A method for screening rice SNP loci and a method for identifying rice varieties

Abstract

The present invention discloses a method for screening rice SNP sites and a method for identifying rice varieties, and relates to the field of biomolecular technology. The present application proposes that before screening the SNP sites for identifying rice varieties, the rice samples used to obtain the SNP sites are first filtered, and after filtering out non-core population samples, the SNP sites are screened and detected, which can significantly improve the pertinence and effectiveness of the SNP sites for identifying and analyzing specific rice varieties, and provide a way for the effective identification of specific rice varieties and the construction of high-quality heterogeneous gene maps of rice.

Description

Technical Field

[0001] The present invention relates to the field of biomolecule technology, and in particular to a method for screening rice SNP sites and a method for identifying rice varieties. Background Art

[0002] Indica and japonica are the two most profound and main directions of common cultivated rice differentiation. Generally speaking, indica and japonica are very different in morphology and geographical distribution. In the long process of rice cultivation, the layout of indica in the south and japonica in the north was formed. However, with the acceleration of modern breeding process, the discovery of affinity genes and the continuous use of interspecific hybrid advantages, the boundary between indica and japonica has become increasingly blurred.

[0003] The new situation has put forward new requirements for the identification of indica and japonica rice. At present, there is a difference in the purchase price of rice between indica rice and japonica rice. In the market, indica rice and japonica rice are generally distinguished by observing the shape of the grain, but this is not accurate. For the increasing number of indica and japonica hybrids, it is particularly important to classify them.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for screening rice SNP loci and a method for identifying rice varieties.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a method for screening rice SNP sites, which comprises: obtaining rice SNP candidate sites based on information of N rice sample genomes, N≥2; the N rice samples are samples after removing non-core population samples; and screening the SNP candidate sites to obtain target sites for identifying rice varieties.

[0008] In a second aspect, an embodiment of the present invention provides a method for identifying rice varieties, which includes detecting a target site of a rice sample to be detected, wherein the target site is a target site screened by the method for screening rice SNP sites described in the above embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a kit for identifying rice varieties, comprising: a reagent for detecting the genotype of a target site; the target site is a target site screened by the method for screening rice SNP sites as described in the above embodiment.

[0010] In a fourth aspect, an embodiment of the present invention provides the use of a reagent for detecting the genotype of a target site in preparing a kit for distinguishing or identifying rice varieties, wherein the target site is a target site screened by the method for screening rice SNP sites described in the aforementioned embodiment.

[0011] The present invention has the following beneficial effects:

[0012] The present application proposes to filter the rice samples used to obtain the SNP sites before screening the SNP sites for identifying rice varieties, first filter out the non-core population samples, and then screen and detect the SNP sites. This can significantly improve the pertinence and effectiveness of the SNP sites for identifying and analyzing specific rice varieties, and provide a way for the effective identification of specific rice varieties and the construction of high-quality heterogeneous rice gene maps. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 This is a schematic diagram of sample screening rules at the chromosome level;

[0015] Figure 2 The distribution diagram of each chromosome of the rice sample on the PC1 axis before screening in Example 1;

[0016] Figure 3 The distribution diagram of each chromosome of the rice sample after screening on the PC1 axis in Example 1;

[0017] Figure 4 The distribution diagram of the whole genome level of rice samples on PC1, PC2, and PC3 axes before and after screening in Example 1;

[0018] Figure 5 The difference in Hp distribution curves before and after the rice sample screening in Experimental Example 1;

[0019] Figure 6 The difference in the number of SNPs and genes obtained before and after screening in Experimental Example 1. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The present invention provides a method for screening rice SNP sites, which comprises: obtaining rice SNP candidate sites based on information of N rice sample genomes, N≥2, and the N rice samples are samples after removing non-core population samples; screening the SNP candidate sites to obtain target sites for identifying rice varieties.

[0022] The "N examples of rice chromosome and / or genome data" in this article can be obtained through existing gene databases or self-detection methods. The method of obtaining SNP sites based on sample genome information can be obtained based on existing technology and will not be repeated here.

[0023] Hybrid rice breeding is often used in rice breeding to select new varieties. Due to differences in breeding goals and breeding generations, some samples of the bred rice varieties will have more heterozygous genotypes. The present invention defines such heterozygous genotypes as "non-core population samples", including non-core population indica rice and non-core population japonica rice.

[0024] At present, the SNP candidate sites obtained based on conventional screening methods cannot effectively reflect the specificity of a specific rice variety, and the sites obtained by screening cannot effectively identify and study them. Therefore, it is necessary to conduct a fine screening of rice samples, remove hybrid rice samples, and obtain the core sites of specific rice varieties.

[0025] In a preferred embodiment, N≥10; N≥50; N≥100. The larger the sample size, the more convincing the result.

[0026] In a preferred embodiment, the step of removing non-core population samples comprises: performing PCA analysis on each chromosome of N rice samples, dividing the N samples into two groups, taking the midpoint of the two groups in the PC1 axis as the center (baseline), and marking the chromosomes corresponding to the 1% to 10% of samples closest to the center in the PCA distribution map as mixed types. Rice has 12 chromosomes. After performing PCA analysis on each chromosome, when the number of chromosomes marked in a sample is ≥2 times, the sample is filtered out and not used for subsequent SNP site screening.

[0027] In a preferred embodiment, the SNP candidate site is a non-synonymous SNP site. SNPs include intergenic region SNPs, gene non-coding region SNPs and gene coding region SNPs. Gene coding region SNPs include non-synonymous mutations and synonymous mutations. Those that do not cause changes in the encoded amino acids are synonymous mutations, and those that cause amino acid changes are non-synonymous mutations. Such SNPs are more representative. Changes in SNPs lead to changes in the amino acids encoded by codons, thereby leading to changes in protein sequences and three-dimensional structures, which have a direct impact on gene function.

[0028] In a preferred embodiment, the step of screening the SNP candidate sites includes: calculating the heterozygosity score Hp of the SNP candidate sites in the population, and marking sites that are less than a set threshold value of Hp as initial target sites.

[0029] The heterozygosity fraction Hp is a parameter used to evaluate gene polymorphism and study overall genetics. Its calculation formula is as follows:

[0030] H p =2*Σn MAJ Σn MIN / (Σn NAI +Σn MIN ) 2 ;

[0031] Among them, Σn MAJ is the sum of the maximum genotype frequencies in the observation window, which is the proportion of the maximum genotype in the population; Σn MIN The observation window can be a region of a specific length on the genome, such as 100-150 kb. In the embodiment of the present invention, the observation window is 1 bp, which is the location of the SNP site.

[0032] The range of Hp is 0 to 0.5. When Hp is 0.5, it means Σn MAJ and Σn MIN When the Hp value is equal, it means that the probability of the two genotypes appearing in the population is the same, and when the Hp value is equal to 0, it means that there is only one genotype (homozygous).

[0033] Preferably, the Hp threshold is set at 0.001 to 0.05; more preferably, the Hp threshold is set at 0.001 to 0.005. Compared with other Hp values, the SNP sites obtained by screening based on the Hp threshold set in this application can more stably and accurately identify specific rice varieties, providing a way to effectively identify specific rice varieties and construct a genetic map of excellent rice varieties.

[0034] In a preferred embodiment, after obtaining the initial target site, the step of screening the SNP candidate sites also includes: marking genes whose SNP sites in the coding region are all the initial target sites as target genes, and obtaining the SNP sites in the coding region of the target gene as the final target sites. The advantage of this arrangement is that more representative sites and genes in different rice varieties can be screened out, and genes containing target sites and common SNP sites are still not representative for the identification of rice varieties. In this case, SNPs may not be sufficient to cause changes in gene function. Genes whose SNPs in the coding region are all initial target sites are marked, so as to obtain the initial target sites in all target genes as the final target sites, which can significantly improve the effectiveness and pertinence of the SNP sites finally obtained. In a preferred embodiment, the rice is selected from at least one of indica rice and japonica rice.

[0035] The embodiment of the present invention further provides a method for identifying rice varieties, which comprises detecting the genotype of a target site of a rice sample to be detected, wherein the target site is a target site screened by the method for screening rice SNP sites described in the above embodiment.

[0036] In a preferred embodiment, the rice variety includes at least one of indica rice and japonica rice.

[0037] The embodiment of the present invention further provides a kit for identifying rice varieties, which comprises: a reagent for detecting the genotype of a target site; the target site is a target site screened by the method for screening rice SNP sites described in any of the above embodiments.

[0038] In an optional embodiment, the type of the reagent may be at least one of a primer pair, a probe and a gene chip.

[0039] In addition, an embodiment of the present invention also provides the use of a reagent for detecting the genotype of a target site in preparing a kit for distinguishing or identifying rice varieties, wherein the target site is a target site screened by the method for screening rice SNP sites described in any of the aforementioned embodiments.

[0040] In a preferred embodiment, the rice is selected from at least one of indica rice and japonica rice.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] Example 1

[0043] A method for screening core SNP sites in rice is provided, which comprises the following steps.

[0044] 1. Data acquisition

[0045] The rice genome DNA sequencing data published by NCBI (NCBI public sequencing data database, website: https: / / www.ncbi.nlm.nih.gov / sra / ) were selected, among which 44697 sequencing data included in the Illumina sequencing platform were selected, accounting for 99.62% of the total data volume.

[0046] 2. SNP detection

[0047] SNP detection was performed using bwa (any version, http: / / bio-bwa.sourceforge.net / ) alignment software to align the double-end sequences, with the aln strategy or mem strategy selected and the default parameters selected. Samtools (any version, http: / / samtools.sourceforge.net / ) was used to sort the aligned sequences, and GATK (any version, https: / / software.broadinstitute.org / gatk / ) was used for SNP detection, and a total of 3.07M SNP sites were detected.

[0048] 3. PCA analysis and sample screening

[0049] PCA analysis was performed using PLINK (version v1.9 or higher, http: / / www.cog-genomics.org / plink / 1.9 / ) and Genome-wide Complex Trait Analysis (GCTA) (version not limited, http: / / cnsgenomics.com / software / gcta / ) to analyze the detected SNP sites. After filtering the SNP sites with MAF < 0.05 (MAF is the minimum allele frequency), PCA analysis was performed on 12 rice chromosomes. At the chromosome level, the PCA analysis results divided rice into two groups on the PC1 coordinate axis. Combined with the rice sample information, the two groups were indica rice group and japonica rice group. According to the sample distribution on the PC1 coordinate axis, some rice varieties were scattered between indica rice and japonica rice groups. Combined with the evolutionary tree analysis, these rice varieties were considered to be mixed types (hybrid varieties, non-core group indica rice or non-core group rice). In order to reduce the interference of mixed rice varieties on subsequent analysis, the varieties were filtered using a python program. The filtering / screening rules can be found in Figure 1Based on the analysis results of the PCA distribution map at the chromosome level of rice samples, the midpoint of the PC1 axis in the PCA distribution of indica and japonica rice is defined as the center (dashed line), and the 10% of samples closest to the center are defined as non-indica and / or non-japonica core population samples of the chromosome. If a sample has more than 2 chromosomes marked as non-core population types, the rice sample is filtered and not used for subsequent analysis. After the above sample filtering strategy, 1205 core indica rice population varieties and 655 japonica rice core sample population varieties were selected.

[0050] In this embodiment, the distribution diagram of each chromosome of the rice sample on the PC1 axis before screening can be referred to Figure 2 The distribution of each chromosome of rice samples on the PC1 axis after screening is shown in Figure 2. Figure 3 The distribution of the whole genome level of rice samples on PC1, PC2, and PC3 axes before and after screening is shown in Figure 3 .

[0051] 4. Hp and Fst analysis

[0052] Hp stands for pooled heterozygosity score: H p =2*Σn MAJ ∑n MIN / (∑n MAJ +Σn MIN ) 2 ; The python program was used to perform analysis and calculation according to the statistical method of Hp, and the Hp values ​​of the indica rice population and the japonica rice population were calculated respectively.

[0053] Fst (fixation coefficient) was calculated using the R language software package hierfstat (version: 0.04-22, https: / / cran.r-project.org / web / packages / hierfstat / index.html). The Fst value ranges from 0 to 1, where 0 means that the genotype is not fixed at all and the base ratios in different populations are the same (corresponding to Hp of each population being equal to 0.5). Fst value equal to 1 means that the genotypes of the two populations are completely different (corresponding to Hp of each population being equal to 0, and the genotypes of the two populations are different).

[0054] 5. Marking target sites and target genes

[0055] The screening of coding genes was based on the gene annotation information of Nipponbare (MSU7.0), and the Hp values ​​of non-synonymous mutation SNP sites in the coding region of each gene were counted. If the Hp values ​​of all non-synonymous mutation SNP sites of the gene in the indica rice population or japonica rice population were less than 0.001 (Hp set threshold) and the Fst value was greater than or equal to 0.95, the gene was determined to be selected in both indica rice and japonica rice (marked as the target gene); if the Hp value of all amino acid-changing SNP sites of the gene was less than 0.001 and the Fst value was less than 0.95 only in japonica rice, it was determined to be a japonica rice selected gene; if the Hp value of all amino acid-changing SNP sites of the gene was less than 0.001 and the Fst value was less than 0.95 only in indica rice, it was determined to be an indica rice selected SNP site (target site).

[0056] A total of 2459 SNP sites selected in both indica and japonica rice (in 1226 genes), 1788 japonica rice selected SNP sites (in 1389 genes) and 828 indica rice selected SNP sites (in 828 genes) were screened, totaling 5636 SNP sites, corresponding to 3224 coding genes.

[0057] 6. Design of specific primers for gene chip

[0058] According to the flanking sequence of the SNP site and the principle of complementary base pairing, a specific short DNA sequence is designed for each SNP site. The method is as follows: Primer3 is used to design primer sequences on both sides of the SNP site, requiring that one of the primer sequences is less than 150bp away from the SNP site, and the distance between the SNP site and the 3' end of the primer is greater than 10bp, to ensure that the amplified sequence can be detected by high-throughput sequencing with a read length of 150bp and is not interfered by the primer sequence. The amplified length is no more than 500bp.

[0059] After the primers are designed, the primer sequences are aligned to the reference genome using blastn (any version) alignment software. When the similarity is greater than 95% and the last three bases at the 3' end are completely matched, at least one of the upstream and downstream primers specifically binds to the SNP target region. If not, the primers are redesigned.

[0060] Example 2

[0061] A method for screening core SNP sites in rice is provided, which is substantially the same as Example 1, except that step 3: PCA analysis and sample screening is omitted.

[0062] Example 3

[0063] A method for screening core SNP sites in rice is provided, which is substantially the same as Example 1, except that: the Hp threshold is set to <0.05.

[0064] Example 4

[0065] A method for screening core SNP sites in rice is provided, which is substantially the same as Example 1, except that: the Hp threshold is set to <0.01.

[0066] Example 5

[0067] A method for screening core SNP sites in rice is provided, which is substantially the same as Example 1, except that: the Hp threshold is set to <0.1.

[0068] Example 6

[0069] A method for screening core SNP sites in rice is provided, which is substantially the same as Example 1, except that in step 5, all SNP sites (synonymous and non-synonymous mutations) in the gene coding region are analyzed and screened for Hp values, and the sites that meet the set threshold are used as the final selected sites / target sites.

[0070] Example 1

[0071] Experiments were conducted based on the screening methods of the core SNP sites provided in Example 1 (after screening) and Example 2 (before screening or without screening). The Hp value distribution curves of the number of SNP sites of indica rice and japonica rice in Example 2 and Example 1 were compared. In the Hp distribution curves before sample screening, the maximum values ​​of the Hp distribution curves of indica rice and japonica rice were not equal to 0, which was mainly caused by the interference of non-core population samples, resulting in the maximum value shifting to the right, as shown in Figure 1. Figure 5 ,The results showed that the curves before and after screening were ,significantly different from those before screening.

[0072] The difference between the number of selected SNP sites detected and the corresponding genes is shown in Figure 6 The results showed that before the screening, only a very small number of genes were selected by both indica and japonica, and a small number of genes were selected by either indica or japonica. After the sample screening, the number of genes selected by both indica and japonica or genes selected by both indica and japonica increased significantly, and the main reason for the increase was the removal of the interference of non-core indica and japonica samples.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening rice SNP loci, characterized in that: It includes: Based on the information of N rice sample genomes, obtain rice SNP candidate sites, N≥2; The N rice samples are samples after removing non-core population samples; Screening the SNP candidate sites to obtain target sites for identifying rice varieties; The step of removing non-core population samples comprises: PCA analysis was performed on each chromosome of the rice sample, and the rice samples were divided into two groups, with the midpoint of the two groups in the PC1 axis as the center, and the chromosomes corresponding to the 1% to 10% samples closest to the center in the PCA distribution map were marked as mixed types; When the number of chromosomes marked in a sample was ≥2 times, the rice sample was removed.

2. The method for screening rice SNP loci according to claim 1, characterized in that: The SNP candidate site is a SNP site of a non-synonymous mutation.

3. The method for screening rice SNP loci according to claim 1, characterized in that: The step of screening the SNP candidate sites includes: calculating the heterozygosity score Hp of the SNP candidate sites in the rice population, and marking sites that meet a set threshold value less than Hp as initial target sites.

4. The method for screening rice SNP loci according to claim 3, characterized in that: The calculation formula of Hp is as follows: H p =2*∑n MAJ ∑n MIN / (∑n MAJ +∑n MIN ) 2 ; Among them, ∑n MAJ is the sum of the maximum genotype frequencies in the observation window, which is the proportion of the maximum genotype in the population; ∑n MIN is the sum of the minimum genotype frequencies in the observation window, where the observation window is 1 bp.

5. The method for screening rice SNP loci according to claim 3, characterized in that: The Hp setting threshold is 0.001 to 0.

05.

6. The method for screening rice SNP loci according to claim 5, characterized in that: The Hp setting threshold is 0.001 to 0.

005.

7. The method for screening rice SNP loci according to claim 3, characterized in that: After obtaining the initial target site, the step of screening the SNP candidate site also includes: marking genes whose SNP sites in the coding region are all the initial target sites as target genes, and obtaining the SNP sites in the coding region of the target gene as the final target site.

8. The method for screening rice SNP loci according to any one of claims 1 to 7, characterized in that: The rice is selected from at least one of indica rice and japonica rice.

9. The method for screening rice SNP loci according to any one of claims 1 to 7, characterized in that: The N≥10.

10. The method for screening rice SNP loci according to claim 9, characterized in that: Said N≥100.

11. A method for identifying rice varieties, characterized in that: The method comprises detecting a target site of a rice sample to be detected, wherein the target site is a target site screened by the method for screening rice SNP sites according to any one of claims 1 to 10.

12. The method for identifying rice varieties according to claim 11, characterized in that: The rice variety includes at least one of indica rice and japonica rice.

13. A kit for identifying rice varieties, characterized in that: It includes: Reagents for detecting the genotype of the target locus; The target site is a target site obtained by screening using the method for screening rice SNP sites according to any one of claims 1 to 10.

14. Use of a reagent for detecting the genotype of a target site in preparing a kit for distinguishing or identifying rice varieties, characterized in that: The target site is a target site obtained by screening using the method for screening rice SNP sites according to any one of claims 1 to 10.

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