Brassica napus 1.6 k whole genome molecular marker combination and application thereof

Abstract

The invention relates to the technical field of brassica napus gene detection, in particular to a brassica napus 1.6 k whole genome molecular marker combination and application thereof. According to the invention, SNP loci of resequencing data of 624 important rape germplasm resources are analyzed, 1597 high-quality whole genome molecular markers and gene function linkage markers with high polymorphism, high detection rate and uniform distribution on chromosomes are screened, specific primer design is carried out on the screened SNP / INDEL loci, and the SNP / INDEL molecular markers can be used for screening the whole genome molecular markers and gene function linkage markers. Specific amplification is simultaneously carried out on thousands of target sites in the same PCR reaction by utilizing a specific primer through multiple PCR, and then sequencing library construction and next-generation sequencing are carried out on PCR amplicons, so that genotypes of different target sites are obtained. The kit is used for detecting the SNP locus of the brassica napus, is high in sample detection flexibility, low in cost and short in period, and can be popularized to seed production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection of Brassica napus, and in particular to a Brassica napus 1.6k whole genome molecular marker combination and application thereof. Background Art

[0002] Rapeseed is one of the important oil crops in my country, with an annual planting area of ​​7 million hectares. 2 The annual oil production is 5.2 million tons, accounting for about 47% of the total domestic vegetable oil production, and is the largest domestic edible vegetable oil source. my country has a long history of rapeseed cultivation and utilization, but the yield level is low and the quality is poor. By effectively utilizing excellent rapeseed resources at home and abroad and continuously cultivating high-yield, disease-resistant and high-quality new varieties, the yield level has increased from 487.5kg / hm2 in 1949 to 2 Increased to 1995.2kg / hm2 in 2017 2 The quality of rapeseed has been improved from "high erucic acid and high glucosinolate" to "low erucic acid and low glucosinolate", and the nutritional quality is comparable to olive oil, with significantly improved nutritional and utilization value. In the development of this series of rapeseed, on the one hand, the discovery and utilization of excellent germplasm played a fundamental role; on the other hand, the rapid development of molecular breeding technology promoted the rapid development of rapeseed breeding.

[0003] In recent years, the molecular marker-assisted selection (MAS) technology has been increasingly used in rapeseed breeding, especially molecular markers based on single nucleotide polymorphism (SNP). SNP markers have the characteristics of wide distribution, high stability, and low mutation rate, and are suitable for genome-wide association analysis and trait positioning. However, in practical applications, the existing SNP marker system still faces some challenges, such as insufficient number of markers, uneven distribution, insufficient specificity and sensitivity, etc., especially in rapeseed breeding for specific environmental conditions in Jiangsu and Zhejiang regions, these problems are more prominent.

[0004] The existing technologies that support whole-genome SNP selection in rapeseed breeding are mainly sequencing and solid-phase chip detection. Sequencing is a four-step process: DNA extraction, library construction, on-machine sequencing, and data analysis to detect the entire genome sequence of a sample. The cost is relatively high, generally more than 500 yuan per sample, and the cycle is relatively long, 30 days. In addition, the amount of data generated is large, and professional analysts are required to handle it, making it difficult to popularize and apply it to seed breeding production selection. The chips are mainly imported Illumina solid-phase chips or Affymetrix chips. Through four steps: DNA extraction, library construction, chip hybridization scanning, and data analysis, the SNP genotype of the designed site is obtained, usually with a density of 10K-60K and a specification of 96 format or 384 format. The cost is relatively high, more than 400 yuan per sample, and the flexibility is poor. The format must be met for detection, and the cycle is short, 15 days.

[0005] In response to these problems, the Chinese invention patent with publication number CN115992292A proposed a SNP molecular marker combination and its application for Brassica napus. The marker combination consists of 1,407 SNP markers, which show high quality and high polymorphism after screening, have good specificity, sensitivity and resolution, are not affected by environmental factors, and can be applied to different plant tissues and sample types. The 1.5K SNP chip developed by it can achieve rapid, high-throughput, and low-cost detection, which is suitable for SNP analysis and assisted breeding of the whole genome of Brassica napus.

[0006] Although the above technical solutions have provided important progress in the whole-genome molecular marker detection of Brassica napus, with the increasing demand for rapeseed breeding, especially the increasing demand for screening varieties with stronger adaptability in Jiangsu and Zhejiang, there is still a need to further improve the coverage of markers and their selectivity for soil and climate adaptability in Jiangsu and Zhejiang. Therefore, it is necessary to construct a set of 1.6k whole-genome molecular marker combinations of Brassica napus suitable for Jiangsu and Zhejiang based on existing technologies, so as to more comprehensively cover the key areas in the rapeseed genome, thereby improving the selection accuracy of markers and breeding efficiency. This new marker combination will not only provide a more efficient auxiliary selection method for the optimization of rapeseed varieties in Jiangsu and Zhejiang, but also help promote the rapid cultivation of new varieties of rapeseed with high adaptability, high yield and high quality. Summary of the invention

[0007] In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a 1.6k whole genome molecular marker combination of Brassica napus, by analyzing the SNP sites of the resequencing data of 624 important rapeseed germplasm resources, screening high polymorphism, high detection rate, high quality whole genome molecular markers and gene function linkage markers uniformly distributed on chromosomes 1597, the screened SNP / INDEL sites are subjected to specific primer design, and specific primers are used to simultaneously carry out specific amplification of thousands of target sites in the same PCR reaction by multiple PCR, and then the PCR amplicon is subjected to sequencing library construction and second generation sequencing to obtain the genotypes of different target sites. The 1.6K site of the present invention is high in quality, single copy, and high in polymorphism (the MAF values ​​in 624 rapeseed varieties are all higher than>=0.1), and has a wide range of application universality. It is used for SNP site detection of Brassica napus, and the detection sample flexibility is large, the cost is low, and the cycle is short, which can be popularized to seed industry production and application.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A 1.6k whole-genome molecular marker combination for Brassica napus, which is suitable for Brassica napus in Jiangsu and Zhejiang regions of China, and consists of 1597 marker loci. The molecular marker combination is designed based on the reference genome Brana_Dar_V10 of rapeseed, and the locus information is specifically shown in Table 1.

[0009] Furthermore, the present invention also discloses a SNP chip suitable for Brassica napus detection in Jiangsu and Zhejiang regions, wherein the Brassica napus SNP chip comprises probes and / or primers for detecting the molecular marker combination.

[0010] Furthermore, the present invention also discloses a kit, which comprises probes and / or primers for detecting the molecular marker combination.

[0011] Furthermore, the present invention also discloses the use of any one of the molecular marker combination, the SNP chip and the kit in genotyping of Brassica napus.

[0012] Furthermore, the present invention also discloses the use of any one of the molecular marker combination, the SNP chip and the kit in genome-wide association analysis of Brassica napus.

[0013] Furthermore, the present invention also discloses the use of any one of the molecular marker combination, the SNP chip and the kit in cluster analysis of Brassica napus.

[0014] Furthermore, the present invention also discloses the use of any one of the molecular marker combination, the SNP chip and the kit in the identification of kinship of Brassica napus.

[0015] Furthermore, the present invention also discloses the use of any one of the molecular marker combination, the SNP chip and the kit in Brassica napus breeding or assisted breeding.

[0016] Preferably, the breeding or assisted breeding includes at least one of assisted major effect gene selection, backcross breeding and background selection, whole genome selection breeding, molecular assisted breeding, seed purity detection, transgenic component identification, QTL analysis, species evolution analysis and germplasm resource identification.

[0017] Furthermore, the present invention also discloses a method for breeding Brassica napus, which is characterized by comprising the following steps: using the molecular marker combination, the SNP chip and the kit to detect the DNA of the Brassica napus to be tested, and selecting suitable Brassica napus for subsequent breeding.

[0018] The present invention has the following advantages due to the adoption of the above technical solution: 1) The present invention screened out a 1.6K whole gene marker combination for Brassica napus, which is used for background detection of Brassica napus breeding materials. This 1.6K locus is of high quality, single copy, and highly polymorphic (the MAF value in 624 rapeseed varieties is higher than ≥ 0.1), and has a wide range of application universality; 2) The present invention provides a SNP marker detection method based on multiplex PCR + second generation sequencing technology, which is used for SNP site detection in Brassica napus. The detection sample is flexible, low cost, and short cycle, and can be popularized in seed production and application; 3) The detection method provided by the present invention is based on the SNP marker method and has all the inherent advantages of the SNP marker method. The marker is a co-dominant marker with high specificity, sensitivity and resolution; the marker is not affected by environmental conditions and can be used with seeds or any type of plant tissue, and the detection results are accurate, repeatable and stable; different testing laboratories and different data results can be compared and verified with each other, and the data are universally comparable; 4) The technology, reagents, consumables and instruments used in the present invention are all domestically produced and are not affected by domestic and foreign environments, so they can be popularized and promoted more stably and reliably. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the distribution map of 1597 marker loci with the best specificity on rapeseed chromosomes.

[0020] Figure 2 This is a flow chart of the detection technology.

[0021] Figure 3 This is the technical schematic diagram of the detection.

[0022] Figure 4 K value curve for sample cluster analysis.

[0023] Figure 5 This is the sample clustering result diagram. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] 1. Acquisition of Brassica napus germplasm resource data A Brassica napus germplasm resource sequencing database was constructed using 206 high-quality rapeseed germplasm resources from Jiangsu and Zhejiang and the resequencing data of 418 germplasm resources that had been made public online.

[0026] 2. Extraction and screening of SNP loci in Brassica napus The data of 624 core germplasm resources were used to perform SNPCalling based on the reference rapeseed genome Brana_Dar_V10, and the parameters were set as follows: the site missing rate was less than 10%, the heterozygosity rate was less than 0.5%, the depth was greater than 7, and the MAF value was greater than 0.1. Finally, 1645 high-quality whole-genome SNP sites were obtained.

[0027] 3. Synthesis and selection of 1645 whole genome markers of Brassica napus The 1645 high-quality loci were further designed based on the reference genome of rapeseed (Brana_Dar_V10), and sequence-specific alignment was performed. Finally, 1597 marker loci with the best specificity were synthesized. The locus information is shown in Table 1. 88 rapeseed resources were selected for genotyping verification. The markers with a data detection rate of >97% can be used for whole-genome SNP detection of rapeseed samples. Figure 1 This is the distribution of loci on rapeseed chromosomes.

[0028] Table 1 Locus information of 1.6k whole genome molecular marker combination in Brassica napus

[0029] 4. Application of whole genome marker combination in rapeseed 88 rapeseed samples were selected from the existing breeding resources of Zhejiang Academy of Agricultural Sciences for marker combination genotyping detection. The standard detection process was adopted to analyze and verify the rapeseed samples. The average detection rate of the samples was greater than 99.0%. Further cluster analysis was performed, and the samples could be divided into three major categories for subsequent selection of high-quality materials.

[0030] Testing technology process such as Figure 2 As mentioned above, the technical principle of the detection is as follows Figure 3 For detailed methods, please refer to the Chinese invention patent with publication number CN115992292A. The statistical data of the test samples are shown in Table 2.

[0031] Table 2 Statistics of sample data for marker combination genotyping detection

[0032] The results of the sample analysis are as follows Figure 4 , Figure 5 As shown, Figure 4 is the K value curve, Figure 5 This is the sample clustering result diagram.

[0033] The chip of the present invention can be directly applied to the molecular breeding of Brassica napus, and the background of breeding materials can be quickly analyzed through the evenly distributed 1597 sites. Based on the principle of multiplex PCR technology and second-generation sequencing, this chip has many advantages: first, the detection flexibility is large, no matter how many samples can be detected at the same time, which is different from the existing solid phase chip which generally uses 96 or 384 as a fixed sample size; second, the detection cost is less than 20% of the high-density chip, which can be popularized to seed production and application; third, the cycle is short, and the general detection cycle is 15 days, which basically meets the requirements of the breeding selection cycle.

[0034] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A 1.6k whole genome molecular marker combination of Brassica napus, which is suitable for Brassica napus in Jiangsu and Zhejiang regions of China, and consists of 1597 marker loci. The molecular marker combination is designed based on the reference genome Brana_Dar_V10 of rapeseed, and the specific locus information is as follows:

2. A SNP chip suitable for Brassica napus detection in Jiangsu and Zhejiang regions, characterized in that: The Brassica napus SNP chip comprises probes and / or primers for detecting the molecular marker combination according to claim 1.

3. A kit, characterized in that: The kit comprises probes and / or primers for detecting the molecular marker combination according to claim 1.

4. Use of any one of the molecular marker combination according to claim 1, the SNP chip according to claim 2, and the kit according to claim 3 in genotyping of Brassica napus.

5. Use of any one of the molecular marker combination according to claim 1, the SNP chip according to claim 2, and the kit according to claim 3 in genome-wide association analysis of Brassica napus.

6. Use of any one of the molecular marker combination according to claim 1, the SNP chip according to claim 2, and the kit according to claim 3 in cluster analysis of Brassica napus.

7. Use of any one of the molecular marker combination according to claim 1, the SNP chip according to claim 2, and the kit according to claim 3 in identification of kinship of Brassica napus.

8. Use of any one of the molecular marker combination according to claim 1, the SNP chip according to claim 2, and the kit according to claim 3 in Brassica napus breeding or assisted breeding.

9. The use according to claim 8, characterized in that: The breeding or assisted breeding includes at least one of assisted major effect gene selection, backcross breeding and background selection, whole genome selection breeding, molecular assisted breeding, seed purity detection, transgenic component identification, QTL analysis, species evolution analysis and germplasm resource identification.

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

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