Gene for cultivating rape with low erucic acid content, molecular marker and application

Abstract

The invention belongs to the technical field of rape breeding and molecular biology, and particularly relates to a gene for cultivating rape with low erucic acid content, a molecular marker and application. The invention firstly provides a gene and / or a gene composition for reducing the erucic acid content of the rape seeds, wherein the gene and / or the gene composition comprises BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D and / or BnaC03g66430D. The invention further provides a preparation method of the gene and / or the gene composition for reducing the erucic acid content of the rape seeds. Based on the genes, a low-SEAC haplotype is further identified, and meanwhile, a molecular marker capable of assisting low-erucic-acid rape breeding is developed. The rape is cultivated based on the gene, the high-quality haplotype and the molecular marker, the erucic acid content in rape seeds can be effectively reduced, and the quality of rapeseed oil is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of rapeseed breeding and molecular biology, and in particular relates to a gene, a molecular marker and an application thereof for cultivating rapeseed with low erucic acid content. Background Art

[0002] Brassica napus is the world's second largest oil crop and is widely planted worldwide. The erucic acid content (SEAC) of rapeseed seeds is a key factor affecting the quality of rapeseed oil. Excessive SEAC not only greatly reduces the nutritional value of the oil, but also harms human health. Therefore, reducing the erucic acid content in rapeseed seeds is of great significance for improving the nutritional value of rapeseed oil, improving edible quality, meeting market demand, and promoting sustainable agricultural development.

[0003] In recent years, researchers have been exploring the genetic mechanisms that regulate SEAC in Brassica napus, exploring target genes that affect SEAC, and ultimately cultivating low-SEAC varieties through molecular breeding to cultivate high-quality Brassica napus to meet diverse market demands. Genes currently identified as affecting the erucic acid content of rapeseed seeds include the FAE1 gene and the GPAT gene. Existing studies have shown that the erucic acid content of rapeseed mutant seeds is almost zero when the FAE1 gene of rapeseed is knocked out using CRISPR / Cas9 technology; overexpression of the GPAT gene in rapeseed can significantly increase the SEAC in seeds. However, existing studies have also suggested that known genes will reduce the total oil content of rapeseed while reducing the SEAC content. Therefore, further exploration of alleles that affect erucic acid content but do not affect total oil content is crucial for breeding high-quality Brassica napus varieties.

[0004] Genome-wide association analysis (GWAS) is widely used in genetic analysis and gene mining of complex agronomic traits of rapeseed. For example, Wang et al. investigated the SEAC of 300 Brassica napus varieties and conducted GWAS analysis based on 3.82 million single nucleotide polymorphism (SNP) sites developed by simplified genome sequencing; by combining candidate gene association analysis and haplotype analysis, BnaMYB28 was identified as a possible candidate gene, see "Wang B, Wu Z, Li Z, Zhang Q, Hu J, Xiao Y, Cai D, Wu J, King GJ, Li H, Liu K (2018) Dissection of the genetic architecture of three seed-quality traits and consequences for breeding in Brassica napus. Plant Biotechnol J 16: 1336-1348". Transcriptome association analysis (TWAS) can directly identify candidate genes that are significantly associated with specific phenotypes. For example, Tang et al. identified a candidate gene BnaA05.PMT6 that affects the oil content of rapeseed seeds by combining GWAS and TWAS, and used CRISPR technology to construct mutant materials of BnaA05.PMT6. Compared with the wild type, the total oil content of the mutant seeds was 8% higher. For details, see "Tang S, Zhao H, Lu S, Yu L, Zhang G, Zhang Y, Yang QY, Zhou Y, Wang X, Ma W, Xie W, Guo L (2021) Genome-and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus. Mol Plant 14:470-487".

[0005] Some existing technologies have reported on erucic acid-related molecular markers for Brassica napus. For example, patent CN101955930A discloses a low-erucic acid molecular marker for Brassica napus and its preparation method and application. The invention adopts the primer 3' end mismatch strategy to develop and detect SNP polymorphism markers for Brassica napus; and uses the forward primer and reverse primer simultaneous mismatch method to develop SNP markers based on PCR amplification and agarose gel electrophoresis in the allopolyploid crop Brassica napus. Patent CN109234428A discloses a molecular marker and breeding method for a high-erucic acid gene of Brassica napus, comprising: 1) constructing a segregating population; 2) extracting genomic DNA of the material to be tested by the CTAB method; 3) performing PCR amplification; 4) using labeled primer pairs Pb1, Pb2, Fa1, and Fa2 to detect whether individual plants in the segregating population contain a clubroot resistance site PbBa8.1 or a low-erucic acid gene fae1 and classify them; 5) using a Brassica napus plant containing the clubroot resistance site PbBa8.1 and high erucic acid as a parent and hybridizing it with a non-disease-resistant low-erucic acid parent, and then continuously self-pollinating, and combining marker-assisted selection with a new rapeseed variety that is homozygous for the disease resistance site and the low-erucic acid gene. Summary of the invention

[0006] In view of this, one object of the present invention is to provide one or more target gene sequences that can reduce the erucic acid content in rapeseed seeds.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] Genes and / or gene compositions for reducing the erucic acid content of rapeseed seeds, the gene composition comprising any one or more of the following genes: BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D, BnaC03g66430D.

[0009] As a combination method 1, it can be: BnaA08g10230D and BnaA08g11470D;

[0010] As a second combination, it can be: BnaA08g10230D, BnaA08g11470D and BnaA08g11580D;

[0011] As a third combination: it can be: BnaA08g10230D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D and BnaC03g66430D.

[0012] This is not an exhaustive list of all permutations and combinations.

[0013] Furthermore, the nucleotide sequences of BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D, and BnaC03g66430D are shown in SEQ ID NO: 1 to SEQ ID NO: 8, respectively.

[0014] Another object of the present invention is to provide a method for reducing the erucic acid content in rapeseed seeds, which method is suitable for planned use.

[0015] To achieve the above object, the technical solution of the present invention is:

[0016] A method for reducing the erucic acid content in rapeseed seeds, using the aforementioned gene and / or gene combination as a marker, reducing the erucic acid content in rapeseed seeds by knocking out and / or overexpressing the gene and / or gene combination, wherein the expression level of the gene and / or gene combination is correlated with the SEAC content.

[0017] This correlation can be either positive or negative.

[0018] Furthermore, the gene expression levels of the BnaA09g10040D, the BnaA08g11470D, the BnaA08g11580D, and the BnaC03g66430D were significantly positively correlated with SEAC; the gene expression levels of the BnaA08g10230D, the BnaA08g11590D, the BnaA08g12110D, and the BnaC03g66110D were significantly negatively correlated with SEAC.

[0019] An object of the present invention is to provide a class of molecular markers that can specifically detect the BnaC03g66110D gene. At the same time, a method for obtaining the molecular marker and its application are also provided.

[0020] To achieve the above object, the technical solution of the present invention is:

[0021] Based on the molecular marker of the aforementioned gene and / or gene combination, the gene is BnaC03g66110D, and the detection primers of the molecular marker include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:10.

[0022] Furthermore, a method for preparing molecular markers of the aforementioned genes and / or gene combinations is provided, wherein the sequences of the aforementioned genes and / or gene combinations and their homologous genes are subjected to multiple sequence alignment to identify whether the SNP within the gene that is significantly associated with SEAC is located at the restriction site, and the specific recognition sequence located at the restriction site is screened out; and then the AmplifX software is used to design SNP upstream and downstream specific primers for developing molecular markers.

[0023] Furthermore, a method for reducing the erucic acid content in rapeseed seeds based on the molecular marker is provided, comprising the following steps:

[0024] (1) Extracting DNA from Brassica napus;

[0025] (2) using the DNA obtained in step (1) as a template and using the detection primers of the aforementioned molecular marker to perform PCR amplification to obtain an amplified product;

[0026] (3) digesting the amplified product obtained in step (2) with a restriction endonuclease corresponding to the target SNP, performing electrophoresis analysis on the digested product, and identifying the rapeseed variety with low erucic acid content; if the digested product contains fragments of 559 bp and 135 bp, it indicates that the single strain of Brassica napus is a rapeseed variety with high erucic acid content; if the digested product contains a band of 694 bp but does not contain bands of 559 bp and 135 bp, it indicates that the single strain of Brassica napus is a rapeseed variety with low erucic acid content;

[0027] (4) Using the rapeseed seeds identified as having low erucic acid content in step (3) for breeding.

[0028] One object of the present invention is to provide a method for identifying low SEAC haplotypes, and a biological sequence obtained using the method.

[0029] To achieve the above object, the technical solution of the present invention is:

[0030] The method for identifying low SEAC haplotypes using the aforementioned genes or gene markers is based on the SNP sites within the aforementioned genes and / or gene combinations, and through association analysis and haplotype analysis of the aforementioned genes and / or gene combinations, low SEAC haplotypes are identified.

[0031] Further, seeds that meet the requirements are used for breeding and / or large-scale planting.

[0032] Furthermore, a biological sequence obtained using the method is provided, wherein the biological sequence is CCTTTT.

[0033] The biological sequence was voluntarily named LSEACHap.

[0034] The Brassica napus variety carrying the biological sequence has lower SEAC content and sulfhydryl content, higher oleic acid and linoleic acid content, and its yield, thousand-grain weight, plant height and branch number are not affected.

[0035] Another object of the present invention is to provide an application of the aforementioned gene / gene combination, the aforementioned molecular marker and / or the aforementioned biological sequence in identifying rapeseed varieties with low erucic acid content and / or cultivating rapeseed with low erucic acid content.

[0036] The beneficial effects of the present invention are:

[0037] (1) The present invention conducted genome-wide association analysis and transcriptome-wide association analysis on the erucic acid content of seeds of 393 Brassica napus varieties, and identified 8 candidate genes that affect the erucic acid content of seeds: BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D and BnaC03g66430D. The expression levels of these genes in Brassica napus seeds showed a significant correlation with SEAC.

[0038] (2) Based on the SNP sites in the candidate genes, the present invention identified six low SEAC haplotypes (LSEACHap) through candidate gene association analysis and haplotype analysis. The SEAC of the Brassica napus varieties carrying the low SEAC haplotype was significantly lower than that of the Brassica napus varieties carrying the high SEAC haplotype, but the seed quality traits and single plant yield were not affected.

[0039] (3) Based on SNP chrC03_55857214 (G / A) in the exon of BnaC03g66110D, the present invention developed a molecular marker Bn_C3_SEAC_marker that can be used to assist low-erucic acid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the SEAC correlation analysis diagram for different locations of the association analysis group;

[0041] Figure 2 This is the genome-wide association analysis map of SEAC of Brassica napus in Wuhan, where WH stands for Wuhan;

[0042] Figure 3 This is the genome-wide association analysis map of Brassica napus SEAC of Sansheng, where SS stands for Sansheng;

[0043] Figure 4 This is the genome-wide association analysis map of SEAC of Brassica napus of Xiema, where XM represents Xiema;

[0044] Figure 5 This is the mean statistical diagram of the genome-wide association analysis of Brassica napus SEAC in Wuhan, Sansheng and Xiema, where MV represents the mean;

[0045] Figure 6 This is the result diagram of the identification of superior alleles, where: Figure 6 -A is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the T allele in SNP chrA08_10194057 (T / C) and the erucic acid content of rapeseed varieties carrying the C allele, Figure 6 -B is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the T allele in SNP chrA09_3050950 (T / C) and the erucic acid content of rapeseed varieties carrying the C allele, Figure 6 -C is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the A allele in SNP chrC03_49325942 (A / T) and the erucic acid content of rapeseed varieties carrying the T allele, Figure 6 -D is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the G allele in SNP chrC03_55739409 (G / A) and the erucic acid content of rapeseed varieties carrying the A allele, Figure 6 -E is a differential analysis diagram of the erucic acid content of rapeseed varieties carrying the T allele in SNP chrC04_2880837 (T / C) and the erucic acid content of rapeseed varieties carrying the C allele, Figure 6 -F is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the A allele in SNP chrC06_22913987 (A / C) and the erucic acid content of rapeseed varieties carrying the C allele, Figure 6 -G is a differential analysis diagram of the erucic acid content of rapeseed varieties carrying the C allele in SNP chrC06_33011759 (C / T) and the erucic acid content of rapeseed varieties carrying the T allele, Figure 6 -H is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the C allele in SNP chrC07_16707304 (C / A) and the erucic acid content of rapeseed varieties carrying the A allele, Figure 6 -I is a difference analysis diagram of the erucic acid content of rapeseed varieties carrying the C allele in SNP chrC07_23476082 (C / A) and the erucic acid content of rapeseed varieties carrying the A allele, Figure 6 -J is a differential analysis diagram of the erucic acid content of rapeseed varieties carrying the C allele in SNP chrC09_20079791 (C / A) and the erucic acid content of rapeseed varieties carrying the A allele;

[0046] Figure 7 This is the result of the correlation analysis between the number of superior alleles carried by rapeseed varieties in the association analysis population and SEAC;

[0047] Figure 8 Manhattan plot of the whole transcriptome association analysis at 20 DAF;

[0048] Fig. 9 Manhattan plot of the whole transcriptome association analysis at 40 DAF;

[0049] Fig.10 This is the correlation analysis result diagram between candidate gene expression and SEAC at 20 DAF, where: Fig.10 -A is a correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA09g10040D gene in the association analysis population, Fig.10 -B is a correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA08g10230D gene in the association analysis population, Fig.10 -C is a correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA08g11470D gene in the association analysis population, Fig.10 -D is the correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA08g11580D gene in the association analysis population, Fig.10 -E is the correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA08g11590D gene in the association analysis population, Fig.10 -F is the correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaA08g12110D gene in the association analysis population, Fig.10 -G is a correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaC03g66110D gene in the association analysis population, Fig.10 -H is a correlation analysis diagram of the TPM value in seeds 20 days after flowering and the erucic acid content in seeds of the BnaC03g66430D gene in the association analysis population;

[0050] Fig.11 This is the correlation analysis result diagram between candidate gene expression and SEAC at 40DAF, where: Fig.11 -A is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA09g10040D gene in the association analysis population; Fig.11-B is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA08g10230D gene in the association analysis population; Fig.11 -C is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA08g11470D gene in the association analysis population; Fig.11 -D is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA08g11580D gene in the association analysis population; Fig.11 -E is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA08g11590D gene in the association analysis population; Fig.11 -F is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaA08g12110D gene in the association analysis population; Fig.11 -G is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaC03g66110D gene in the association analysis population; Fig.11 -H is a correlation analysis diagram of the TPM value in seeds 40 days after flowering and the erucic acid content in seeds of the BnaC03g66430D gene in the association analysis population;

[0051] Fig.12 This is the result of gene enrichment analysis of candidate genes significantly associated with SEAC;

[0052] Fig.13 This is the result of the association analysis between the SNP site in the candidate gene BnaA08g11580D and the candidate gene of SEAC, where: Fig.13 -A is the association analysis diagram of the candidate gene between the SNP in the BnaA08g11580D gene and the erucic acid content in seeds, Fig.13 -B is a diagram showing the difference in erucic acid content in seeds of rapeseed varieties carrying two haplotypes of the BnaA08g11580D gene;

[0053] Fig.14 This is the result of the association analysis between the SNP site in the candidate gene BnaA08g11590D and the candidate gene of SEAC, where: Fig.14 -A is the association analysis diagram of the candidate gene between the SNP in the BnaA08g11590D gene and the erucic acid content in seeds, Fig.14 -B is a diagram showing the difference in erucic acid content in seeds of rapeseed varieties carrying two haplotypes of the BnaA08g11590D gene;

[0054] Fig.15This is the result of the association analysis between the SNP site in the candidate gene BnaA08g12110D and the candidate gene of SEAC, where: Fig.15 -A is the association analysis diagram of the candidate gene between the SNP in the BnaA08g12110D gene and the erucic acid content in seeds, Fig.15 -B is a diagram showing the difference in erucic acid content in seeds of rapeseed varieties carrying two haplotypes of the BnaA08g12110D gene;

[0055] Fig.16 This is the result of the association analysis between the SNP site in the candidate gene BnaA08g10230D and the candidate gene of SEAC, where: Fig.16 -A is the association analysis diagram of the candidate gene between the SNP in the BnaA08g10230D gene and the erucic acid content in seeds, Fig.16 -B is a diagram showing the difference in erucic acid content in seeds of rapeseed varieties carrying two haplotypes of the BnaA08g102300D gene;

[0056] Fig.17 This is the result of the association analysis between the SNP site in the candidate gene BnaC03g66430D and the candidate gene of SEAC, where: Fig.17 -A is the association analysis diagram of the candidate gene between the SNP in the BnaC03g66430D gene and the erucic acid content in seeds, Fig.17 -B is a diagram showing the difference in erucic acid content in seeds of rapeseed varieties carrying two haplotypes of the BnaC03g66430D gene;

[0057] Fig.18 This is the result of the association analysis between the SNP site in the candidate gene BnaC03g66110D and the candidate gene of SEAC, where: Fig.18 -A is the association analysis diagram of the candidate gene between the SNP in the BnaC03g66110D gene and the erucic acid content in seeds, Fig.18 -B is a diagram showing the difference in erucic acid content in seeds of two haplotype rapeseed varieties carrying the BnaC03g66110D gene;

[0058] Fig.19 This is a graph comparing the differences in erucic acid content, total oil content, seed protein content, oleic acid, linoleic acid and single plant yield between rapeseed varieties carrying LSEACHap and HSEACHap. Fig.19 -A is a diagram showing the difference in erucic acid content in seeds between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -B is a graph showing the difference in glucosinolate content in seeds between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19-C is a diagram showing the difference in seed oleic acid content between rapeseed varieties carrying LSEACHap and HSEACHap;

[0059] Fig.19 -D is the difference analysis diagram of seed linoleic acid content between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -E is the difference analysis diagram of seed oil content between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -F is a graph showing the difference in seed protein content between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -G is a difference analysis diagram of seed thousand-grain weight between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -H is the difference analysis diagram between the single plant yield of rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -I is a diagram showing the difference in plant height between rapeseed varieties carrying LSEACHap and HSEACHap; Fig.19 -J is the difference analysis diagram of the branch number between rapeseed varieties carrying LSEACHap and HSEACHap;

[0060] Fig. 20 The difference analysis results of SEAC of two alleles of chrC03_55857214 (G / A) under different environments are shown in Figure 2. Fig. 20 -A is the difference analysis result of two alleles of chrC03_55857214 (G / A) in SEAC of Wuhan City, Fig. 20 -B is the difference analysis result of two alleles of chrC03_55857214 (G / A) in SEAC of Xiema, Fig. 20 -C is the difference analysis result of two alleles of chrC03_55857214 (G / A) in SEAC of Sansheng, Fig. 20 -D is the difference analysis result of the average SEAC of the three locations of the two alleles of chrC03_55857214 (G / A);

[0061] Fig.21 A map showing the position of chrC03_55857214 (G / A) in BnaC03g66110D;

[0062] Fig. 22 The electrophoresis diagram of 10 cabbage varieties with different genes after SpeI digestion. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0064] In order to enhance the understanding of the present invention, some key technical and scientific terms are clearly defined below. Unless otherwise specifically defined in this article, all other technical and scientific terms follow the meanings generally accepted and understood in the field to which the present invention belongs. It should be emphasized that the scope of the present invention is not limited to the specific methods, compositions or applications described, but allows reasonable flexibility and adjustment in these aspects. At the same time, please understand that the terms used in this article are intended to illustrate specific embodiments, rather than to be interpreted restrictively.

[0065] In addition, all literature cited herein, including but not limited to patents, patent applications, academic papers, textbooks and further citations therein, are deemed to be incorporated herein by reference as a whole to the extent not directly cited. If there is any inconsistency or conflict between the contents of these cited documents or similar materials and this application, especially in terms of term definitions, term usage, technical descriptions, etc., the contents of this application shall prevail.

[0066] In the embodiments of the present invention, some key technical and scientific terms are defined as follows:

[0067] Genome-wide association study (GWAS): A high-throughput, unbiased genetic research method that aims to identify genetic loci that are significantly associated with complex traits by comparing the correlation between genetic variations (such as SNPs) and specific phenotypes (such as disease states, physiological indicators, etc.) at the genome-wide level in different individuals. GWAS is based on the principle of linkage disequilibrium (LD), that is, adjacent genetic variants tend to be inherited together, so pathogenic variants can be captured indirectly through marker SNPs.

[0068] SNP site: Single Nucleotide Polymorphism (SNP) site refers to the DNA sequence polymorphism caused by the variation of a single nucleotide within the gene sequence.

[0069] Coding region (exon): SNPs located in the coding region of a gene may directly affect the amino acid sequence of the protein, thereby changing the structure and function of the protein.

[0070] Non-coding regions (introns, regulatory sequences, etc.): Although SNPs in non-coding regions do not directly change the amino acid sequence of proteins, they may indirectly affect the function of genes by affecting gene expression regulation (such as transcription factor binding sites, splicing sites, etc.).

[0071] Haplotype: A group of DNA variations (or polymorphisms) that tend to be inherited together along a chromosome. These variations tend to be inherited together because they are located close to each other on the chromosome and recombination between them is rare. A haplotype can include one or more genes that are located closely together on the chromosome and whose alleles (different forms of the gene that control the same trait) are arranged in sequence.

[0072] In the embodiment of the present invention, the sequence information involved is shown in Table 1.

[0073] Table 1. List of biological sequences

[0074] serial number name Involved rights SEQ ID NO: 1 BnaA08g10230D Right 2 SEQ ID NO: 2 BnaA08g11470D Right 2 SEQ ID NO: 3 BnaA08g11580D Right 2 SEQ ID NO: 4 BnaA08g11590D Right 2 SEQ ID NO: 5 BnaA08g12110D Right 2 SEQ ID NO: 6 BnaA09g10040D Right 2 SEQ ID NO: 7 BnaC03g66110D Right 2 SEQ ID NO: 8 BnaC03g66430D Right 2 SEQ ID NO: 9 BnaC03g66110D-F Right 4 SEQ ID NO: 10 BnaC03g66110D-R Right 4

[0075] Example 1. Rapeseed planting and SEAC phenotype investigation

[0076] 393 accessions of Brassica napus were planted in Wuhan, Hubei, Sansheng, Chongqing, and Xiema, Chongqing, and SEAC was investigated after maturity. Three rows of each variety were planted, with 12 plants in each row, and the row and plant spacing was 20 cm. After harvest, the seeds were scanned three times using a near-infrared spectrometer, and the average of the three scans was used as the final SEAC phenotypic value for subsequent GWAS analysis.

[0077] The results are as follows Figure 1 As shown in Table 2, the SEAC of this population has a wide range of phenotypic variation. In Wuhan, SEAC ranges from 0.01% to 44.25%; in Chongqing Sansheng, SEAC ranges from 0.01% to 56.89%; in Chongqing Xiema, SEAC ranges from 0.01% to 51.25%. At the same time, the coefficient of variation of SEAC among the three planting sites ranges from 153.35% to 184.19%. In addition, the SEAC of this association analysis population at different planting sites also has extremely significant correlations. For example, the correlation coefficient of SEAC between Wuhan and Sansheng is 0.93, and the correlation coefficient of SEAC between Sansheng and Xiema is 0.91.

[0078] Table 2. Statistical analysis of erucic acid content in seeds of association analysis population

[0079]

[0080] Example 2. Genome-wide association analysis

[0081] We previously performed whole-genome resequencing on 505 samples of Brassica napus, and developed more than 10 million SNP sites based on the Darmor reference genome, as detailed in “Tang S, Zhao H, Lu S, YuL, Zhang G, ZhangY, Yang QY, ZhouY, Wang X, MaW, Xie W, Guo L (2021) Genome-and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seedoil content in Brassica napus. Mol Plant 14: 470-487”. In the present invention, 393 samples of rapeseed were selected for subsequent whole-genome association analysis. First, the VCFtools software was used to filter the above SNP sites according to the screening criteria of near-allele frequency > 0.05 and missing rate < 0.2, and 4.6 million SNP sites were obtained for this GWAS analysis. The Fast-LMM model was used to conduct GWAS analysis on 393 rapeseed samples from Wuhan, Xiema, and Sansheng. -10 As the threshold, SNP sites significantly associated with SEAC were screened.

[0082] The results are as follows Figure 2 to Figure 5 As shown in Table 3, a total of 28 genetic regions significantly associated with SEAC were identified. Figure 2 to Figure 5 These QTLs are distributed on chromosomes A01, A05, A07, A08, A09, C02, C03, C04, C06, C07, C08, and C09, and can explain 16.32% to 47.66% of the phenotypic variation. For details, see Figure 2 to Figure 5 and Table 2. Based on the lead SNPs in the above QTLs, 10 low SEAC alleles were identified. For example, the T allele in chrA08_10194057 (T / C) and the A allele in chrC03_49325942 (A / T) are both low SEAC alleles. Figure 6 At the same time, there is a very significant negative correlation between the number of low SEAC alleles carried by the variety and SEAC (p value: 2.2e-16, R: -0.641), see Figure 7 .

[0083] Table 3. Genetic regions significantly associated with SEAC identified by GWAS

[0084]

[0085]

[0086] Note: In Table 3, WH stands for Wuhan; SS stands for Sansheng; XM stands for Xiema; MV stands for mean.

[0087] Example 3. Whole transcriptome association analysis

[0088] Tang et al. previously sequenced the transcriptome of seeds 20 days after flowering (20 DAF) and 40 days after flowering (40 DAF) in the association analysis population; the data have been uploaded to the BnIR database, see "Tang S, Zhao H, Lu S, Yu L, Zhang G, Zhang Y, Yang QY, Zhou Y, Wang X, Ma W, Xie W, Guo L (2021) Genome-and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus. Mol Plant 14:470-487" and "Yang Z, Wang S, Wei L, Huang Y, Liu D, Jia Y, Luo C, Lin Y, Liang C, Hu Y, Dai C, Guo L, Zhou Y, Yang Q (2023) BnIR: A multi-omics database with various tools for Brassica napus research and breeding. Mol Plant 16:775-789". The present invention used the BnIR database to perform TWAS analysis on the SEAC of this population, with a 1×10 -5 The candidate genes significantly associated with SEAC were screened as a threshold. The BnIR database was used to calculate the correlation between the gene expression of candidate genes at 20 DAF and 40 DAF and SEAC. Gene enrichment analysis was performed using the OmicShare online website (https: / / www.omicshare.com / ).

[0089] The results of the whole transcriptome association analysis of 393 rapeseed varieties SEAC are as follows Figure 8 to Figure 11 As shown in FIG, the present invention identified 133 and 233 candidate genes significantly associated with SEAC at 20 DAF and 40 DAF, respectively. Figure 8. The GO enrichment analysis of the above candidate genes showed that these candidate genes were mainly enriched in secondary cell wall biogenesis (GO:0009834), cell wall biogenesis (GO:0042546), glycyl-tRNA aminoacylation (GO:0006426), positive regulation of cyclin-dependent protein serine / threonine kinase activity (GO:0045737) and positive regulation of secondary cell membrane biogenesis (GO:1901348), see for details. Fig.12 .

[0090] The present invention compares the candidate genes significantly associated with SEAC identified by TWAS with the genetic regions significantly associated with SEAC identified by GWAS, and combines the correlation between gene expression levels and SEAC to identify a total of 8 candidate genes: BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D, BnaC03g66430D, for example For example, at 20 DAF, the gene expression of BnaA0811470D in the association analysis population was significantly positively correlated with SEAC (p: 2.76e-17; R: 0.327), and the gene expression of BnaA09g10040D, BnaA08g11580D, and BnaC03g66430D was also significantly positively correlated with SEAC; the gene expression of BnaA08g10230D, BnaA08g11590D, BnaA08g12110D, and BnaC03g66110D was significantly negatively correlated with SEAC, see for details. Figures 9 to 11 The above genes may play an important role in the accumulation of SEAC in Brassica napus.

[0091] Example 4. Identification of superior haplotypes

[0092] (1) Candidate gene association analysis and haplotype analysis

[0093] In order to identify the low SEAC allele variation in the above candidate genes and explore their breeding value, the present invention extracted the SNPs in the promoter region and coding region of the above candidate genes, and performed candidate gene association analysis based on the general linear model to identify the SNP sites significantly associated with SEAC in the genes. Specifically, VCFtools software was used to extract SNP variations in candidate genes BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D, and BnaC03g66430D. The GLM model in Tassel 5.0 software was used to perform candidate gene association analysis on the SNP variation of the above candidate genes and the SEAC of the rapeseed association analysis population, see "Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23: 2633-2635" for details. Haplotype typing was performed on the significant SNP sites using Haploview.4.2 software.

[0094] The results of association analysis between SNP sites in the 6 candidate genes and SEAC candidate genes are as follows: Figures 13 to 18 As shown, 17 SNP loci significantly associated with SEAC were identified within the BnaA08g11580D gene, and haplotype typing was performed based on the lead SNP chrA08_10479573 (C / G). The SEAC of the Brassica napus varieties carrying BnaA08g11580DHap1 was much lower than that of the varieties carrying BnaA08g11580DHap2. Similar to BnaA08g11580D, haplotype analysis was performed on the remaining five candidate genes, and five low SEAC haplotypes were identified.

[0095] According to statistics, 49 materials carried all low SEAC haplotypes, and 76 materials carried all high SEAC haplotypes.

[0096] (2) Analysis of differences in seed quality and yield traits between HSEACHap and LSEACHap

[0097] 49 rapeseed varieties carrying six low SEAC haplotypes were defined as LSEACHap, and 76 rapeseed varieties carrying six high SEAC haplotypes were defined as HSEACHap. These varieties were planted in Meichuan Town, Wuhan City, Hubei Province. After harvest, the yield per plant was investigated, and the total oil content, fatty acid content, protein content and yield per plant of each rapeseed variety were measured using a DS2500 near-infrared analyzer. The differences in the above traits between LSEACHap and HSEACHap were compared using the Student t-test.

[0098] The LSEACHap haplotype consists of low SEAC alleles in 6 SNPs, namely: C allele in chrA08_10479573 (C / G), C allele in chrA08_10488109 (C / A), T allele in chrA08_10839268 (T / G), T allele in chrA08_9528484 (T / C), T allele in chrC03_55855485 (T / C), T allele in chrC03_56246200 (T / C). Therefore, the nucleotide sequence of LSEACHap is CCTTTT.

[0099] The results are as follows Fig.19 As shown in Table 4, the average SEAC of LSEACHap was 0.58%, significantly lower than 32.84% of HSEACHap. The average sulfur content of LSEACHap was 30.42 μmol / g, significantly lower than 102.91 μmol / g of HSEACHap. At the same time, the oleic acid and linoleic acid contents of LSEACHap were significantly higher than those of HSEACHap. More importantly, there was no significant difference in yield between LSEACHap and HSEACHap. The above results show that LSEACHap can be used as a high-quality haplotype for improving Brassica napus varieties.

[0100] Table 4. Comparison of seed quality traits and seed yield between rapeseed varieties carrying LSEACHap and HSEACHap

[0101]

[0102]

[0103] Note: In Table 4, SEAC means seed erucic acid content; SGC means sulfhydryl content; SOC means seed oil content; C18:1 means oleic acid content; C18:2 means linoleic acid content; C18:3 means linolenic acid content; SY means yield; SW means 1000-grain weight; PH means plant height; BN means branch number.

[0104] Example 5. Molecular marker development

[0105] The sequences of the candidate gene (BnaC03g66110D) and its homologous genes were downloaded from the BnIR database, and multiple sequence alignment was performed using DNAMAN software (https: / / www.lynnon.com / dnaman.html). SnapGene software (https: / / www.genoscope.cns.fr / brassicanapus / ) was used to identify whether the SNP significantly associated with SEAC within the gene was located at the restriction site. AmplifX software was used to design specific primers BnaC03g66110D-F and BnaC03g66110D-R about 500 bp upstream and downstream of the SNP used to develop molecular markers. Five varieties with LSEACHap and HSEACHap haplotypes were selected for verification. After DNA extraction, PCR amplification was performed using the above primers, and the PCR product was digested with the restriction endonuclease corresponding to the target SNP to verify the accuracy of the newly developed molecular marker.

[0106] Among the SNP sites significantly associated with SEAC in the above candidate genes, chrC03_55857214 (G / A) is located in the specific recognition sequence of the restriction endonuclease SpeI. The position of chrC03_55857214 (G / A) in BnaC03g66110D is as follows: Fig.21 As shown. Among the 393 varieties in the association analysis population, 41 varieties carried the A allele in chrC03_55857214(G / A), and 323 varieties carried the G allele in chrC03_55857214(G / A). The SEAC of the two types of B. napus varieties was significantly different in different locations, see Fig. 20 Importantly, chrC03_55857214 (G / A) is located in the exon of gene BnaC03g66110D and causes a non-synonymous mutation. Therefore, the present invention selects chrC03_55857214 (G / A) to develop a molecular marker.

[0107] As shown in Table 5, the B. napus varieties L60, L198, L226, L262 and L340 carry the G allele in chrC03_55857214 (G / A), and the SEAC of these varieties ranges from 0.01% to 3.94%. The B. napus varieties L732, L984, L994, L976 and L1014 all carry the A allele in chrC03_55857214 (G / A), and the SEAC of these varieties ranges from 32.22% to 42.36%. First, primers BnaC03g66110D-F and BnaC03g66110D-R were used to amplify the above varieties, and then the amplified fragments of the above varieties were digested with SpeI. The amplified fragments of the five high SEAC varieties were cut into two fragments of 559 bp and 135 bp by SpeI, while the amplified fragments of the five low SEAC varieties could not be cut by SpeI. The electrophoresis diagram of 10 cabbage varieties with different genes after SpeI digestion is shown in Fig. 22 .

[0108] In summary, the present invention developed a molecular marker based on chrC03_55857214 (G / A) that can be used to identify low SEAC varieties and named it Bn_C3_SEAC_marker.

[0109] Table 5. Information on 10 varieties used for molecular marker development

[0110] Variety name Alleles of chrC03_55857214(G / A) Erucic acid content L60 G 1.37％ L198 G 3.94％ L226 G 3.21％ L262 G 3.44％ L340 G 0％ L732 A 42.36％ L984 A 37.73％ L994 A 38.64％ L976 A 39.26％ L1014 A 32.22％

Claims

1. A gene and / or gene combination for reducing the erucic acid content of rapeseed seeds, characterized in that: The gene composition includes any one or more of the following genes: BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D, and BnaC03g66430D.

2. The gene and / or gene combination according to claim 1, characterized in that: The nucleotide sequences of BnaA08g10230D, BnaA08g11470D, BnaA08g11580D, BnaA08g11590D, BnaA08g12110D, BnaA09g10040D, BnaC03g66110D and BnaC03g66430D are shown in SEQ ID NO: 1 to SEQ ID NO: 8 respectively.

3. A method for reducing the erucic acid content of rapeseed seeds, characterized in that: The gene and / or gene combination according to any one of claims 1 to 2 is used as a marker, and the erucic acid content of rapeseed seeds is reduced by knocking out and / or overexpressing the gene and / or gene combination, and the expression level of the gene and / or gene combination is correlated with the SEAC content.

4. A molecular marker based on the gene and / or gene combination according to any one of claims 1 to 2, characterized in that: The gene is BnaC03g66110D, and the detection primers of the molecular marker include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

10.

5. The method for preparing a molecular marker of a gene and / or a gene combination according to any one of claims 1 to 2, characterized in that: The sequences of the gene and / or gene combination and homologous genes thereof according to any one of claims 1 to 2 are subjected to multiple sequence alignment to identify whether the SNP significantly associated with SEAC in the gene is located at the restriction site, and the specific recognition sequence located at the restriction site is screened out; then, the AmplifX software is used to design SNP upstream and downstream specific primers for developing molecular markers.

6. The method for reducing the erucic acid content in rapeseed seeds based on the molecular markers according to claim 4, characterized in that: The steps include: (1) Extracting DNA from Brassica napus; (2) using the DNA obtained in step (1) as a template and using the molecular marker detection primers described in claim 4 to perform PCR amplification to obtain an amplified product; (3) digesting the amplified product obtained in step (2) with a restriction endonuclease corresponding to the target SNP, performing electrophoresis analysis on the digested product, and identifying the rapeseed variety with low erucic acid content; if the digested product contains fragments of 559 bp and 135 bp, it indicates that the single strain of Brassica napus is a rapeseed variety with high erucic acid content; if the digested product contains a band of 694 bp but does not contain bands of 559 bp and 135 bp, it indicates that the single strain of Brassica napus is a rapeseed variety with low erucic acid content; (4) Using the rapeseed seeds identified as having low erucic acid content in step (3) for breeding.

7. A method for identifying a low SEAC haplotype using the gene or gene marker according to any one of claims 1 to 2, characterized in that: Based on the SNP sites in the gene and / or gene combination according to any one of claims 1 to 2, low SEAC haplotypes are identified through association analysis and haplotype analysis of the gene and / or gene combination.

8. The method according to claim 7, characterized in that Seeds that meet the requirements are used for breeding and / or large-scale planting.

9. The biological sequence obtained by the method of claim 7, characterized in that The biological sequence is CCTTTT.

10. Use of the gene / gene combination according to claim 1 or 2, the molecular marker according to claim 4 and / or the biological sequence according to claim 9 in identifying rapeseed varieties with low erucic acid content and / or cultivating rapeseed with low erucic acid content.

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