CN115867667A - Cabbage type rape high-density whole genome SNP chip and application thereof - Google Patents

Abstract

The invention discloses a cabbage type rape high-density whole genome SNP chip and application thereof, the chip is named as Bnapus50K, and comprises 42,090 SNP marked probes, the SNPs are uniformly distributed on 19 chromosomes of a cabbage type rape genome, the average density is 100Kb 5 SNPs, and the probes have abundant polymorphism in all tested varieties. Compared with the existing SNP chip of the cabbage type rape, the SNP marker of the Bnapus50K chip is derived from the re-sequencing result of 510 varieties of the cabbage type rape, has better variety representativeness, is added with a new functional gene probe, and can be more effectively used for functional gene identification or positioning, genetic diversity analysis and variety analysis.

Description

High-density whole genome SNP (single nucleotide polymorphism) chip for brassica napus and application of chip Technical Field

The invention relates to the fields of molecular biology, genomics, bioinformatics and plant molecular breeding, in particular to a high-density whole genome SNP chip for brassica napus and application thereof.

Background

The development of SNP markers is based on DNA sequencing (Davey, et al, genome-wide genetic marker discovery and genetic-sequencing Nat Rev Genet.12: 499-510.), more than ten years since the appearance of 454 sequencer in 2005, the second-generation sequencing technology is continuously improved, the Genome sequencing efficiency is greatly improved, the sequencing cost is greatly reduced, the whole Genome sequence of a large number of species is completed, and the development of functional Genome research is greatly promoted.

Brassica napus is a brassica herbaceous plant, is also the species with the highest grain yield in three kinds of oil rapes (brassica napus, brassica juncea and brassica napus), and is a compound species formed by double-diploidization evolution of brassica napus (AA, n = 10) and brassica oleracea (CC, n = 9) after natural interspecific hybridization. Performing re-sequencing on different varieties, constructing a high-density Brassica napus haplotype map (HapMap) by using SNP markers discovered by re-sequencing, performing Association analysis on important agronomic traits by using Genome-Wide Association Mapping (GWAS), determining candidate gene sites related to the important agronomic traits, and establishing a set of efficient, rapid, stable-maturing, low-cost and high-throughput genotype identification methods (WEI DaYong and the like, genome-Wide Association Study of the Fertility Restorer Loci for polar CMS in Rapeseed (Brassica napus L.). Scientific Agricutural Sinica.2017, issue (5): 802-810), which are directions preferentially considered by molecular biological researchers.

<xnotran> SNP , Illumina InfiniumBrassica 60K (Wayne E.Clarke , A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome.Theoretical and Applied Genetics,2016,1887-1899), Illumina Infinium Rice6K (Yu , A whole genome SNP array (RICE 6K) for genomic breeding in rice.Plant Biotech J,2014,12:28-37), cornell _6K_Array_Infinium_Rice (Thomson , large-scale deployment of a rice6K SNP array for genetics and breeding applications.Rice (NY), 2017,10:40), illumina GoldenGate (Parida , SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure.BMC Genomics.2012, 13:426-443;), affymetrix GeneChip Rice 44K (Zhao , genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa.Nat Commun.2011, 13:467), illumina Infinium RiceSNP50 (Chen , A High-Density SNP Genotyping Array for RiceBiology and Molecular Breeding.Molecular Plant,2014,7 (3): 541-553) , , ; </xnotran> Ganal et al (A large mail (Zea Mays L.) SNP genetic array: reduction and geloplasm genetic, and genetic mapping to complex with the B73reference gene. Plos one.2011, 6; in the acclimatization history study of soybean, affymetrix Axiom genome-wide SNP chip NJAU 355K soysnpp analyzed 105 wild and 262 cultivars, concluding that soybean cultivars originated from the middle and north of china (Wang et al, development and application of a novel genome-wide SNP array discovery in soybean rep.2016,6 20728-20737; to construct a high-density genetic linkage map of wheat, a wheat 90KINFINIUM iSelect SNP chip was used to scan 4 wheat populations and to map 29692 SNP markers to 21 chromosomes of 6-fold wheat (Wen et al, A high-diversity consensus map of common world mapping synthesized by the 90K SNP array, front Plant Sci.2017, doi.

The present invention designed 50K DNA chips based on 32,216,304 SNPs extracted from the re-sequencing data of 510 inbred lines of rape. Considering the application in breeding, 1,618 functional probes against published functional genes, such as mitochondrial specific genes and corresponding restorer genes from the cytoplasmic male sterile mitochondrial genome, and specific probes for detecting transgenic material were designed. The experimental result shows that compared with the Brassica napus 60K chip, the probes of the Bnapus50K chip have more abundant polymorphism in the tested variety, so that the Bnapus50K chip has better application prospect in genome breeding and genome research of Brassica napus.

Disclosure of Invention

The invention aims to provide a high-density whole genome SNP chip for Brassica napus, which comprises 42,090 SNP chips

The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1-42,090.

The invention also aims to provide application of the high-density whole genome SNP chip for the Brassica napus.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant utilizes 510 natural population second-generation re-sequencing data, wherein 89.8% of varieties come from China, 10.2% of varieties come from countries such as east Asia, europe, america, australia and the like, and have very good representativeness to Chinese rape varieties, and identifies 32,216,304 original variation sites (SNP + INDEL) in total, and retains 11,249,037 sites with the frequency of the next-allele being more than 0.05 and the mass fraction being more than or equal to 30; removing SNPs with the distance less than 50bp from the left and right SNPs, and remaining 1,040,415; extracting the only aligned SNPs: the site with one side uniquely matched (the matching degree of the sequence at other positions is less than 85%) is 286,921 in the 50bp sequences at the left and right sides of the SNP; these 286,921 sites were submitted to a scoring system for scoring and integration of QTL sites (including flowering time, oil content, self-incompatibility, etc.) reported in 25 rape GWAS documents consulted, and finally screened to yield 45,707 polynucleotides. Submitting the sequences to Illumina, manufacturing an SNP chip by using an Infinium chip manufacturing technology, and successfully synthesizing 42,090 high-quality probes named as Bnapus50K according to the condition requirements of the Illumina on the probes; the 42,090 polynucleotides are shown in SEQ ID NO. 1-42,090.

The application of the high-density whole genome SNP chip for cabbage type rape comprises the steps of carrying out functional gene identification or positioning on cabbage type rape by utilizing the SNP chip provided by the invention; the SNP chip provided by the invention is used for carrying out genetic diversity analysis on the brassica napus; the SNP chip provided by the invention is used for carrying out variety analysis on the brassica napus.

Compared with the prior art, the invention has the following advantages:

compared with a set of cabbage type rape Brassica60K chips which are published by 16 international academies and commercial institutions of the same type and integrate 52157 SNP markers, the invention has better variety representativeness. The SNP locus of the Brassica60K chip is identified and screened from more than 70 cabbage type rape sequencing data, and the SNP locus of the Bnapus50K cabbage type rape whole genome chip is derived from the heavy sequencing result of 510 cabbage type rape varieties. The Bnapus50K probe of the invention was more evenly distributed across the genome than Brassica60K (fig. 1, left).

In addition, with Brassica60K, researchers can only effectively use approximately 40% of the probes in GWAS analysis; the invention optimizes the markers, has more excellent markers, more reasonable marker density distribution, increases the published functional gene probes, and can more effectively identify the functional genes and analyze the genetic background of breeding materials.

Drawings

FIG. 1 is a schematic diagram of the distribution of all SNP sites on the whole genome of a Brassica60K chip and a Bnapus50K whole genome breeding chip, wherein the left image is the Brassica60K chip, and the right image is the Bnapus50K chip.

FIG. 2 is a typing map of Brassica napus P4, 1L238 and its progeny P4/1L 238.

Figure 3 is a manhattan diagram showing GWAS correlation results.

FIG. 4 shows the results of BSA localization.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

obtaining a cabbage type rape whole genome breeding chip Bnapus 50K:

the method utilizes 510 parts of brassica napus varieties (89.8 percent of the varieties are from China, 10.2 percent of the varieties are from countries such as east Asia, europe, america, australia and the like, and have very good representativeness to the Chinese rape varieties) to re-sequence results, identifies 32,216,304 sites with original variation sites (SNP + INDEL), reserves two genotypes, and 11,249,037 sites with the frequency of the next-allele greater than 0.05 and the mass fraction greater than or equal to 30; removing SNPs with the distance less than 50bp from the left and right SNPs, and remaining 1,040,415; extracting the only aligned SNPs: the site with one side uniquely matched (the matching degree of the sequence at other positions is less than 85%) is 286,921 in the 50bp sequences at the left and right sides of the SNP; these 286,921 sites were submitted to a scoring system for scoring and integration of QTL sites reported from the 25 rape GWAS documents referred to, and finally screened for 45707 polynucleotides. These sequences were submitted to Illumina, and SNP chips were fabricated using the Infinium chip fabrication technique, in which 42,090 high quality probes, named Bnapus50K, were successfully synthesized; the nucleotide sequences of the 42,090 probes are shown in SEQ ID NO. 1-42,090.

The selection procedure for SNP sites is as follows:

1) Identifying original variation sites by using 510 parts of the re-sequencing results of the cabbage type rape varieties;

2) Reserving loci with the frequency of the minor allele being more than 0.05 and the mass fraction being more than or equal to 30, wherein only two genotypes exist;

3) Removing sites with the distance of less than 50bp from the SNP on the left side and the SNP on the right side;

4) Extracting the only aligned SNPs: the 50bp sequences on the left and right sides of the SNP have sites with one side matched uniquely (the matching degree of the sequences at other positions is less than 85 percent);

5) Dividing the Darmor genome into segments of every 100kb, calculating the correlation coefficient between SNP pairs in each segment, and calculating the correlation coefficient (R) ² Not less than 0.65) as a threshold value;

6) Constructing a comprehensive scoring strategy, and evaluating the residual SNP efficiency, wherein the comprehensive scoring strategy specifically comprises the following steps:

(1) From QTL sites reported in GWAS literature (see also literature 25), 304 very significant sites were extracted, including the following traits: flowering time, precocity, plant type (plant height, branching angle, etc.), yield (thousand kernel weight, etc.), seed quality (oil content, erucic acid content, thioglycoside content, etc.), disease resistance (clubroot, black stalk disease, sclerotinia), resistance to abiotic stress (water, heat, salt, cadmium); plan to increase 3 probes in each site up and down 150K;

(2) Determining the mark density: less than 6 probes per 100K on chr, and less than 2 probes per 100K on random;

(3) Counting the number of probes required to be increased in every 100K, and screening 286,921 SNPs according to the following priority: within every 100K, the score is more than or equal to 0.6; MAF is higher; as far as possible from different bins;

(4) Finally, 45707 SNP sites are selected.

These sequences were submitted to Illumina and SNP chip Bnapus50K was produced by Infinium chip manufacturing technology. The distribution of all SNP loci of Bnapus50K on the whole genome is shown in figure 1 (right), the SNP loci are uniformly distributed on the whole genome, and are densely distributed in important functional gene regions, so that the genetic diversity analysis and genetic relationship analysis of cabbage type rape germplasm resources, the functional gene identification and gene positioning of the cabbage type rape, and the requirements of the authenticity identification of the cabbage type rape variety and the genetic background analysis of cabbage type rape breeding materials can be met.

Example 2:

the method for detecting the cabbage type rape sample by using the Bnapus50K cabbage type rape whole genome breeding chip comprises the following steps:

1) Preparation of cabbage type rape sample DNA: extracting DNA of different tissues, organs or individuals according to a standard flow of a kit by using a plant genome DNA extraction kit of Tiangen Biochemical technology (Beijing) Co., ltd according to the detection requirement;

2) Quality control of cabbage type rape sample DNA: detecting the integrity of the genome DNA by using 1% agarose gel electrophoresis; according to the instructions of the instrument, the concentration of DNA is measured by using a Nanodrop2000 spectrophotometer, and the pollution degree of protein and RNA is judged;

3) Detection of a gene chip: operating according to an Illumina Infinium gene chip detection standard flow, and scanning the chip by using an Illumina HiScan chip scanner;

4) Gene chip data analysis: results of Illumina HiScan scanning genotype was analyzed using Genome Studio software and genotype comparisons were obtained using R language programming.

Example 3:

the Bnapus50K chip is applied to the genetic background analysis of the cabbage type rape:

using the method described in example 2, the Bnapus50K chip was used to test P4/1L238 and its parents P4 and 1L238 to determine if it could be used for background selection of progeny population of the above mentioned cross combinations.

The analysis result shows that the P4 and 1L238 parents are different homozygous locus regions, and the hybrid progeny P4/1L238 shows a heterozygous state in the regions, thereby conforming to the hybridization phenomenon; and the differential markers are uniformly distributed on the whole genome, so that the marker requirement for accurately selecting the background in molecular breeding can be effectively met (figure 2).

Example 4:

the Bnapus50K chip is applied to Genome-wide association analysis (GWAS):

the whole genome association analysis is a new strategy for finding out genetic variation affecting complex traits by using millions of Single Nucleotide Polymorphisms (SNPs) in a genome as molecular genetic markers, performing control analysis or correlation analysis on the whole genome level and comparing.

356 parts of brassica napus material were tested using the method described in example 2 using Bnapus50K chips, and the same quantitative trait RT2 (weight of seedling roots) of the corresponding material was determined, and the p-value of each locus associated with the phenotype was calculated and the corresponding manhattan plot was drawn according to the p-value.

The analysis results show that the phenotype has strong correlation with the 53Mb region of the chromosome C03, and the genes controlling the phenotypic traits are presumed to be possibly located in the section of the chromosome C03, so that the novel genes can be effectively identified and identified (FIG. 3).

Example 5:

the Bnapus50K chip was applied to mixed packet analysis (Bulkedsegregationanalysis; BSA):

BSA (bulked segregationanalysis), also known as segregating population grouping analysis, is a method of analyzing by picking individual constituent mixed pools of extreme or representative traits in a population. By studying the difference in allele/molecular marker frequency between pools, loci associated with the trait are mapped on the genome.

Using the method described in example 2, two pools of Brassica napus with extreme phenotypes (flowering and closed flowers) (20 individual samples mixed) were tested using the Bnapus50K chip, and the genotypes at each of the tested loci were compared to mark the positions of the different loci in the Brassica napus genome.

The analysis result shows that the two mixed pools have difference in the 8Mb region of the chromosome A01, and the gene for controlling the phenotypic character is presumed to be located in the section of the A01 chromosome, so that the novel gene can be effectively identified and identified (figure 4).

Claims (4)

1. The high-density whole genome SNP chip for Brassica napus is characterized by comprising 42090 SNP molecular markers, and the nucleotide sequence of the SNP molecular markers is shown as SEQID No. 1-42090.
2. The SNP chip of claim 1, wherein the SNP chip is used for functional gene identification or localization of Brassica napus.
3. The use of the SNP chip of claim 1 for genetic diversity analysis of Brassica napus.
4. The SNP chip of claim 1, in use for variety analysis of Brassica napus.

Images