MAJ+Σn_MIN)²(ii) a Wherein, Σ n_MAJThe sum of the maximum genotype frequencies in the observation window, the maximum genotype frequency is the ratio of the maximum genotype to the population; sigma-n_MINIs the sum of the minimum genotype frequencies in an observation window, which is 1 bp;

preferably, the set threshold value of Hp is 0.001-0.05;

preferably, the Hp is set to a threshold value of 0.001 to 0.005.

5. The method for screening SNP loci of rice according to claim 4, wherein the step of screening SNP candidate loci further comprises: and marking the gene with the SNP sites in the coding region as the initial target site as a target gene, and acquiring the SNP site in the coding region of the target gene as a final target site.

6. The method for screening SNP loci of rice according to any one of claims 1 to 5, wherein the rice is selected from at least one of indica rice and japonica rice;

preferably, N is more than or equal to 10;

preferably, N.gtoreq.100.

7. A method for identifying rice varieties, which comprises detecting target sites of a rice sample to be detected, wherein the target sites are obtained by screening according to the method for screening rice SNP sites as claimed in any one of claims 1 to 5.

8. The method of identifying a rice variety as claimed in claim 7, wherein the rice variety comprises at least one of indica and japonica rice.

9. A kit for identifying a rice variety, comprising: reagents for detecting the genotype of the target site; the target site is obtained by screening the rice SNP site according to any one of claims 1 to 5.

10. Use of a reagent for detecting the genotype of a target site in the preparation of a kit for distinguishing or identifying rice varieties, wherein the target site is obtained by screening according to the screening method of the rice SNP site as claimed in any one of claims 1 to 5.