Brassica napus generic genome liquid phase chip and application thereof

Abstract

The invention discloses a brassica napus generic genome liquid phase chip and application thereof. The invention provides a liquid chip for detecting a whole genome of brassica napus. The liquid chip comprises a probe composition and a hybridization capture reagent, the probe composition is a probe group for specifically capturing a locus combination of the whole genome of the brassica napus; the locus combination of the whole genome of the brassica napus is composed of 10414 loci, and the 10414 loci comprise 9901 SNP (Single Nucleotide Polymorphism) loci and 513 Indel loci; the chip disclosed by the invention is high in cost performance and can be applied to academic research, such as genetic map construction and gene localization, population layering, population evolution, whole genome association analysis (GWAS) and the like; the method can be applied to breeding practices, such as molecular marker-assisted selection (MAS), whole genome selective breeding (GS), seed purity and authenticity identification, variety right protection and the like, and can meet the requirements of large-scale commercial breeding of the brassica napus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Brassica napus pan-genome liquid phase chip and an application thereof. Background Art

[0002] Brassica napus L., AACC, is one of the three major types of rapeseed. It originated in the Mediterranean region about 7,500 years ago. It is an allotetraploid plant formed by natural hybridization between Brassica rapa (AA, n = 10) and Brassica oleracea (CC, n = 9). Its chromosome set is AACC, containing 19 chromosomes. This crop can not only produce edible oil, but also its cake is rich in protein and can be used as animal feed. Brassica napus has undergone extensive variation during the long-term domestication and cultivation process. According to the low temperature requirement for flowering, rapeseed is divided into three ecological types: winter rapeseed (requires long-term low-temperature vernalization), spring rapeseed (does not require low-temperature vernalization or short-term low-temperature vernalization) and semi-winter rapeseed (requires a certain period of low-temperature vernalization).

[0003] With the rapid development of sequencing technology and the substantial reduction in costs, the whole genome sequences of a large number of species have been completed, which has greatly promoted the development of genome breeding. At present, gene chips have been developed for multiple crop species and applied in breeding. The known rapeseed chips all use the winter rapeseed "Darmor-bzh" as the reference genome, which is not suitable for China, which produces a large number of semi-winter rapeseed types. Liquid phase chips are based on resequencing technology to perform specific capture sequencing on each target site. Compared with solid phase chips, they have the advantages of high flexibility, low detection cost, high throughput detection capability, small sample usage, simple and fast operation, high sensitivity and accuracy.

[0004] The pan-genome of a species contains almost all the genetic information of the species. Two versions of the rapeseed pan-genome have been constructed. One is based on the genome Damor v8.1, which was obtained by second-generation sequencing and iterative assembly of 33 non-synthetic rapeseed germplasms and 20 artificial synthetic germplasms (reference: Hurgobin B, Golicz AA, Bayer PE, et al. Homoeologous exchange is a major cause of gene presence / absence variation in the amphidiploid Brassica napus. Plant Biote chnol J. 2018; 16(7): 1265-1274. doi: 10.1111 / pbi.12867); the other is based on the genome ZS11 v0, including the pan-genome constructed by the third-generation sequencing assembly sequences of 8 Brassica napus varieties (including winter, spring and semi-winter varieties), with a size of about 1.8 Gb and containing about 150,000 genes (reference: Song JM, Guan Z, Hu J, et al. Eight high-quality genomes rev eal pan-genome architecture and ecotype differentiation of Brassica napus. Nat Plants. 2020;6(1):34-45. doi:10.1038 / s41477-019-0577-7). Compared with the second generation sequencing, the third generation sequencing assembly provides higher assembly quality and more comprehensive genetic variation information in the construction of pan-genome. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a detection chip containing semi-winter, winter and spring rapeseed varieties, thereby solving the problem of insufficient basic population diversity of existing rapeseed chips.

[0006] In a first aspect, the present invention provides a locus combination of the whole genome of Brassica napus, wherein the locus combination of the whole genome of Brassica napus consists of 10414 loci, and the 10414 loci include 9901 SNP loci and 513 Indel loci;

[0007] The physical positions of the 10414 sites are determined based on the pan-genome sequence alignment of the semi-winter Brassica napus variety ZS11, and the pan-genome sequence based on the semi-winter Brassica napus variety ZS11 is the pan-referenc e.v0.fa in the Brassica napus pan-genome database BnPIR; the specific sites are as shown in the claims.

[0008] The website of the pan-genome sequence based on the semi-winter Brassica napus variety ZS11 is http: / / cbi.hzau.edu.cn / rape / downlo ad_ext / panrefgenome.fa.

[0009] In a second aspect, the present invention provides a probe composition, which is a probe set that specifically captures a combination of sites in the entire genome of Brassica napus.

[0010] In the above, the design process of the probe is: the probe length is 110bp, the probe GC content is between 30% and 70%, the number of homology regions is ≤5, and the selected region does not contain SSR and Gap regions as much as possible; two nucleotide sequences with 60% to 70% overlap and covering the SNP site are designed according to the SNP site obtained by screening; single-stranded nucleotide synthesis is performed according to the designed nucleotide sequence to synthesize two DNA nucleotide sequences with a length of 110bp and a biotin group modified at the 5' end. The above two synthesized probes are mixed with equal molar mass, and the probe mixture is fixed to 3pmol / mL using EDTA and Tris HCl mixed solution.

[0011] In a third aspect, the present invention provides a liquid phase chip for detecting the whole genome of Brassica napus, which comprises the probe composition described in the second aspect and a hybridization capture reagent.

[0012] In the above, the hybridization capture reagent in the embodiments of the present invention comes from the GenoBaits DNA seq Library Prep kit of Shijiazhuang Boridi Biotechnology Co., Ltd., including independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2×Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2×Beads Wash Buffer, GenoBaits 10×Wash Buffer I, GenoBaits 10X WashBuffer II, GenoBaits 10X Wash Buffer III, and GenoBaits 10X Str ingent Wash Buffer.

[0013] In a fourth aspect, the present invention provides the use of the site combination described in the first aspect or the probe combination described in the second aspect in the design, development or preparation of a gene chip for detecting whole genome sites of Brassica napus.

[0014] In the application described above, the gene chip is a liquid phase chip.

[0015] In a fifth aspect, the present invention provides the use of the site combination described in the first aspect, the probe combination described in the second aspect, or the liquid phase chip described in the third aspect in any of the following or in the preparation of a product having any of the following functions:

[0016] 1) Detection of genome-wide loci in Brassica napus;

[0017] 2) Detect the genotype of the target material;

[0018] 3) Differentiate between rapeseed types;

[0019] 4) Genetic map construction and gene localization;

[0020] 5) Group stratification

[0021] 6) Group evolution;

[0022] 7) Genome-wide association analysis;

[0023] 8) Marker-assisted selection;

[0024] 9) Whole genome selection breeding;

[0025] 10) Seed purity and authenticity identification;

[0026] 11) Protection of variety rights.

[0027] In a sixth aspect, the present invention provides a method for detecting whole genome loci of Brassica napus, comprising the following steps: using the liquid phase chip described in the first aspect to detect the Brassica napus to be detected, thereby detecting the whole genome loci of Brassica napus.

[0028] The existing rapeseed chips have insufficient basic population diversity, and winter rapeseed varieties are used as reference genomes, with few functional markers, high marker density, and high detection costs. Therefore, it is more ideal to build a pan-genome based on the representative semi-winter rapeseed variety "Zhongshuang 11 (ZS11)" as the development of Chinese Brassica napus breeding chips, which not only takes into account most semi-winter rapeseed populations, but also other types of rapeseed populations.

[0029] The present invention uses 2774 samples of Brassica napus (including winter, spring and semi-winter rapeseed) from around the world as the basic population, and the pan-genome constructed based on the semi-winter rapeseed ZS11 as the reference genome for breeding chip development. It uses a variety of site screening algorithms to extract core key sites, and integrates all reported important agronomic trait candidate genes and their functional sites. Finally, after multiple rounds of verification, the Brassica napus 10K liquid phase chip "BnaPan10K" is formed. The chip is cost-effective and can be applied to academic research, such as: genetic map construction and gene positioning, population stratification, population evolution, whole genome association analysis (GWAS), etc.; it can also be applied to breeding practices, such as: molecular marker-assisted selection (MAS), whole genome selection breeding (GS), seed purity and authenticity identification, variety rights protection, etc., which can meet the needs of large-scale commercial breeding of Brassica napus.

[0030] Compared with the prior art, the beneficial effects mainly include:

[0031] 1) The present invention uses whole genome sequencing data of 2774 different types of Brassica napus from all over the world, and uses the pan-genome constructed based on Double 11, a representative rapeseed variety in China, as the reference genome, making full use of all the genetic information of Brassica napus, taking into account both specificity and comprehensiveness.

[0032] 2) The prospective marker sites selected by the gene chip of the present invention are all functional sites, preferably cloned genes and their functional sites, GWAS significant association sites and functional sites in known chips, which can be used for molecular marker-assisted selection breeding.

[0033] 3) The background marker sites selected by the gene chip of the present invention are core markers carefully selected by integrating multiple algorithms such as site scoring system and LD block, and evenly cover the whole genome, which is equivalent to the effect of whole genome resequencing.

[0034] 4) The gene chip of the present invention adopts targeted genotyping by sequencing (GBTS) technology, and after three rounds of testing and verification, the core 10K loci are screened out, which has the advantages of low cost, high accuracy, and high detection sensitivity.

[0035] 5) The gene chip of the present invention takes into account both the foreground and background selection in rapeseed breeding, and can accelerate the breeding process when applied to rapeseed molecular marker-assisted breeding and whole genome selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Developed flow chart for BnaPan10K.

[0037] Figure 2Characteristics of BnaPan10K marker; (a) Chromosome distribution statistics of BnaPan10K marker; the horizontal axis is the chromosome / genome number; the left vertical axis represents the number of sites and segments; the right vertical axis represents the chromosome length; (b) MAF distribution statistics; the horizontal axis represents the different ranges of MAF values, and the vertical axis represents the number of sites within each MAF range; the larger the MAF value, the better the polymorphism of the site; (c) BnaPan10K marker type statistics; the horizontal axis represents the marker type classification, which is divided into 7 types including C / G, A / C, G / T, A / T, A / G, C / T and InDel; the vertical axis represents the number of sites under this type; (d) Distribution of BnaPan10K marker in gene structure; the horizontal axis represents the gene structure type, and the vertical axis represents the number of sites under this type.

[0038] Figure 3 The density distribution of different panels of rapeseed on genome chromosomes; (a) distribution of core markers on the ZS11 pan-genome; (b) distribution of all markers on the ZS11 pan-genome; (c) distribution of core markers mapped to the Darmor v4.1 genome; (d) distribution of chip Bnap us50K on Darmor v4.1; (e) distribution of core markers mapped to the Darmorv8.1 genome; (f) distribution of chip BnaPan50T on Darmor v8.1.

[0039] Figure 4 Figure 2. PCA of 1560 rapeseeds from different panels; (a) PCA results obtained from core markers; (b) PCA results obtained from all markers; the shapes of different points represent winter, semi-winter, spring, mixed and unknown types of rapeseed, and different colors represent clustering results.

[0040] Figure 5 Phylogenetic trees of 1560 rapeseed accessions were constructed for different panels; (a) Phylogenetic tree constructed using core markers; (b) Phylogenetic tree constructed using all markers; different colors represent winter, semi-winter, spring, mixed and unknown types of rapeseed.

[0041] Figure 6 The figure below shows information on 296 rapeseed varieties. The figure on the left shows the distribution statistics of different rapeseed types. The figure on the right shows the top 13 countries and regions with the most rapeseed varieties.

[0042] Figure 7 This is the distribution map of the detected sites in the BnaPan10K population; the figure shows the site types (reference sites, variant sites, heterozygous sites and missing sites) and their proportions of 21 samples among 296 rapeseed samples.

[0043] Figure 8PCA of 296 rapeseed materials using BnaPan10K and WGS data; (a) PCA results obtained using BnaPan10K; (b) PCA results obtained using WGS; different colors represent winter, semi-winter, and spring rapeseed, respectively.

[0044] Fig. 9 The phylogenetic tree of 296 rapeseed materials was constructed based on BnaPan10K and WGS data; (a) phylogenetic tree constructed by BnaPan10K; (b) phylogenetic tree constructed by WGS; different colors represent winter, spring, and semi-winter rapeseed. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0046] 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.

[0047] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0048] The synthesis of the probes and the liquid phase chip detection in the following examples were completed by Shijiazhuang Boredy Biotechnology Co., Ltd.

[0049] Example 1. Acquisition of loci combination of the whole genome of Brassica napus and preparation of rapeseed gene chip BnaPan10K for detecting the whole genome of rapeseed

[0050] 1. Acquisition of SNP and Indel target sites

[0051] First, we downloaded 2774 whole-genome resequencing data of rapeseed (about 23.5Tb) from all research reports (as of May 2023), and used the rapeseed pan-genome as the reference genome. The pan-genome sequence is pan-reference.v0.fa in the Brassica napus pan-genome database BnPIR (download URL: http: / / cbi.hzau.edu.cn / rape / download_ext / panrefgenome.fa, ​​referred to as the reference genome "ZS11-pan"; from this article: Song JM, Guan Z, Hu J, et al. Eight high-quality genomes reveal pa n-genome architecture and ecotype differentiation of Brassica napus. Nat Plants.2020;6(1):34-45.doi:10.1038 / s41477-019-0577-7), ZS11-pan is ~1.77Gb, the sequence size of 19 chromosomes mounted on ZS11 is 960.82Mb, the sequence size of unmounted chromosomes is 50.07Mb, the sequence size from other 3 semi-winter rapeseed (Gangan, Zheyou7, Shengli), 2 winter rapeseed (Tapidor, Quinta) and 2 spring rapeseed (Westar, synthetic rapeseed No2127) is 761.40Mb, and there are significant differences in flowering period, plant type and seeds among the eight rapeseed varieties. GATK best practice process was used for variation detection. The alignment rate, average depth, coverage and other indicators in the analysis process were statistically analyzed, and materials with an average sequencing depth greater than 3x and an average alignment rate greater than 98% were selected for subsequent chip site screening, and a total of 1,560 rapeseed materials were retained.

[0052] (1) Background site screening

[0053] First, the original variation of 1560 materials was hard filtered and population filtered, and the biallelic sites were retained. The sites were filtered according to the conditions of site missing rate <0.2, minor allele frequency (MAF)>0.05, and heterozygosity rate <0.1, and 1272484 sites were retained. Secondly, the ZS11-pan genome was masked for repeated sequences, and the sequences of 150bp upstream and downstream of each site were extracted. If there was a gap, the site was filtered. The GC content of the 150bp upstream and downstream of the remaining sites was calculated, and the target sites with GC content of 30-70% were retained. The sequences of 150bp upstream and downstream of the remaining sites were selected, and the ZS11-pan genome was aligned by BLAST. The sites with consistency, coverage greater than the threshold, and alignment results of no more than 5 homologous sequences were retained, and 424324 sites were retained. Then, a site scoring system was created to grade the importance of sites according to polymorphism information content (PIC) and SNP annotation information. PIC classification is based on the Anderson method (reference: Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet. 1980; 32 (3): 314-331.) The PIC value of each SNP is weighted. SNP annotation classification is based on the position of SNP in coding region and non-coding region. The weights obtained by combining the above two classification standards are added together to finally obtain the score value of each SNP. Finally, the haploid block (LD block) of the whole genome of all materials is calculated based on the remaining SNPs, and the SNPs with linkage disequilibrium with the adjacent sites are analyzed and calculated. According to the LD block interval threshold setting and the principle of uniform distribution of sites, the SNP with the highest score in the above site scoring system is selected as the tag SNP in the interval, and 10099 tag SNPs are finally selected as the background sites for screening by the sliding window method.

[0054] (2) Screening of prospect sites

[0055] For the screening of prospect sites of rapeseed gene chip, the present invention selects from two aspects: known functional sites and candidate gene variations. Functional sites are mainly derived from gene mining studies reported in rapeseed. After a large number of literature searches, 1249 sites associated with 28 GWAS studies based on Brassica60K chip were selected. 1489 sites (including 743 SNPs and 745 Indels) were found in 4 GWAS studies based on whole genome resequencing (WGS). In addition, functional sites in Bnapus50K were also comprehensively adopted. Damor v4.1 sites were converted to the ZS11-pan genome through genomic coordinate conversion.

[0056] Candidate genes include cloned genes and GWAS candidate genes. Through literature search and collation, a total of 150 cloned genes related to important agronomic traits such as rapeseed yield, quality, and resistance were collected, including important genes that have been verified by experiments or populations. There are a total of 1579 candidate genes (mostly unverified) reported in all GWAS studies. Find the homologous genes on Darmor v4.1 corresponding to ZS11-pan, and select all the variants in the transcription region of each cloned gene that have been verified; for unverified candidate genes, only the coding region variant sites are selected. Finally, there are 1888 sites that have been successfully converted to genomic coordinates, including 1375 SNPs and 513 Indels, which are prospect sites.

[0057] 2. Synthesis of probe

[0058] Probes are designed and synthesized for the background sites and foreground sites obtained in 1 above, with at least 2 capture probes for each site.

[0059] The design process of the probe is as follows: the probe length is 110 bp, the probe GC content is between 30% and 70%, the number of homology regions is ≤5, and the selected region does not contain SSR and Gap regions as much as possible; two nucleotide sequences with 60% to 70% overlap and covering the SNP sites are designed according to the SNP sites obtained by screening; single-stranded nucleotide synthesis is performed according to the designed nucleotide sequence to synthesize two DNA nucleotide sequences with a length of 110 bp and a biotin group modified at the 5' end. The above two synthesized probes are mixed with equal molar mass, and the probe mixture is fixed to 3 pmol / mL using EDTA and Tris HCl mixed solution.

[0060] The hybrid capture reagents were from the GenoBaits DNA seqLibrary Prep Kit of Shijiazhuang Boredy Biotechnology Co., Ltd., including independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2×Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2×Beads WashBuffer, GenoBaits 10×Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits 10XWash Buffer III, and GenoBaits 10X Stringent Wash Buffer. The sites were also verified during the probe evaluation, including the lowercase repetitive sequences in the genome 50 bp upstream and downstream of the core site, and the target sites with GC content of 30%-70% in the upstream and downstream 50 bp were retained. Based on the probe synthesis test data, the sites with poor detection performance were deleted, and finally 10414 sites were retained as the rapeseed gene chip BnaPan10K capture sites, including 9901 SNP sites and 513 Indel sites.

[0061] According to the different sources of the 10,414 sites screened, they are divided into levels 1-3 according to their importance: Level 1 sites mainly come from important sites in known rapeseed chips and functional sites of cloned genes in rapeseed, including 735 SNPs and 355 Indels; Level 2 sites mainly come from significant association sites reported in rapeseed whole genome association study GWAS studies, including 640 SNPs and 158 Indels; Level 3 sites mainly come from SNPs in candidate genes in GWAS studies and background sites of carefully selected populations, totaling 8,526 SNPs.

[0062] Distribution of 10414 loci in the rapeseed pan-genome: 4068 loci in the A subgenome (3732 SNPs and 336 Indels), 5487 loci in the C subgenome (5333 SNPs and 154 Indels), and 859 loci in the non-ZS11 genome (836 SNPs and 23 Indels).

[0063] Figure 1 Developed flow chart for BnaPan10K.

[0064] The 10414 site information of the BnaPan10K chip is separated by genome and chromosome and expressed in the format of "position_polymorphic site". The position in the site is the position of the polymorphic site in the reference genome, the base before the " / " is the base of the polymorphic site in the reference genome (i.e., the reference genotype), and the base after the " / " is the base of the polymorphic site after mutation (i.e., the variant genotype). For example, 124713 [T / G] in the ZS11 chromosome scaffoldA01 indicates the 124713th position of the chromosome scaffoldA01 in the ZS11 genome, the reference genotype is T, and the variant genotype is G. If the variant genotype of Indel is multiple types of variation, each variation type is separated by ","; such as 2316110[CTTTCTT / C,CTTCTT] in ZS11 chromosome scaffoldA01, which means that the Indel site is formed from position 2316110 of the reference genome ZS11 chromosome scaffoldA01, and the reference genotype of this site is CTTTCTT. After the variation, there are two variation forms: C (variant genotype) or CTTCTT (variant genotype); * in the variant genotype indicates that there may be other types of variation. In addition to the 19 chromosomes of the ZS11 genome (scaffoldA01-scaffoldA10, scaffoldC01-scaffoldC09), there are other sites from the pan-genome composed of eight genomes (damor.v4.chromosomes, ganganF73.genome, no2127.genome, quintaA.genome, shengli3.genome, tapidor3.genome, westar.genome, zheyou73.genome).

[0065] Therefore, the information of 10414 sites is as follows:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] 3. Probe testing

[0082] The first round of probe testing was performed on the 10414 loci marked by BnaPan10K. The testing process was as follows: leaves of 12 different Brassica napus varieties (from the literature: Wu D, Liang Z, Yan T, et al. Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence. Mol Plant. 2019; 12(1): 30-43. doi: 10.1016 / j.molp.2018.11.007) were taken and placed in the laboratory to dry naturally; DNA extraction, library construction and sequencing were performed according to the liquid phase chip experimental operation steps, and 10414 target loci were obtained through the standard variation detection process.

[0083] The site detection rate is one of the important indicators for measuring the quality of gene chips. In plants, it is generally measured based on the ratio of the number of product detection sites to the number of development sites.

[0084] Detection rate = percentage of detected sites / total number of sites

[0085] The results are shown in Table 1. It can be seen that, after testing and analysis, the average detection rate of the 12 samples tested in this round is 96.983%, indicating that the probe of 10414 sites of the present invention can be used for efficient detection of genotypes.

[0086] Table 1 shows the detection rate of the first round of probe testing sites

[0087]

[0088]

[0089] 4. Obtaining the rapeseed gene chip BnaPan10K

[0090] The rapeseed gene chip BnaPan10K includes probes (probe mixture) for detecting 10414 site combinations and hybridization capture reagents. Figure 2 Marking features for BnaPan10K.

[0091] 5. Comparative analysis of 10414 loci and 1560 rapeseed whole genome sequencing data

[0092] The 10,414 loci screened from the resequencing of 1,560 rapeseed genomes were compared with all the markers after hard filtering (about 18 million loci, or 18M) to evaluate the representativeness of the core loci. The distribution of about 10K core loci, all loci, and different types of rapeseed chips on chromosomes was compared to determine the uniformity of the loci.

[0093] The results are as follows Figure 3 As shown, the distribution of core sites on the pan-genome ZS11-pan is similar to that of all sites, and the Gap on ZS11 is related to its highly repetitive sequences; the solid phase chip Bnapus50K uses Darmor v4.1 as the reference genome. When the core sites on ZS11-pan are converted to Darmor v4.1, the marker density is evenly distributed; the liquid phase chip BnaPan50T uses the pan-genome constructed based on Darmor v8.1 as the reference genome. When the core sites on ZS11-pan are converted to Darmor v8.1, the marker density is evenly distributed.

[0094] Therefore, the 10,414 core loci markers 10K screened by the gene chip of the present invention can represent the marker density of 50K or even whole genome resequencing.

[0095] The smartpca tool of EIGENSOFT software was used to perform principal component analysis (PCA) on 10414 core loci and all markers of 1560 rapeseed samples. The results are as follows: Figure 4 As shown, the results obtained from the two analyses are basically consistent.

[0096] The neighbor-joining method was used to construct phylogenetic trees for the core loci and all markers of 1560 rapeseed samples. The results are as follows: Figure 5 As shown, the results obtained from the two analyses are basically consistent.

[0097] Example 2: Group testing and comparative analysis of BnaPan10K

[0098] A total of 296 Brassica napus varieties (such as Figure 6 shown), reference: Wu D, Liang Z, Yan T, et al. Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals theGenetic Basis of Ecotype Divergence. Mol Plant. 2019; 12(1): 30-43. doi: 10.1016 / j.molp.2018.11.007.

[0099] DNA extraction, library construction and sequencing were performed on the samples.

[0100] The liquid phase chip BnaPan10K constructed in Example 1 was used for genotype detection and whole genome resequencing at the same time, and the analysis results of the two were compared.

[0101] All markers of BnaPan10K were detected in 296 rape samples, with Q30>94% for all samples, average site detection rate>90%, and average correct alignment rate of about 90%. The site type results of 21 materials were as follows Figure 7 shown.

[0102] The smartpca tool of EIGENSOFT software was used to perform principal component analysis on the BnaPan10K and WGS data of 296 rapeseed samples. The results are as follows: Figure 8 As shown, the results of the two methods are similar, and both can clearly distinguish the three different types of rapeseed: spring, winter and semi-winter.

[0103] The phylogenetic tree was constructed using the neighbor-joining method for the BnaPan10K and WGS data of 296 rapeseed accessions. Fig. 9 As shown in the figure, the results of the two analyses are almost consistent and can basically classify different rapeseed types.

[0104] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A genome-wide locus combination of Brassica napus, which consists of 10,414 loci, including 9,901 SNP loci and 513 Indel loci; The physical positions of the 10414 loci are determined based on the pan-genome sequence alignment of the semi-winter Brassica napus variety ZS11, and the pan-genome sequence based on the semi-winter Brassica napus variety ZS11 is pan-referenc e.v0.fa in the Brassica napus pan-genome database BnPIR; The information of 9901 SNPs and 513 Indel sites in the 10414 sites is as follows:

2. A probe composition, which is a probe set that specifically captures the site combination of claim 1.

3. A liquid phase chip for detecting the whole genome of Brassica napus, comprising the probe composition according to claim 2 and a hybridization capture reagent.

4. Use of the site combination according to claim 1 or the probe combination according to claim 2 in designing, developing or preparing a gene chip for detecting whole genome sites of Brassica napus.

5. The use according to claim 4, characterized in that: The gene chip is a liquid phase chip.

6. Use of the site combination according to claim 1, the probe combination according to claim 2, or the liquid phase chip according to claim 3 in any of the following or in the preparation of a product having any of the following functions: 1) Detection of genome-wide loci in Brassica napus; 2) Detect the genotype of the target material; 3) Differentiate between rapeseed types; 4) Genetic map construction and gene localization; 5) Group stratification 6) Group evolution; 7) Genome-wide association analysis; 8) Marker-assisted selection; 9) Whole genome selection breeding; 10) Seed purity and authenticity identification; 11) Protection of variety rights.

7. A method for detecting whole genome sites of Brassica napus, comprising the following steps: using the liquid phase chip according to claim 3 to detect the Brassica napus to be detected, thereby detecting the whole genome sites of Brassica napus.

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