A japonica rice whole genome SNP site combination, gene chip and its application

Abstract

The present invention belongs to the field of plant germplasm resource evaluation and molecular plant breeding, and specifically relates to a japonica rice whole genome SNP site combination, gene chip and its application. The SNP site combination includes SNP sites numbered 1-69554. The present invention deeply analyzes the japonica rice variant group big data, extracts the site information that is directly related to the evaluation of japonica rice germplasm resources and molecular breeding, and designs it into a high-density liquid phase capture genotyping chip with high cost performance. It can be used as a japonica rice fingerprint map for homogenization identification of rice germplasm resources and molecular pedigree detection; detect the genotype and aggregation of known favorable genes; the dominant haplotype of the genomic response site of the important agronomic trait constituent factor of japonica rice; detect the indica-japonica blood relationship ratio and chromosome interval of rice samples; for the construction of genetic map of QTL mapping population and haplotype map of GWAS natural population; detect whether rice has transgenic components.

Description

Technical field

[0001] The invention belongs to the fields of plant germplasm resource evaluation and molecular plant breeding, and specifically relates to a japonica rice whole genome SNP site combination, a gene chip and its application. Background technique

[0002] Existing locus combinations and genotyping chips for rice genotyping include:

[0003] (1) The 56K rice chip invented by the Institute of Crop Science, Chinese Academy of Agricultural Sciences, is based on the Affymetrix Axiom platform. Technical effect: This 56K SNP molecular marker combination can conduct molecular marker fingerprint analysis of rice variety resources, genotype identification of hybrid population offspring, identification of variety authenticity, analysis and screening of genetic background of breeding materials, and agronomic traits. Correlation Analysis.

[0004] (2) The 44K SNP rice genotyping chip developed by Cornell University in the United States is based on the Affymetrix Axiom platform. Technical effect: Used for classification of rice germplasm resources, identification of varieties and analysis of genetic variation.

[0005] (3) The 50K rice genotyping chip developed by the Indian Academy of Agricultural Sciences is based on the Affymetrix Axiom platform. Technical effect: The marker detection rate is relatively high, the technology is reproducible, and 194 agronomic trait genes can be typed in 50K SNP.

[0006] (4) The rice 8K breeding chip invented by Zhongyujin Marker is based on the Affymetrix Axiom platform. Technical effects: Genetic map construction, QTL analysis, variety purity identification, germplasm resource genotype identification, etc.

[0007] (5) The Rice6K and Rice60K rice genome-wide SNP breeding chips invented by China Seed Group Co., Ltd. are based on the Illumina Infinium platform. Technical effects: variety authenticity detection and genetic background analysis, genetic relationship analysis of rice germplasm resources, and gene mapping analysis.

[0008] (6) A 1536 SNP site chip, a 96 SNP site chip, and 6 384 SNP site chips designed by Cornell University in the United States, based on the Illumina GoldenGate platform. Technical effect: These low-density chips can be used for classification, identification and genetic analysis of rice samples.

[0009] The above-mentioned (1)-(4) rice chips are all based on the Axiom GeneChip biochip system and detection solution of Affymetrix, which uses in-situ photolithography technology to synthesize gene hybridization detection probes on the substrate of the solid-state chip. The above (5) and (6) Rice6K and Rice60K series breeding chips are based on the Infinium SNP chip system and detection solution of single base extension technology.

[0010] The shortcomings of existing rice genotyping site combinations and genotyping chips are as follows:

[0011] Existing rice microarray selection targets all rice types, but the genetic diversity of japonica rice is narrow, so the success rate and genetic analysis power of japonica rice typing are low. Contents of the invention

[0012] In view of the shortcomings of the existing technology, the present invention provides a japonica rice whole genome SNP site combination, a gene chip and its application.

[0013] In order to achieve the above objects, the present invention adopts the following technical solutions:

[0014] The first aspect of the present invention provides a japonica rice genome-wide SNP site combination. The SNP site combination includes SNP sites numbered 1-69554, which are 8785 SNP sites located on chromosome 1 of japonica rice. , 8035 SNP sites on chromosome 2, 5141 SNP sites on chromosome 3, 5789 SNP sites on chromosome 4, 3879 SNP sites on chromosome 5, and 7233 SNP sites on chromosome 6. There are 9089 SNP sites on chromosome 7, 4975 SNP sites on chromosome 8, 4673 SNP sites on chromosome 9, 6220 SNP sites on chromosome 10, and 4982 SNP sites on chromosome 11. 3956 SNP sites on chromosome 12.

[0015] The variation information of the SNP sites is expressed in the form of number: physical location reference genotype / allele type. The 69554 SNP sites are as follows:

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] Chromosome 7,9089

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] Chromosome 12, 3956 SNP sites

[0028]

[0029] A second aspect of the present invention provides a gene chip for detecting SNP site combinations in the whole genome of japonica rice.

[0030] The third aspect of the present invention provides the application of the japonica rice whole genome SNP chip as a japonica rice fingerprint for homogenization identification of rice germplasm resources and molecular pedigree detection.

[0031] A fourth aspect of the present invention provides the application of the gene chip in detecting whether rice has genetically modified components.

[0032] The fifth aspect of the present invention provides that the gene chip can detect the genotype and aggregation status of known "stuck" genes such as disease resistance, quality, heading period and adaptability, and assist early generations in eliminating bad families or single plants. applications in.

[0033] A sixth aspect of the present invention provides the application of the gene chip in detecting dominant haplotypes of response sites in the genome of factors constituting important agronomic traits of japonica rice.

[0034] A seventh aspect of the present invention provides the application of the gene chip in detecting the ancestry ratio of indica and japonica and the chromosome interval of rice samples.

[0035] The eighth aspect of the present invention provides the application of the gene chip in the construction of genetic maps of QTL mapping populations and the construction of GWAS natural population haplotype maps.

[0036] A ninth aspect of the present invention provides the application of the gene chip in whole genome selective breeding of japonica rice.

[0037] Compared with the prior art, the present invention has the following distinctive effects:

[0038] 1. The present invention conducts an in-depth analysis of the genetic characteristics, population differentiation and dominant functional genes of japonica rice, refines key variation information, and specifically designs a typing site combination based on the functional variation of the entire japonica rice genome and Its gene chip application has strong genetic analysis power for japonica rice.

[0039] 2. The present invention can detect the variation of tens of thousands of target functional sites in rice at one time. Compared with traditional high-throughput sequencing to detect the genotype of functional genes, there is no need to re-extract the complex process of target information from high-throughput sequencing data. The output of the invention is user-friendly results, without the need for users to have a biometric foundation, and it directly provides users with genotype results and downstream analysis results, greatly reducing detection and analysis costs.

[0040] 3. The present invention applies "liquid phase capture precise positioning sequencing and typing technology" without the need to collect 96 or 384 samples, any integer multiple of 12 samples can be used, and the cost performance is greatly improved compared with the existing solid-state chip detection solution.

[0041] 4. Compared with existing solid-state chip detection solutions, all variations in the target detection site interval of the rice genome can be detected, so unknown alleles and haplotypes can be discovered.

[0042] 5. It can simultaneously detect and test whether rice contains genetically modified ingredients. Description of the drawings

[0043] figure 1 This is a schematic flow diagram of Embodiment 1 of the present invention.

[0044] figure 2 This is a phylogenetic tree based on the SNP chip analysis of 60 japonica rice varieties in Example 1. In the figure, A: comes from Chinese japonica rice farmer varieties, B: comes from Japanese japonica rice farmer varieties, C: comes from Liaoning modern cultivated rice varieties. , D: comes from the japonica rice varieties cultivated in Yunnan, and E comes from the variety collection composed of japonica rice cultivated in Ningxia, Liaoning and Japan.

[0045] image 3 This is a diagram based on the SNP chip analysis of 9 varieties of indica rice introgression patterns in Example 1.

[0046] Figure 4 The SNP chip of Example 1 is used to analyze rice milk viscosity, a component of quality traits of 192 japonica rice test varieties, and the results of genome-wide association analysis are used. Detailed ways

[0047] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limitations of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources, unless otherwise specified.

[0048] Example 1

[0049] In this example, the japonica rice genome-wide functional variation typing site combination data set (ie, 69554 SNP sites) was derived from the integration and in-depth mining of independent sequencing data and database data. High-throughput data including NGS data of China's high-yield, China's high-quality, and farm-grown japonica rice varieties independently sequenced by Shenyang Agricultural University. As well as Rice 3K (https: / / snp-seek.irri.org / ), NGDC (https: / / bigd.big.ac.cn / ), DDBJ (https: / / www.ddbj.nig.ac.jp / ) database, the NGS data of China's high-yield, China's high-quality, and farmhouse japonica rice constitute the Sino-Japanese high-quality japonica rice NGS data set. In-depth exploration process of data sets ( figure 1 )as follows:

[0050] 1. Perform core SNP screening on the japonica rice germplasm resources in the Sino-Japanese high-quality japonica rice NGS data set. The parameters are the minimum allele frequency of 0.3, heterozygosity less than 0.025, uniform chromosome distribution, and 1 SNP per 200 bp physical distance, and then obtain 30,000 highly informative core SNP variation sites in japonica rice.

[0051] 2. Calculate the population differentiation coefficient Fst based on the high-yield japonica rice in China and high-quality japonica rice in Japan in the Sino-Japanese japonica rice NGS data set, and obtain 1,000 highly differentiated variant SNP sites between high-yield japonica rice in China and high-quality japonica rice in Japan. The population differentiation coefficient Fst was further calculated by combining two groups of Chinese japonica rice populations from Liaoning, Jilin, and Heilongjiang, and weighted screening was performed based on the proportion of the number of loci with a genetic differentiation index (Fst) value >= 0.4 in each chromosome. , 3,000 highly differentiated SNP sites in the japonica rice genome from the three northeastern provinces were obtained.

[0052] 3. Conduct a selective elimination analysis on japonica rice from the three northeastern provinces of China and Japan, and analyze the genomic regions that respond to modern genetic improvements. iSAFE software was used to analyze the 250 kb upstream and downstream of the peak sites in these regions, and the results were sorted and filtered from high to low, and a total of 5,500 SNP sites responding to the genetic improvement of modern japonica rice were obtained.

[0053] 4. Conduct genome-wide association analysis on yield and quality component traits, and select peak SNP sites. At the same time, Candihap software was used to analyze candidate genes in the peak area and extract haplotype variation sites of candidate genes. At the same time, the literature was searched to obtain genome-wide association loci that contribute to important agronomic traits of japonica rice. Finally, 16,763 mutation sites were obtained that contributed to yield and quality.

[0054] 5. Conduct subspecies-specific SNP frequency analysis on the resequencing (NGS) data in the NGS database of indica rice and japonica rice farm varieties in the RiceHAP2 database (http: / / www.ncgr.ac.cn / RiceHap2 / index.html). The species-specific SNP frequencies were sorted from high to low, and a total of 20,000 SNP sites with significant effects on indica and japonica rice differentiation were obtained.

[0055] 6. Obtain all known functional genes from the rice genome MBK database (http: / / www.mbkbase.org / rice), use 300bp as a window to screen effective mutations, and select according to the frequency of effective mutations, finally obtaining 5665 known functional gene haplotype loci.

[0056] 7. Extract the resistance and characteristic element sequence information of the transgenic vector and incorporate it into the japonica rice genome-wide functional variation typing site combination.

[0057] Specific capture probes were designed for the above 81,928 japonica rice genome-wide functional variation typing sites. The probes were used to capture and enrich the target variant regions of 192 japonica rice test materials. The captured and enriched target genome sequences were sequenced and library constructed. High-throughput sequencing. Based on the final yield of effective mutation sites, 69,554 typing site combinations of functional variations in the entire japonica rice genome were finally screened, and the invention of the chip was completed. The physical locations of the 69554 SNP sites were determined based on the comparison of the whole genome sequence of the rice variety Nipponbare. The version number of the whole genome sequence of the rice variety Nipponbare is IRGPS1.0 (https: / / rapdb.dna.affrc .go.jp) genomic region.

[0058] Application examples

[0059] 1. Based on the SNP chip of Example 1, the gene sequence files of 60 japonica rice varieties were analyzed and MEGA5.0 was used to construct a phylogenetic tree of the genetic relationships of the varieties. Open the gene sequence files of 60 japonica rice varieties through MEGA5.0, compare the gene sequences of 60 japonica rice varieties, and save the comparison results. The gene sequence alignment results of 60 japonica rice varieties were opened again with MEGA5.0, and the NJ (Neighbor-Joining) method was used to construct a phylogenetic tree of the genetic relationships of 60 japonica rice varieties. The result is as follows figure 2 shown.

[0060] 2. Based on the SNP chip analysis of 9 varieties of indica rice kinship introgression results in Example 1, use the RCircos installation package in R language to draw the 9 varieties of indica rice kinship introgression pattern diagrams. The result is as follows image 3 As shown, each circle from the inside to the outside represents a variety, and the outermost circle is the density of subspecies differentiation sites in this area. Among the 12 chromosomes, the white color represents the japonica blood background, and the colored genome region represents the introgression region of the indica variety.

[0061] 3. Based on the SNP chip analysis in Example 1, the rice milk viscosity results of 192 japonica rice test varieties that constitute quality traits, use TASSEL5.0 to conduct genome-wide association analysis, and save the analysis results. Use the qqman installation package in R language to draw a Manhattan plot on the genome-wide association analysis results. The results are as follows: Figure 4 As shown in the figure, it can be clearly seen from the figure that there is a significant peak on chromosome 6, which is determined to be the variation in rice milk viscosity in the test variety caused by the genetic effect of the Waxy gene through the functional gene information on the chip.

[0062] Although the preferred embodiments of the invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A genome-wide SNP locus combination for japonica rice, characterized in that, The SNP locus combination includes SNP loci numbered 1-69554, which are located on chromosome 1 of japonica rice: 8785 SNP loci, chromosome 2: 8035 SNP loci, chromosome 3: 5141 SNP loci, chromosome 4: 5789 SNP loci, chromosome 5: 3879 SNP loci, chromosome 6: 7233 SNP loci, chromosome 7: 9089 SNP loci, chromosome 8: 4975 SNP loci, chromosome 9: 4673 SNP loci, chromosome 10: 6220 SNP loci, chromosome 11: 4982 SNP loci, and chromosome 12: 3956 SNP loci.

2. A gene chip for detecting the combination of SNP sites in the whole genome of japonica rice as described in claim 1.

3. The application of the gene chip described in claim 2 as a fingerprint of japonica rice in the homogenization identification and molecular pedigree detection of rice germplasm resources.

4. The application of the gene chip according to claim 2 in detecting whether rice contains transgenic components.

5. The application of the gene chip according to claim 2 in detecting the genotype and aggregation of known "bottleneck" genes such as disease resistance, quality, heading date and adaptability, to assist in the early generation elimination of undesirable families or individual plants.

6. The application of the gene chip according to claim 2 in detecting the dominant haplotypes of genomic response sites of important agronomic traits in japonica rice.

7. The application of the gene chip according to claim 2 in detecting the indica-japonica lineage ratio and chromosome intervals in rice samples.

8. The application of the gene chip according to claim 2 in the construction of genetic maps of QTL mapping populations and the construction of haplotype maps of natural GWAS populations.

9. The application of the gene chip according to claim 2 in whole-genome selection breeding of japonica rice.

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