SNP (Single Nucleotide Polymorphism) site combination for distinguishing wild rice and indica cultivated rice and application of SNP site combination

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site combination for distinguishing wild rice from indica type cultivated rice and application of the SNP site combination. The SNP site combination comprises 180 SNP sites which are uniformly distributed on a rice whole genome. The genotypes of the 180 SNP loci in rice to be detected are detected, if the genotypes of at least 60% of the SNP loci in the rice to be detected are consistent with the genotype of the wild rice, the rice to be detected is identified as the wild rice, and if the genotypes of at least 60% of the SNP loci in the rice to be detected are consistent with the genotype of the indica type cultivated rice, the rice to be detected is identified as the indica type cultivated rice. By utilizing the 180 SNP site combination provided by the invention, wild rice and indica type cultivated rice can be quickly distinguished, so that the wild rice resource can be more sufficiently utilized.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular breeding, and particularly relates to a SNP site combination for distinguishing wild rice from indica cultivated rice and an application thereof. Background Art

[0002] Rice is one of the three most important food crops, and it is the largest food crop in my country. With the continuous advancement of commercial breeding, the homogeneity of rice varieties promoted on a large scale has become increasingly serious. The narrow genetic diversity between cultivated varieties used in modern production has become a bottleneck restricting the further improvement of rice breeding. Compared with modern cultivated rice, wild rice has richer genetic diversity and gene resources. The rapid exploration and utilization of wild rice diversity and specific resources is of great significance.

[0003] After years of research, it is possible to introduce excellent wild rice genes into cultivated rice through gene introgression, thus achieving genetic improvement of cultivated rice and broadening the germplasm resources of the genetic basis of cultivated rice. Using the recently developed rice high-density gene chip and breeding accelerator, a series of wild rice chromosome single small fragment introduction lines can be quickly constructed. All the introduction line fragments can cover the entire wild rice genome, that is, any chromosome segment has a corresponding introduction line for research and breeding applications.

[0004] SNP (single nucleotide polymorphism) is a third-generation DNA molecular marker with higher genetic stability, stronger reliability and more comprehensive coverage. At present, the main forms of application of SNP markers in plant genetic research and breeding are gene chips and resequencing technology. Gene chip is a highly integrated, high-throughput, miniaturized and automated SNP detection method. It can screen thousands of sites with just one hybridization. It is an effective technical means for research on crop genetic diversity, population structure, QTL positioning, etc. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP locus combination that can distinguish wild rice from indica cultivated rice and its application. Specifically, a 180 SNP locus combination that can distinguish wild rice from indica cultivated rice is provided, and it is evenly distributed on the whole rice genome.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention obtains 22637 SNP sites based on literature analysis; among the above SNP sites, 180 SNP sites with large differences between wild rice and indica cultivated rice are screened out by Euclidean distance, that is, 180 SNP site combinations that can distinguish wild rice from indica cultivated rice are obtained, and the SNP site names, positions and corresponding genotypes are shown in Table 2. Through the genotype analysis of wild rice and indica cultivated rice at 180 SNP sites, the differentiation results are as follows: among 966 varieties, 92.86% of wild rice and indica cultivated rice have at least 60% of SNP sites that meet the corresponding genotype; 86.75% of wild rice and indica cultivated rice have at least 70% of SNP sites that meet the corresponding genotype. Therefore, when at least 60% of the SNP sites meet the corresponding genotype, most wild rice and indica cultivated rice can be distinguished.

[0008] The application of SNP loci combination in distinguishing wild rice from indica cultivated rice, the SNP loci are shown in Table 2.

[0009] A rice chip for distinguishing wild rice from indica cultivated rice, the chip comprising probes for detecting the SNP site combination shown in Table 2, wherein the probes are synthesized based on the SNP site and a 50 bp sequence on one side thereof.

[0010] A method for distinguishing wild rice from indica cultivated rice comprises the following steps:

[0011] (1) detecting the SNP locus genotype of the rice to be tested, wherein the SNP locus is shown in Table 2;

[0012] (2) Counting the genotypes of each site, if the genotype of at least 60% of the SNP sites of the rice to be tested is consistent with the genotype of wild rice, it is identified as wild rice; if the genotype of at least 60% of the SNP sites of the rice to be tested is consistent with the genotype of indica cultivated rice, it is identified as indica cultivated rice.

[0013] Beneficial effects of the present invention: The present invention provides a set of SNP site combinations that accurately and evenly cover the entire genome for distinguishing wild rice from indica cultivated rice. The SNP site combination can be widely used to distinguish wild rice from indica cultivated rice, which is of great significance to the utilization of wild rice resources. DETAILED DESCRIPTION

[0014] The present invention is described in detail below in conjunction with specific embodiments. The given embodiments are only for illustrating the present invention but not for limiting the scope of the present invention.

[0015] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0016] Example 1: Obtaining a SNP locus combination for distinguishing wild rice from indica cultivated rice

[0017] 1. SNP data analysis of wild rice and indica cultivated rice (indica rice): Through literature research, 22,637 loci were extracted from the SNP variation data of 446 wild rice populations and 520 indica rice populations.

[0018] 2. Screening of whole genome SNP sites: The differences of the above SNP sites in wild rice and indica rice were calculated by Euclidean distance, and 180 SNP sites with large differences between wild rice and indica cultivated rice were screened according to the data of wild rice genotype, indica genotype and proportion of samples with undetected genotype in wild rice population and indica rice population. That is, 180 SNP site combinations that can distinguish wild rice from indica cultivated rice were obtained. The SNP site name, location (based on the rice reference genome Nipponbare version 7) and corresponding genotype are shown in Table 2. The screening criteria are as follows:

[0019] 2.1 Screening based on Euclidean distance: For the 22637 SNP sites obtained in the previous step, screen out sites with Euclidean distance greater than 0.8485, totaling 2624; the calculation formula of Euclidean distance is as follows:

[0020] Where ρ is the Euclidean distance between the points (x2, y2) and (x1, y1). In this embodiment, the point (x2, y2) is The point (x1, y1) is Where ww is the number of loci of wild rice genotype in wild rice population, wi is the number of loci of indica rice genotype in wild rice population, iw is the number of loci of wild rice genotype in indica rice population, and ii is the number of loci of indica rice genotype in indica rice population;

[0021] 2.2 Screening based on the proportion of samples with undetected genotypes: The number of samples with undetected genotypes for each remaining SNP locus in wild rice and indica rice populations was counted, and the average value was calculated for each chromosome (Table 1); sites with the number of undetected genotypes lower than the average were screened, totaling 1794;

[0022] Table 1 The average number of samples with undetected genotypes on each chromosome in wild rice and indica rice populations

[0023]

[0024] 2.3 Screening out abnormal sites: Screening out sites where the proportion of wild rice corresponding genotypes in wild rice populations is less than 50%, and screening out sites where the proportion of indica rice corresponding genotypes in indica rice populations is less than 50%, leaving 1484 sites;

[0025] 2.4 Screening based on the distribution on the genome: 15 SNP sites with relatively even distribution were screened for each chromosome, and a total of 180 SNP sites were screened for 12 chromosomes.

[0026] Table 2 180 SNP loci names, positions and corresponding genotypes

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[0032] 3. SNP site combination differentiation method: The genotypes of the above 180 SNP sites in 446 wild rice and 520 indica rice varieties were investigated (see Table 3). The results showed that: among all 966 varieties, 92.86% of wild rice and indica rice samples had at least 60% of SNP sites that matched the corresponding genotypes; 86.75% of wild rice and indica rice samples had at least 70% of SNP sites that matched the corresponding genotypes. Therefore, when at least 60% of the SNP sites matched the corresponding genotypes, most wild rice and indica cultivated rice can be distinguished.

[0033] Table 3. Genotype statistics of 180 SNP loci in 446 wild rice and 520 indica rice samples

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[0058] Note: The ratio of characteristic loci of wild rice = the number of loci of wild rice genotype / (the number of loci of wild rice genotype

[0059] =Number of loci in the indica rice genotype + Number of loci in the indica rice genotype), the ratio of characteristic loci in the indica rice = Number of loci in the indica rice genotype

[0060] =Number of loci in wild rice genotype + number of loci in indica rice genotype.

[0061] Example 2: Application of 180 SNP loci combination in distinguishing wild rice from indica cultivated rice

[0062] In order to test the application effect of the combination of 180 SNP loci developed in the present invention in distinguishing wild rice from indica cultivated rice, 6 wild rice and 6 indica rice samples were randomly selected, and the genotypes of the combination of 180 SNP loci in these samples were detected. The results are shown in Table 4. When at least 60% of the SNP loci meet the corresponding genotypes, wild rice and indica cultivated rice can be accurately distinguished.

[0063] Table 4 Genotypes of 6 randomly selected wild rice and 6 indica rice varieties

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[0065] Note: The proportion of characteristic loci of wild rice = the number of loci of wild rice genotype / the number of loci of wild rice genotype + the number of loci of indica rice genotype); the proportion of characteristic loci of indica rice = the number of loci of indica rice genotype / the number of loci of wild rice genotype + the number of loci of indica rice genotype.

[0066] As shown in Table 4, the combination of 180 SNP sites developed by the present invention can quickly and accurately distinguish wild rice from indica cultivated rice, making it easier to fully utilize wild rice resources.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of SNP locus combination in distinguishing wild rice from indica cultivated rice, characterized in that: The SNP sites are shown in Table 2.

2. A probe combination, characterized in that: The probe is used to detect the SNP site described in claim 1.

3. Rice chip, characterized in that, The chip comprises probes for detecting the SNP site combinations shown in Table 2.

4. The probe according to claim 2 or the rice chip according to claim 3, characterized in that: The probes used to detect the SNP sites shown in Table 2 were synthesized based on the SNP site and the 50 bp sequence on one side.

5. A method for distinguishing wild rice from indica cultivated rice, characterized in that: The following steps are involved: (1) Detecting the SNP locus genotype of the rice to be tested, wherein the SNP locus is shown in Table 2; (2) Count the genotypes of each site. If the genotypes of at least 60% of the SNP sites of the rice to be tested are consistent with the genotypes of wild rice, it is identified as wild rice; if the genotypes of at least 60% of the SNP sites of the rice to be tested are consistent with the genotypes of indica cultivated rice, it is identified as indica cultivated rice.

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