Brassica napus molecular marker combination, chip and application thereof

Abstract

The invention discloses a brassica napus molecular marker combination, a chip and application of the brassica napus molecular marker combination. The brassica napus molecular marker combination comprises at least one of 52,444 SNP (Single Nucleotide Polymorphism) / InDel molecular markers, and the physical positions of the 52,444 molecular markers are determined by carrying out sequence alignment on the basis of a brassica napus reference genome bnatusdarmorbzhv10 (GCA905183035.1). The brassica napus molecular marker combination provided by the scheme of the invention can be effectively used for molecular marker breeding of brassica napus, and the brassica napus whole genome liquid phase chip is prepared from the brassica napus molecular marker combination provided by the scheme of the invention; the application cost of genetic diversity analysis, QTL positioning, GWAS analysis and the like of the brassica napus in scientific research is effectively reduced, the problem that no applicable product exists in molecular assisted breeding and whole-genome selective breeding of the brassica napus is solved, and the process of basic research and breeding of the brassica napus is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Brassica napus molecular marker combination, a chip and applications thereof. Background Art

[0002] Rapeseed has multiple uses such as feed, vegetable, honey, sightseeing, and saline-alkali land improvement. At present, rapeseed breeding work mainly focuses on high yield, high quality, multi-resistance, suitable growth period, and suitable mechanized harvesting. However, the traditional breeding method of improving rapeseed target traits through phenotypic selection has a long breeding cycle, low efficiency, and poor controllability, which cannot meet the development needs of the rapeseed industry. With the widespread application of genome sequencing and molecular marker technology, more and more important agronomic trait genes such as rapeseed yield, quality, flowering, plant type and resistance have been located and cloned. However, traditional marker-assisted selection methods such as RFLP and SSR are expensive, inefficient, and time-consuming, and cannot be widely used in rapeseed molecular breeding. Compared with traditional molecular markers, SNP markers have the characteristics of high density, wide distribution range, and simple typing. In related technologies, solid-phase chips have the disadvantages of high detection cost, low flexibility, and difficulty in customization; and the disclosed rapeseed-related liquid-phase chips are difficult to be widely promoted due to low chip density, low detection rate, poor site representativeness, poor versatility, and single function. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a Brassica napus molecular marker combination.

[0004] The present invention also provides a chip for detecting the above-mentioned Brassica napus molecular marker combination.

[0005] The present invention also provides a kit for detecting the above-mentioned Brassica napus molecular marker combination.

[0006] The present invention also provides a method for screening the above-mentioned Brassica napus molecular marker combination.

[0007] The present invention also provides an application of the above-mentioned Brassica napus molecular marker combination, chip or kit.

[0008] The invention also provides a breeding method for Brassica napus.

[0009] According to one aspect of the present invention, a Brassica napus molecular marker combination is proposed, including 52,444 molecular markers, the physical positions of the molecular markers are determined based on sequence alignment of the Brassica napus reference genome bnapus_darmor_bzh_v10 (GCA_905183035.1), and the site information is specifically shown in Table 1 below:

[0010] Table 1

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[0034] In some embodiments of the present invention, the information of the molecular marker is expressed in the form of "physical position + ref / alt (reference base / variant base or insertion, deletion base sequence)".

[0035] In a second aspect of the present invention, a chip is provided, wherein the chip comprises a primer set and / or a probe for detecting the above-mentioned Brassica napus molecular marker combination.

[0036] In a third aspect of the present invention, a kit is provided, which comprises a primer set and / or a probe for detecting the above-mentioned Brassica napus molecular marker combination.

[0037] In a fourth aspect of the present invention, a method for screening the above-mentioned Brassica napus molecular marker combination is provided, and the screening method comprises the following steps:

[0038] S1. Compare the whole genome sequencing data of Brassica napus samples and detect variant sites, perform preliminary hard filtering, and obtain a file containing SNP / InDel variant information of all samples; screen the file to obtain candidate sites; the screening parameters include: MAF ≥ 0.1, missing rate < 0.1, heterozygosity rate ≤ 0.2, sequencing depth ≥ 8×;

[0039] S2. Functionally annotating and screening the candidate sites to obtain gene region sites; the screening is based on the mutation position of the candidate sites and the degree of their influence on gene function;

[0040] S3, obtaining loci in the gene region that are significantly associated with the trait;

[0041] S4, obtaining QTL loci;

[0042] S5. Integrate the molecular marker sites obtained in steps S1 to S4 to screen and obtain the Brassica napus molecular marker combination.

[0043] In some embodiments of the present invention, in step S1, the whole genome sequencing data of the Brassica napus sample is aligned and the variant sites are detected using Sentieon software.

[0044] In some embodiments of the present invention, step S1 further comprises selecting molecular markers that are evenly distributed on the chromosomes of Brassica napus.

[0045] In some embodiments of the present invention, the reference genome used for comparison is bnapus_darmor_bzh_v10 (GCA_905183035.1). Compared with other reference genomes, it is better in assembly quality, accuracy, completeness, gene annotation and repeated sequence processing, and the physical location of the sites obtained based on it is more realistic and the probe sequence is more accurate.

[0046] In some embodiments of the present invention, in step S1, the standard of hard filtering is as follows: QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0.

[0047] In some embodiments of the present invention, in step S1, the screening further comprises the following steps: designing probes to remove molecular marker sites corresponding to probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences.

[0048] In some embodiments of the present invention, in step S2, the priority of screening according to the mutation position of the candidate site is: exonic>splicing>UTR>intronic>upstream, downstream>intergenic.

[0049] In some embodiments of the present invention, in step S2, the priority of screening according to the degree of influence of the candidate sites on gene function is: HIGH>MODERATE>LOW.

[0050] In some embodiments of the present invention, in step S3, the traits include at least one of yield, growth and development, flowering period, quality, disease resistance, stress resistance, oil content, nutrient utilization, fertility, growth period, and flower color.

[0051] In some embodiments of the present invention, in step S4, a quantitative trait locus (QTL) positioning method is used to perform QTL positioning analysis on the trait to obtain a SNP site co-segregated with the trait or QTL; the trait includes at least one of oleic acid content, rapeseed quality, sterility, herbicide resistance, and disease resistance. The disease resistance includes at least one of resistance to black shank, resistance to clubroot, resistance to sclerotinia, and resistance to Fusarium.

[0052] In some embodiments of the present invention, the screening criteria in step S5 include at least one of quality index, annotated position of the site on the gene, site probe design quality, and site spacing.

[0053] In the fifth aspect of the present invention, the application of the above-mentioned Brassica napus molecular marker combination, chip and kit is proposed.

[0054] In some embodiments of the present invention, the application is in whole-genome selection breeding of Brassica napus, whole-genome association analysis of Brassica napus, germplasm resources and variety identification of Brassica napus, cluster analysis and kinship identification of Brassica napus, genetic background analysis of Brassica napus, genotyping detection of Brassica napus, molecular design breeding of Brassica napus or variety purity identification of Brassica napus.

[0055] In some embodiments of the present invention, the application can be specifically achieved by the following method: D1, using at least one of the Brassica napus molecular marker combination, chip and kit to genotype the Brassica napus to be tested to obtain a genotyping result; D2, analyzing the genotyping result obtained in step D1.

[0056] In the sixth aspect of the present invention, a breeding method for Brassica napus is proposed, comprising the following steps: using at least one of the above-mentioned Brassica napus molecular marker combination, chip and kit to detect the DNA of the Brassica napus to be tested, and selecting the desired Brassica napus for subsequent breeding.

[0057] In some embodiments of the present invention, the detection is performed based on liquid phase probe capture sequencing typing technology.

[0058] The present invention has at least the following beneficial effects:

[0059] The molecular marker combination of Brassica napus in the embodiment is developed based on 1,409 rapeseed germplasm resources data with rich global diversity. The developed population is more numerous and more representative. It has the advantages of strong representativeness, high polymorphism, good versatility, high coverage rate on the genome (average coverage rate 99.7%) and uniform distribution. It covers 1,981 gene function markers and trait / gene linkage markers related to important traits such as rapeseed growth and development, yield, quality, oil content, flowering period, fertility, growth period, disease resistance, and stress resistance, including 1,195 high-quality markers co-segregated with oleic acid content, rapeseed quality, sterility, herbicide resistance, disease resistance (black leg resistance, clubroot resistance, sclerotinia resistance, and Fusarium resistance) traits or QTLs; gene region sites are rich (gene region sites account for 43.2%). Compared with low-density chips, it can be used for molecular marker-assisted selection, directional improvement, important trait gene mining and identification, and functional analysis.

[0060] The Brassica napus 50K liquid phase chip based on the Brassica napus molecular marker combination of the embodiment has excellent overall performance, high genotype detection rate (99.29%), is designed based on a reference genome (bnapus_darmor_bzh_v10) with high assembly quality, has high accuracy and stability of genotyping results, and has a high amount of detection information. The existing low-density rapeseed chip can perform initial gene positioning, but also has great limitations in whole genome selection and whole genome association analysis. The Brassica napus 50K liquid phase chip of the present invention is conducive to more accurate identification of rapeseed germplasm resources, purity identification, genetic background analysis of breeding materials, genetic map construction and QTL positioning, molecular marker-assisted selection, directional improvement, whole genome association analysis, whole genome selection breeding, intelligent design breeding and other different application scenarios. It is a high-throughput, high-accuracy, low-cost, multi-functional molecular marker chip with a wide range of application scenarios. Compared with the solid-phase array of Brassica napus, it has lower detection cost and higher flexibility. The array sites can be increased as needed, the number of samples to be tested is more flexible, the amount of detection information is high, and all genetic variation sites within the target interval of the genome can be tested at one time. Genotyping of rapeseed materials based on high-throughput sequencing technology has the advantages of high detection throughput, large amount of one-time output data, and can cover the detection of nearly a thousand materials at the same time; it is also suitable for mainstream second-generation sequencing platforms such as Illumina and MGI, and has wide platform adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a flow chart for the preparation of a 50K liquid phase chip for Brassica napus;

[0062] Figure 2 is the statistical result of the functional sites in the Brassica napus molecular marker combination in Example 1 of the present invention;

[0063] Figure 3 The site annotation result of the Brassica napus 50K liquid phase chip in Example 1 of the present invention;

[0064] Figure 4 This is a locus chromosome density distribution map of the Brassica napus 50K liquid phase chip in Example 1 of the present invention;

[0065] Figure 5 This is a schematic diagram of the cGPS liquid chip process detection in Example 2 of the present invention;

[0066] Figure 6 is the detection result of the Brassica napus sample site detection rate in Example 3 of the present invention;

[0067] Figure 7 The average consistency rate test result of the repeated sample genotypes in Example 3 of the present invention;

[0068] Figure 8The phylogenetic tree analysis results of 21 rapeseed materials in Example 4 of the present invention;

[0069] Fig. 9 This is a cluster analysis diagram of 154 rapeseed materials in Example 5 of the present invention. DETAILED DESCRIPTION

[0070] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0071] Example 1

[0072] In this embodiment, a Brassica napus molecular marker combination and a Brassica napus 50K liquid phase chip designed based on the Brassica napus molecular marker combination are prepared. The preparation flow chart of the Brassica napus 50K liquid phase chip is as follows: Figure 1 shown.

[0073] 1. The screening process of the Brassica napus molecular marker combination is as follows:

[0074] S1. Acquisition of whole genome background sites:

[0075] 1,409 rapeseed germplasm resources and variety materials from China, Germany, France, Canada, Japan, the United States and other countries were collected. Among them, there are 787 winter rapeseed, 332 semi-winter rapeseed, and 290 spring rapeseed. The resequencing data of 1,409 rapeseed germplasm resources with rich global diversity were analyzed. The analysis process is as follows:

[0076] Sentieon was used to align reads to the corresponding rapeseed reference genome bnapus_darmor_bzh_v10 (GCA_905183035.1), position sorting and marking duplicate reads. The variant sites were detected for each sample to obtain the variant information of each sample. The gVCF of all samples was jointly analyzed to obtain the variant results of each individual in the population. To ensure the accuracy of SNP / InDel, the SNP / InDel sites obtained after the joint analysis were initially hard filtered (SNP hard filtering standard: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0"). The MAF value, detection rate, heterozygosity rate and sequencing depth of the loci were calculated and counted from the vcf file containing all sample variation information, and polymorphic molecular marker loci with minimum allele frequency MAF ≥ 0.1, deletion rate < 0.1, heterozygosity rate ≤ 0.2, and sequencing depth ≥ 8× were selected as initial candidate loci. All the above candidate loci were used for probe design. The probe design method was to design probes within 100 bp upstream and downstream of each locus, and the GC content of the probes ranged from 30% to 70%. The probes that could not be uniquely aligned on the genome and contained repetitive sequences in the flanking sequences were removed, and 879,733 molecular marker loci that could be used for chip development were screened. According to the principle of uniform distribution of loci on chromosomes, molecular marker loci that were evenly distributed on rapeseed chromosomes were selected as whole genome background loci.

[0077] S2. Gene region site screening: Use snpEff annotation software to annotate all candidate sites for site function, and screen candidate sites based on marker mutation positions and the degree of impact on gene function. The priority of screening molecular marker sites on gene mutation positions is: exonic>splicing>UTR>intronic>upstream, downstream>intergenic; the priority of molecular marker site impact is: HIGH>MODERATE>LOW. Screen sites from high to low according to the priority of site annotation.

[0078] S3. Screening of important functional loci: Collect the functional genes and significant trait-associated loci reported for important traits such as yield (20.5%), growth and development (18.6%), flowering period (14.4%), quality (12.2%), disease resistance (11.6%), stress resistance (11.3%), oil content (4.4%), efficient nutrient utilization (2.8%), fertility (1.7%), growth period (1.5%), and flower color (1.0%), extract the population index of gene region loci and significant trait-associated loci from the resequencing data, and retain 1,981 high-quality loci, including 1,270 significant trait-associated loci and 711 functional gene loci. See details. Figure 2 .

[0079] S4. QTL site screening: using Brassica 60K Illumina Infinium TM The SNP markers of the RIL population were analyzed by array genotyping chip, and a high-density genetic linkage map was constructed. The SNP markers were distributed on 19 linkage groups of the A and C genomes of Brassica napus. The QTL positioning method was used to locate the QTLs for oleic acid content, rapeseed quality, sterility, herbicide resistance, and disease resistance (resistance to black leg, clubroot, sclerotinia, and fusarium). A total of 1,195 high-quality markers co-segregating with these traits or QTLs were identified, and the sequences before and after the markers were aligned to the rapeseed reference genome bnapus_darmor_bzh_v10 to obtain the physical positions of the markers on the new reference genome. These markers can be used for marker-assisted selection of rapeseed.

[0080] S5. Integrate the molecular marker sites screened in steps S1 to S4. According to the quality indicators of these sites, the annotation positions of the sites on the gene, the design quality of the site probes, and the spacing between the sites, the high-quality molecular marker sites are preferentially retained according to the screening methods of steps S1 to S4. Finally, 52,444 molecular markers with strong representativeness, high polymorphism, good versatility, and uniform distribution on the chromosome are screened. The specific site information is shown in Table 1. Among them, there are 1,981 functional markers and trait / gene linkage markers related to important traits such as rapeseed growth and development, yield, quality, oil content, flowering period, fertility, growth period, disease resistance, and stress resistance, and 1,195 high-quality QTL markers associated with oleic acid content, rapeseed quality, sterility, herbicide resistance, disease resistance (resistance to black shank, clubroot, sclerotinia, and fusarium) traits and genes. There are 22,657 gene region sites (accounting for 43.2%).

[0081] 2. Preparation of Brassica napus 50K liquid phase chip:

[0082] Genotyping by Pinpoint Sequencing ofliguid captured target (cGPS) is based on an optimized thermodynamic stability algorithm model to design specific probes for target interval sequences, and uses synthetic specific probes to capture and enrich multiple different target sequences located at different genomic locations by liquid phase hybridization. The captured and enriched target intervals are then subjected to library construction and second-generation sequencing to obtain the genotypes of the SNP / InDel sites in the target region. The 52,444 selected molecular markers were developed into the Brassica napus 50K liquid phase chip using cGPS liquid phase chip technology. The site annotation results of the Brassica napus 50K liquid phase chip are shown below. Figure 3 As shown, the density distribution is Figure 4 The average coverage of 50K liquid phase microarray loci on the chromosome of Brassica napus is 99.7%, the average spacing is 16.6Kb, and the 50K loci are evenly distributed on each chromosome.

[0083] Example 2 Method for using the Brassica napus 50K liquid phase chip

[0084] This example provides a method for using the Brassica napus 50K liquid phase chip prepared in the above-mentioned Example 1. Figure 5 The specific steps are as follows:

[0085] (1) Extraction and quality control of genomic DNA: The sample DNA was extracted using the magnetic bead method and the quality of the DNA sample was tested. The quality test included measuring the DNA concentration using a Qubit fluorescence quantifier and testing the integrity of the DNA using 1% agarose gel electrophoresis. Samples that passed the quality test were used for library preparation.

[0086] (2) Construction and quality control of cGPS library: a. Use fragmentase to digest DNA samples, repair the digestion ends, add A base to the 3' end, and use agarose gel electrophoresis to detect the fragment size. b. Use T4 ligase to connect sequencing adapters and DNA fragments, and use magnetic beads to purify the ligation products. The concentration of the purified products is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. c. Perform PCR amplification on the purified ligation products, and use magnetic beads to screen the fragments of the amplified products. Use Qubit fluorescence quantification instrument to detect the concentration of the fragment screening products, and use agarose gel electrophoresis to detect the fragment size. d. Take 200 ng of the constructed library, add probes and hybridization reagents, and incubate at 50°C for 16 to 24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to wash the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. The library concentration was detected by Qubit fluorescence quantification instrument, and the fragment size was detected by agarose gel electrophoresis. After the concentration and fragment size were qualified, the construction of cGPS sequencing library was completed. The prepared library was sequenced by BGI sequencer for high-throughput sequencing, and the sequencing strategy was PE150.

[0087] (3) Data analysis: The raw data after high-throughput sequencing was processed by quality control filtering, and the adapter fragments and low-quality reads were removed using FASTP software to obtain high-quality Clean Reads. The obtained Clean reads were aligned with the reference genome using BWA software, and the positions were sorted to obtain the bam file after the sample was sorted. The sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target sites.

[0088] Example 3 Evaluation of genotyping effect of Brassica napus 50K liquid phase chip

[0089] To verify the genotyping effect of the Brassica napus 50K liquid phase chip, the Brassica napus 50K liquid phase chip prepared in Example 1 was used to perform genotyping detection on 60 rapeseed materials (including 3 duplicate materials) (see Example 2 for the specific operation method).

[0090] The results are as follows Figure 6 and Figure 7 As shown. After sequencing and data analysis, the site detection rate of 60 rapeseed materials was between 97.62% and 99.90%, and the average detection rate was 99.29%. The genotype consistency rate of repeated samples was between 99.80% and 99.84%, and the average consistency rate was 99.82%. This shows that when the Brassica napus 50K liquid phase chip is used for genotyping of the materials to be tested, the target site detection rate is high, the stability is good, and the typing results are accurate and reliable.

[0091] Example 4 Application of Brassica napus 50K liquid phase chip in analysis of genetic differences in Brassica napus

[0092] The Brassica napus 50K liquid phase chip prepared in Example 1 and the genotyping process of Example 2 were used to identify the genotypes of 21 local Brassica napus materials in Hunan with small genetic differences to determine whether they can be used for genetic difference analysis between similar Brassica napus materials. The genetic distance matrix of the 21 Brassica napus materials was calculated using Plink software and a phylogenetic tree was drawn. The length of the branches in the phylogenetic tree can indicate the degree of genetic change during the evolution of the material. Figure 8 As shown in the figure, there are different genetic differences among the 21 Brassica napus materials, indicating that the 50K molecular marker combination of Brassica napus can effectively distinguish the genetic differences between similar Brassica napus materials.

[0093] Example 5 Application of Brassica napus 50K liquid phase chip in the analysis of phylogenetic relationships of Brassica napus materials

[0094] The Brassica napus 50K liquid phase chip prepared in Example 1 was used to perform genotype identification on 154 Brassica napus materials (the specific operation method is shown in Example 2), and the genetic distance matrix of the 154 Brassica napus materials was calculated using Plink software and cluster analysis was performed.

[0095] The results are as follows Fig. 9 As shown in the figure, the test materials are mainly divided into five subgroups. This shows that the SNP molecular marker combination contained in the Brassica napus 50K liquid phase chip is highly representative and can be used to determine the kinship, evolutionary relationship, composition structure, etc. of different materials.

[0096] Example 6 Application of Brassica napus 50K Liquid Phase Chip in Brassica napus Backcross Breeding

[0097] The Brassica napus 50K liquid phase chip prepared in Example 1 was used to perform genotyping on two rapeseed materials (see Example 2 for the specific operation method), one of which was the recurrent parent and the other was the BC2 recurrent strain. The background recovery rate was calculated by comparing the genotype data of the two rapeseed materials to be 87.37%, and the background recovery rate = 1-the number of differential sites between the recurrent progeny and the recurrent parent / the number of differential sites between the donor parent and the recurrent parent. The Brassica napus 50K liquid phase chip can effectively distinguish the differential segments between the recurrent parent and the recurrent strain, thereby achieving targeted improvement of the target segment.

[0098] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A Brassica napus molecular marker combination, characterized in that: It includes 52,444 molecular markers, the physical positions of which are determined by sequence alignment based on the rapeseed reference genome bnapus_darmor_bzh_v10 (GCA_905183035.1), and the site information is specifically shown in Table 1 in the specification.

2. A chip, characterized in that: The chip comprises a primer set and / or a probe for detecting the Brassica napus molecular marker combination as claimed in claim 1.

3. A kit, characterized in that: The kit comprises a primer set and / or a probe for detecting the Brassica napus molecular marker combination as claimed in claim 1.

4. A method for screening a combination of molecular markers for Brassica napus according to claim 1, characterized in that: The screening method comprises the following steps: S1. Compare the whole genome sequencing data of Brassica napus samples and detect variant sites, perform preliminary hard filtering, and obtain a file containing SNP / InDel variant information of all samples; screen the file to obtain candidate sites; the screening parameters include: MAF ≥ 0.1, missing rate < 0.1, heterozygosity rate ≤ 0.2, sequencing depth ≥ 8×; S2. Functionally annotating and screening the candidate sites to obtain gene region sites; the screening is based on the mutation position of the candidate sites and the degree of their influence on gene function; S3, obtaining loci in the gene region that are significantly associated with the trait; S4, obtaining QTL loci; S5. Integrate the molecular marker sites obtained in steps S1 to S4 to screen and obtain the Brassica napus molecular marker combination.

5. The screening method according to claim 4, characterized in that In step S1, the hard filtering standard is as follows: QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.

0.

6. The screening method according to claim 4, characterized in that In step S1, the screening further includes the following steps: designing probes to remove molecular marker sites corresponding to probes that cannot be uniquely aligned on the genome and whose flanking sequences contain repetitive sequences.

7. The screening method according to claim 4, characterized in that In step S2, the priority of screening according to the mutation position of the candidate site is: exonic>splicing>UTR>intronic>upstream, downstream>intergenic; And / or, in step S2, the priority of screening according to the influence degree of the candidate sites on gene function is: HIGH>MODERATE>LOW.

8. The screening method according to claim 4, characterized in that In step S3, the traits include at least one of yield, growth and development, flowering period, quality, disease resistance, stress resistance, oil content, nutrient utilization, fertility, growth period, and flower color.

9. Use of at least one of the Brassica napus molecular marker combination according to claim 1, the chip according to claim 2, and the kit according to claim 3 in any one of A1) to A8): A1) Whole genome selection breeding of Brassica napus; A2) Genome-wide association analysis of Brassica napus; A3) Germplasm resources and variety identification of Brassica napus; A4) Cluster analysis and identification of genetic relationships of Brassica napus; A5) Genetic background analysis of Brassica napus; A6) Genotyping detection of Brassica napus; A7) Molecular design breeding of Brassica napus; A8) Identification of the purity of Brassica napus.

10. A method for breeding Brassica napus, characterized in that: The method comprises the following steps: using the Brassica napus molecular marker combination as claimed in claim 1, the chip as claimed in claim 2 and at least one of the kits as claimed in claim 3 to detect the DNA of the Brassica napus to be detected, and selecting the desired Brassica napus for subsequent breeding.

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