Development and application of brassica napus 1.2 K liquid phase chip
By developing a gene chip with a 1.2K Brassica napus SNP/InDel site combination and using multiplex PCR and bioinformatics methods, the lag problem of Brassica napus breeding chips was solved, efficient and low-cost genotype detection and agronomic trait positioning were achieved, supporting molecular breeding technology selection.
Patent Information
- Application Number
- CN202510975806.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies lag significantly behind in the development of molecular markers and breeding chips for Brassica napus, are costly, and are difficult to efficiently assist in breeding, variety intellectual property protection, seed purity identification, and hybrid vigor classification.
A 1.2K Brassica napus SNP/InDel locus combination was developed. Using multiplex PCR targeted capture sequencing technology combined with bioinformatics methods, a Brassica napus gene chip was designed and prepared, containing 1,150 SNP/InDel loci for genome-wide association analysis and linkage analysis, achieving efficient detection and precise positioning of agronomic traits.
It realizes efficient and low-cost genotype detection, significantly shortens the breeding cycle, improves detection accuracy and flexibility, is applicable to multiple sequencing platforms, and supports the selection of excellent varieties using molecular breeding technology.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of gene chips, and particularly relates to development and application of a 1.2K Brassica napus liquid chip. BACKGROUND
[0002] Brassica napus is one of the most important oil crops in the world, and genotype detection on a large number of germplasm resources by means of high-efficiency genotyping technology is an important basis for realizing efficient and accurate molecular breeding. With the rapid development of DNA sequencing technology, DNA breeding chips based on SNP markers optimized and screened according to target breeding traits have become a core tool for efficient genotyping. In the field of rice breeding, 44K SNP and 6K SNP breeding chips have been widely used in genetic identification of germplasm resources and background analysis of breeding materials; in the research of melon, through evolutionary analysis of 16K SNP and 2K SNP chip sets, it is clear that the 2K SNP chip can maximize the coverage of genetic diversity while minimizing the number of markers, and accordingly developed Melon2K melon liquid breeding chip provides technical support with high efficiency and accuracy for genetic analysis and molecular breeding of melon.
[0003] Brassica napus is a complex allopolyploid (AACC) with a complicated genome, and its progress in molecular marker development and breeding chip research and application is significantly lagging behind that of diploid crops such as rice, wheat and corn. In recent years, with the rapid development of bioinformatics technology and the continuous reduction of high-throughput sequencing cost, researchers have used liquid target capture technology to construct a Brassica napus liquid breeding chip containing 54,765 SNP markers, BnaPan50T. The chip has a high use cost, therefore, developing a small Brassica napus breeding chip can effectively assist breeders in molecular marker-assisted breeding, variety property protection, seed purity and authenticity identification, and hybrid advantage division. SUMMARY
[0004] The purpose of the present application is to develop a 1.2K Brassica napus SNP / InDel site combination by using the target capture sequencing technology of multiplex PCR, and the site information is shown in Table 1 and Table 2.
[0005] Another purpose of the present application is to provide the application of the above-mentioned SNP site combination.
[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical measures:
[0007] The protection scope of the present application includes:
[0008] A 1.2K SNP / InDel site combination of Brassica napus, wherein the site information is shown in Table 1 and Table 2, wherein ID is site number, A01-A10 and C01-C09 in the site number represent the chromosome where the site is located, the numerical value is the physical position of the site on the chromosome, ref / alt, ref represents the base genotype of the site on the Brassica napus reference genome, alt represents the genotype after base mutation of the site, if alt is blank, the site is a missing base.
[0009] The genome of the present application refers to Zhongshuang 11 v0 genome (https: / / yanglab.hzau.edu.cn / BnIR / germplasm_info?id=ZS11.v0).
[0010] The Brassica napus gene chip prepared from the above SNP / InDel site combination.
[0011] The application of the above SNP / InDel site combination or chip, wherein the application is selected from any one of the following:
[0012] (1) application in Brassica napus genotyping;
[0013] (2) application in Brassica napus genetic map construction;
[0014] (3) application in Brassica napus material genetic relationship analysis;
[0015] (4) application in Brassica napus material variety authenticity identification;
[0016] (5) application in Brassica napus trait-related gene positioning;
[0017] (6) application in Brassica napus population classification;
[0018] (7) application in Brassica napus germplasm improvement;
[0019] (8) application in Brassica napus germplasm resource protection; or
[0020] (9) application in Brassica napus QTL positioning.
[0021] Preferably, in the above-mentioned application, the trait in the Brassica napus trait-related gene positioning includes one or more of yield, oil content, quality, flowering date or disease resistance.
[0022] Compared with the prior art, the present application has the following advantages:
[0023] (1) Using the Brassica napus genome and resequencing data, we used bioinformatics methods to identify specific regions at the genome-wide level and developed a SNP / InDel chip. This chip can be used for genome-wide association and linkage analysis, enabling efficient detection of associations between variant loci and phenotypes, and accurately locating QTLs related to important agronomic traits such as yield and oil content.
[0024] (2) The liquid phase chip is developed based on targeted capture technology. The principle is to design probes and sample DNA to achieve targeted capture and sequencing through base complementary pairing, so as to complete the genetic detection and typing of the target area.
[0025] (3) Compared with solid-phase arrays, the 1.2K Brassica napus liquid-phase array has high detection accuracy, more effective information, high flexibility and diversity, is applicable to a wider range of sequencing platforms, has a shorter test cycle, and significantly reduces detection costs. In addition, the array contains 35 foreground detection sites and 1115 background detection sites, which efficiently integrates and utilizes molecular marker information from different platforms, and mines the variant sites of target traits through genome-wide association and linkage analysis. At the same time, through molecular marker-assisted selection, it can accelerate the screening and improvement of target traits, thereby shortening the breeding cycle. This provides important technical support for the genetic improvement of Brassica napus and helps promote the selection of excellent Brassica napus varieties using molecular breeding technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a distribution diagram of the 1150 SNP / InDel marker detection sites on chromosomes in the present invention.
[0027] Figure 2 This is a statistical diagram of the distribution of SNP / InDel markers in the gene structure.
[0028] Figure 3 Build a pipeline for your library.
[0029] Figure 4 It is a statistical graph of minor allele frequency (Minor Allele Frequency) and polymorphism information content (Polymorphism Information Content).
[0030] Figure 5 This is a statistical chart of the impact caused by SNP / InDel related mutation sites.
[0031] Figure 6 This is a phylogenetic tree analysis of 51 Brassica napus materials using the breeding chip of the present invention.
[0032] Figure 7The correlation between the high and low oil content of 17 rapeseed samples and the actual oil content of the samples is analyzed using the breeding chip of the application. DETAILED DESCRIPTION
[0033] The technical solutions of the application will be described below in connection with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application. The genome of the application refers to the Zhongshuang 11 v0 genome (https: / / yanglab.hzau.edu.cn / BnIR / germplasm_info?id=ZS11.v0).
[0034] Embodiment 1
[0035] Development of Brassica napus 1.2K mGPS liquid chip
[0036] I. Screening of Brassica napus 1.2K genotyping SNP / InDel sites
[0037] 1) Background site screening
[0038] a. Initial selection of candidate sites: The selection of chip sites is based on the resequencing data of 350 diverse Brassica napus germplasm resources. The quality indicators of resequencing sites are calculated, and SNP polymorphic sites that meet the heterozygosity ≤0.1, site deletion frequency ≤0.1, MAF ≥0.3, and sequencing depth ≥30x in all sample populations are selected as initial candidate sites, a total of 457,373 initial candidate sites are screened.
[0039] b. Screening of gene region sites: The initial candidate sites are annotated using snpEff annotation software, and the candidate sites are screened according to the marker mutation position and the influence degree on gene function. The priority of the molecular marker site in the gene mutation position is: exonic>splicing>UTR>intronic>upstream, downstream>intergenic; the priority of the influence degree of the molecular marker site is: HIGH>MODERATE>LOW. The sites are screened according to the priority of site annotation from high to low.
[0040] c. Site density screening: According to the principle of uniform distribution of sites, SNP sites uniformly distributed on chromosomes are selected as whole genome background sites.
[0041] d. Site-specific primer design and copy number analysis: Primer design and copy number analysis were performed on the selected sites. Primers with high copy numbers, dimers, and hairpin structures were removed. Ultimately, 1,115 specific primer pairs were retained for mGPS system development and validation.
[0042] In the table of the present invention, ID is the site number, in which A01 to A10 and C01 to C09 indicate the chromosome where the site is located, and the numerical value is the physical position of the site on the chromosome. In ref / alt, ref indicates the base genotype of the site on the Brassica napus reference genome, and alt indicates the genotype after the base mutation of the site. If alt is blank, the site is a missing base.
[0043] For example, ID: A01-294869 in Table 1 represents base 294869 of chromosome A01 of Brassica napus, which is C in the Zhongshuang 11 v0 genome and becomes G after mutation.
[0044] Table 1 Information of 1115 background sites
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
[0057]
[0058]
[0059] 2) Determination of important functional sites (prospect sites)
[0060] According to the published functional site information, the primers are designed according to the primer design method and principle in 1), and the qualified sites are screened. Finally, 35 functional markers related to important traits of rape yield, oil content, quality, flowering period, disease resistance, etc. are reserved, of which 34 are derived from published patents, and the phenotypes are derived from the data in the published patent texts, and 1 is a new site A09-64849648 related to rape oil content resistance. The site is derived from the CLE20 gene, which is highly expressed in rape seeds. By cloning the CLE20 promoter sequence in rape with different oil contents, it is found that the sequence difference located on the promoter of the gene is related to oil content. Based on this, it is found that the A09 chromosome of ZS11 v0 at the position of 64,849,648 exists a mutation from A to G, thereby affecting the oil content. Randomly select 66 of 350 germplasm resources to detect the site genotype and oil content. When the site is AA (32 germplasm resources), the average oil content of the material is 45.68%, and when the site is GG (34 germplasm resources), the average oil content of the material is 43.66%.
[0061] Table 2 35 prospective site information
[0062]
[0063]
[0064]
[0065] Based on the sites obtained in Tables 1 and 2 above, the applicant obtained a set of 1150 SNP / InDel sites, which were used for the preparation of the following chip.
[0066] II. Technical principle and preparation of Brassica napus 1.2K mGPS liquid chip
[0067] 1) Targeted capture sequencing technology based on multiplex PCR, specific primers are designed for multiple target intervals in the reference genome, and multiple different target sequences are specifically amplified by multiplex PCR, then the PCR amplicon is sequenced library construction and second-generation sequencing, thereby obtaining the genotype of all SNP / InDel sites in the target interval.
[0068] 2) Integrate the whole genome background site and the functional site, synthesize the 1,150 specific primers screened, and develop Brassica napus 1.2K mGPS liquid chip by using the mGPS liquid chip technology independently developed by Huazhi. The specific site information is shown in Table 2. Figure 1
[0069] 3) Analysis of the position of the sites in the chip on the genome using R packages (GenomicRanges, rtracklayer, ChIPseeker, GenomicFeatures and dplyr) showed that 53.6% were located on exons, 14.2% were located upstream of genes, 7.5% were located in intergenic regions Figure 2
[0070] Example 2
[0071] Application of Brassica napus 1.2K mGPS liquid chip in genotyping
[0072] 1) Sample preparation and DNA
[0073] Fifty-two rapeseed germplasms from the 350 natural populations in Example 1 were selected as test materials, four of which were set as a duplicate group, ZS-11 was derived from two different units, so it was marked differently, a total of 56 materials were used as test samples, leaf blades were used to extract genomic DNA by CTAB method, and the concentration, integrity and purity of DNA were detected by agarose gel electrophoresis combined with Nanodrop.
[0074] 2) Library construction and sequencing
[0075] After the extracted DNA samples were qualified, the library construction was carried out according to the library construction process Figure 3 ). After the library construction was completed, Aglient 2100 / Bioanalyzer was used for library fragment size detection, qPCR / Qubit was used for library accurate quantification, and Nanodrop was used for library contaminant detection. After passing the quality inspection, the library was sequenced on the MGI high-throughput sequencing platform, and the sequencing read length was PE150.
[0076] 3) Data analysis
[0077] After the raw data of high-throughput sequencing was filtered and processed, the Reads containing adapter contamination and low-quality Reads were removed. BWA software was used for alignment with the reference genome, and Freebayes software was used for variant site analysis of the sequencing results. IGV was used to compare with the reference genome to view the variation. Freebayes was used to detect and filter SNPs.
[0078] 4) SNP site analysis
[0079] The MAF (Minor Allele Frequency) and PIC (Polymorphism Information Content) of each SNP site were analyzed using vcftools and PowerMarker software, respectively. The results showed that the MAF value was between 0.06 and 0.5, and the MAF value of 80.5.2% sites was greater than 0.3; the PIC value was between 0.1 and 0.4, and the PIC value of 88.6% sites was greater than 0.3 Figure 4 ), which indicated that the diversity of the chip was high. The harmful degree of the variation site was evaluated using snpEFF, and 13.5% of the sites had high impact after mutation Figure 5 ).
[0080] In this embodiment, the test results include various indicators of the target region, various indicators of the probe region, and coverage of different regions, etc., which comprehensively and in detail show the performance of the chip. The average site detection rate of the sites of the chip in 56 rapeseed germplasms was 96.77%, and the detection rate of two samples of Zhongshuang 11 was 100%, and the average consistency rate of the genotypes of the repeated samples was 99.15%. It is proved that the deletion rate of the liquid phase chip is low, the stability is good, and the genotype is accurate.
[0081] Table 3 Verification results of Brassica napus 1.2K mGPS chip
[0082]
[0083]
[0084] Example 3
[0085] Application of Brassica napus 1.2K mGPS liquid phase chip in kinship analysis
[0086] 1) The detection results of the Brassica napus liquid phase chip on 51 rapeseed germplasm materials (350 were randomly selected) in Example 2 were analyzed, the target site genotyping was extracted, and the SNP detection results were annotated using ANNOVAR software.
[0087] 2) The distance matrix was calculated using TreeBeST (http: / / treesoft.sourceforge.net / treebest.shtml) software, and based on this, a phylogenetic tree was constructed by the neighbor-joining method. The bootstrap values were obtained after 1000 calculations, and the phylogenetic tree analysis results showed that the chip could distinguish 51 materials Figure 6 ).
[0088] Example 4:
[0089] Application of Brassica napus 1.2K mGPS liquid chip in identification of oil content of Brassica napus
[0090] Seventeen Brassica napus germplasms with significant difference in oil content (provided by breeding units, not the 350 germplasms used in Example 1) were selected for chip detection, and the oil content level was predicted based on the typing results of the oil content related sites. Subsequently, the actual oil content level and the prediction results were associated for analysis, as shown in Table 4, the two showed a significant positive correlation, indicating that the chip can be applied to the identification of oil content of Brassica napus. Figure 7
[0091] Table 4 Identification results of oil content of 17 Brassica napus varieties
[0092]
[0093]
[0094] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.
Claims
1. A genome-wide 1.2K SNP / InDel locus combination in Brassica napus, wherein the locus information is shown in Tables 1 and 2. ID is the locus number. In the locus number, A01-A10 and C01-C09 indicate the chromosome where the locus is located, and the value is the physical position of the locus on the chromosome. In ref / alt, ref indicates the base genotype of the locus on the Brassica napus reference genome, and alt indicates the genotype after the base mutation of the locus. If alt is blank, the locus is missing a base.
2. A Brassica napus gene chip prepared using the SNP / InDel site combination according to claim 1.
3. Use of the site combination according to claim 1 or the chip according to claim 2, wherein the use is selected from any one of the following: (1) Application in genotyping of Brassica napus; (2) Application in the construction of genetic map of Brassica napus; (3) Application in the analysis of genetic relationships of Brassica napus materials; (4) Application in authenticity identification of Brassica napus varieties; (5) Application in the mapping of genes related to traits in Brassica napus; (6) Application in classification of Brassica napus populations; (7) Application in improving Brassica napus germplasm resources; (8) Application in the protection of Brassica napus germplasm resources; or (9) Application in QTL mapping in Brassica napus.
4. The use according to claim 3, characterized in that The traits targeted by the Brassica napus trait-related genes include one or more of yield, oil content, quality, flowering period or disease resistance.