Brassica napus seed oil content gene soca9 and related molecular markers

Abstract

The application belongs to the technical field of molecular biology and genetic breeding, and discloses a Brassica napus seed oil content gene SOCA9 and related molecular markers, wherein the SOCA9 gene for regulating the seed oil content of Brassica napus has a nucleotide sequence as shown in SEQ ID No. 1 or a complementary sequence thereof, or a derived nucleotide sequence with not less than 95% homology with the nucleotide sequence as shown in SEQ ID No. 1 and the same function due to addition, deletion or replacement of one or more nucleotides. The gene SOCA9 with the nucleotide sequence as shown in SEQ ID No. 1 (or a complementary sequence thereof, a derived nucleotide sequence, etc.) can be used to improve the seed oil content of Brassica napus, reduce the seed lignocellulose content and make the seed coat color lighter, and can be used for high-oil-content Brassica napus transgenic breeding and molecular marker assisted screening breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and genetic breeding technology. More specifically, it relates to a gene SOCA9 for oil content in rapeseed seeds and related molecular markers. This gene and related molecular markers can be applied to rapeseed breeding to improve the oil content trait, and can further improve the lignocellulose content trait and seed coat color at the same time. Background Technology

[0002] Rapeseed is one of my country's most important oilseed crops, accounting for more than 50% of the country's domestic vegetable oil production and serving as a crucial source of edible vegetable oil. Furthermore, rapeseed oil can also be used as a raw material for biodiesel. Currently, my country heavily relies on imports for vegetable oil, with a dependence rate exceeding 60%. Therefore, increasing the oil content of rapeseed seeds is one of my country's urgent scientific research tasks and a primary goal of rapeseed breeding.

[0003] Among rapeseed varieties, Brassica napus (Brassica napus) has the widest planting area and the highest oil yield. It has the advantage of not competing with staple crops for land, and its low-erucic acid rapeseed oil has the lowest saturated fatty acid content among major edible oils, with a fatty acid composition that meets human nutritional and health needs for different fatty acids. Therefore, rapeseed breeding in my country mainly focuses on Brassica napus.

[0004] Currently, marker-assisted selection (MAG) breeding of Brassica napus (Brassica napus) has been widely implemented in my country. Compared to traditional breeding methods, MAG significantly improves selection efficiency and shortens the breeding cycle by using molecular markers linked to QTLs of desirable traits. In recent years, with advancements in genomics, numerous QTLs related to oil content and related traits in Brassica napus have been located, and a large number of molecular markers linked to these QTLs have been developed. However, the accuracy of QTL mapping is limited by population size and marker density, often resulting in mappings to the Brassica napus genome that are several Mbps or even tens of Mbps long. This leads to insufficient linkage between QTL-mapped molecular markers and phenotypes, making them prone to site loss due to gene recombination during breeding. Therefore, ideally, molecular markers should be tightly linked to functional genes, especially those located within functional genes.

[0005] Meanwhile, transgenic breeding has been successfully applied in crops such as soybeans and cotton, and there are no technical barriers to transgenic breeding in rapeseed. Compared with traditional breeding methods, transgenic breeding has the potential to rapidly and significantly improve crop traits. However, transgenic breeding requires a higher level of research foundation, necessitating the fine mapping and cloning of functional genes from QTLs, and verification of gene function through transgenic experiments before it can be carried out. However, currently, the number of genes identified in rapeseed that regulate seed oil content is small, their effects are low, and they often alter the fatty acid composition of rapeseed oil. This significantly limits the potential of transgenic breeding for high-oil-content rapeseed. Currently reported patents for genes that regulate the oil content of rapeseed, such as BnNTT1 (CN113564181A), BnNTT2 (CN113481213A), BnaCCR-LIKE (CN114438122A), BnaPPT1 (CN114438121A), and orf188 (CN111286504A), generally have a weak effect on increasing oil content. They also cannot simultaneously improve lignocellulose content and seed coat color. Furthermore, in addition to regulating oil content, they may alter the fatty acid composition of rapeseed oil, which is not conducive to maintaining the fatty acid composition advantage of superior germplasm resources in application.

[0006] Therefore, in summary, identifying functional genes related to high oil content in Brassica napus and developing molecular markers closely linked to them is of great significance for molecular marker-assisted screening and transgenic breeding of high-oil-content Brassica napus. Summary of the Invention

[0007] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a gene SOCA9 for regulating the oil content of Brassica napus seeds and related molecular markers. Utilizing the gene SOCA9 with the nucleotide sequence shown in SEQ ID No. 1 (or its complementary sequence, derived nucleotide sequence, etc.), the oil content of Brassica napus seeds can be increased, while simultaneously reducing the lignocellulose content and lightening the seed coat color. The gene SOCA9 and related molecular markers obtained by this invention, capable of regulating the oil content of Brassica napus seeds, can be used for transgenic breeding and marker-assisted selection breeding of high-oil-content Brassica napus, providing a powerful tool for improving seed oil content, lignocellulose content, or seed coat color.

[0008] To achieve the above objectives, according to a first aspect of the present invention, an SOCA9 gene for regulating the oil content of rapeseed seeds is provided, characterized in that it has a nucleotide sequence as shown in SEQ ID No. 1 or its complementary sequence, or a derived nucleotide sequence that has at least 95% homology with the nucleotide sequence shown in SEQ ID No. 1 and is functionally identical due to the addition, deletion or substitution of one or more nucleotides.

[0009] According to a second aspect of the present invention, the present invention provides the application of the above-mentioned SOCA9 gene in regulating the oil content, lignocellulose content and / or seed coat color of rapeseed;

[0010] Preferably, the application is in regulating the oil content of rapeseed seeds.

[0011] According to a third aspect of the present invention, the present invention provides the application of the above-mentioned SOCA9 gene in the breeding of Brassica napus;

[0012] Preferably, the application specifically involves genetically modifying, adding back, knocking out, or silencing the SOCA9 gene in rapeseed in breeding to improve seed oil content, lignocellulose content, and / or seed coat color; more preferably, it is to improve the seed oil content.

[0013] According to a fourth aspect of the present invention, the present invention provides an SOCA9 protein that regulates the oil content of rapeseed seeds, characterized in that it has the amino acid sequence shown in SEQ ID No. 4, or a derived protein having the same activity as the amino acid sequence shown in SEQ ID No. 4 due to the addition, deletion or substitution of one or more amino acids.

[0014] According to a fifth aspect of the present invention, the present invention provides the application of the above-mentioned SOCA9 protein in regulating the oil content, lignocellulose content and / or seed coat color of rapeseed.

[0015] Preferably, the application is in regulating the oil content of rapeseed seeds.

[0016] According to a sixth aspect of the present invention, the present invention provides the application of the above-mentioned SOCA9 protein in codon optimization, point mutation or sequence modification.

[0017] According to a seventh aspect of the present invention, the present invention provides an open reading frame of the SOCA9 gene that regulates the oil content of rapeseed seeds, characterized in that the nucleotide sequence of the open reading frame is shown in SEQ ID No. 3.

[0018] According to an eighth aspect of the present invention, the present invention provides a primer pair for amplifying a molecular marker linked to the SOCA9 gene described above, characterized in that it is specifically a first primer pair or a second primer pair, wherein:

[0019] The forward primer of the first primer pair has the nucleotide sequence shown in SEQ ID No. 5, and the reverse primer has the nucleotide sequence shown in SEQ ID No. 6;

[0020] The forward primer of the second primer pair has the nucleotide sequence shown in SEQ ID No. 7, and the reverse primer has the nucleotide sequence shown in SEQ ID No. 8.

[0021] According to a ninth aspect of the present invention, the present invention provides a molecular marker linked to the SOCA9 gene described above, characterized in that the molecular marker is obtained by PCR amplification using specific primers, wherein the specific primers are selected from a first primer pair or a second primer pair, wherein:

[0022] The forward primer of the first primer pair has the nucleotide sequence shown in SEQ ID No. 5, and the reverse primer has the nucleotide sequence shown in SEQ ID No. 6;

[0023] The forward primer of the second primer pair has the nucleotide sequence shown in SEQ ID No. 7, and the reverse primer has the nucleotide sequence shown in SEQ ID No. 8.

[0024] Through the technical solutions conceived in this invention, compared with the prior art, this invention is the first to clone the key gene SOCA9, which regulates the oil content, lignocellulose content, and seed coat color of Brassica napus seeds. Expression of the SOCA9 gene can increase seed oil content while decreasing lignocellulose content and lightening the seed coat color; conversely, knockout of the SOCA9 gene leads to a decrease in seed oil content and an increase in lignocellulose content, while darkening the seed coat color. This gene can be used for transgenic breeding and marker-assisted selection breeding of high-oil-content Brassica napus, providing a tool for improving the quality of Brassica napus.

[0025] This invention yields a gene, SOCA9, that regulates seed oil content, lignocellulose content, and seed coat color. This gene inhibits flavonoid metabolism and lignocellulose synthesis in the seed coat during rapeseed seed development and promotes oil accumulation, making it highly valuable in breeding high-oil-content Brassica napus. Unlike currently reported gene patents regulating oil content in Brassica napus, such as BnNTT1 (CN113564181A) which can only increase oil content by a maximum of 3.5%, BnNTT2 (CN113481213A) which can only increase oil content by a maximum of 3%, BnaCCR-LIKE (CN114438122A) which can only increase oil content by a maximum of 4.2%, BnaPPT1 (CN114438121A) which can only increase oil content by a maximum of 7.9%, orf188 (CN111286504A) which can increase oil content by 3.27%, and BnaTT18 (CN116515859A) which can increase oil content by 2%, Bna... Existing technologies such as TT12 (CN116515859A) increasing oil content by 8.25%, BnaTT10 (CN116515859A) by 3.08%, and BnaTT7 (CN116515859A) by 5.69% generally have weak effects on improving oil content, requiring modification of multiple genes or gene copies simultaneously. Furthermore, besides regulating oil content, these technologies may also alter the fatty acid composition of rapeseed oil (e.g., CN113564181A, CN113481213A, CN116515859A), which is detrimental to maintaining the fatty acid composition advantage of superior germplasm resources in practical applications. The SOCA9 gene obtained in this invention has advantages such as high effect value, multiple effects from a single gene, and no alteration of the fatty acid composition of rapeseed oil, making it more widely applicable. Furthermore, the present invention is more efficient at increasing oil content. As illustrated in the embodiments below, after the improvement of the present invention, the oil content of rapeseed varieties can be increased by up to 11.06%.

[0026] Furthermore, this invention provides two linked molecular markers for the SOCA9 gene. These markers facilitate marker-assisted selection breeding using SOCA9, increasing the oil content of conventional Brassica napus varieties while reducing seed lignocellulose content and lightening the seed coat. Based on Sanger sequencing results, this invention developed two molecular markers linked to SOCA9: A9-M6 (corresponding specific primers, i.e., the first primer pair) and A9-M7 (corresponding specific primers, i.e., the second primer pair). These two markers enable precise identification of dominant SOCA9 haplotypes in segregating populations, providing an effective tool for the application of the SOCA9 gene in marker-assisted selection breeding.

[0027] This invention is the first to develop two molecular markers, A9-M6 and A9-M7, closely linked to SOCA9. These markers facilitate marker-assisted selection breeding using SOCA9, thereby increasing the oil content of conventional Brassica napus varieties. Although numerous molecular markers linked to oil content loci in Brassica napus have been reported, such as CN 114736986A, CN103667484A, CN103667485A, CN101988118A, CN102766627A, and CN105505925A, A9-M6 and A9-M7 are the first reported molecular markers closely linked to SOCA9, a functional gene regulating oil content in Brassica napus. Furthermore, A9-M6 and A9-M7 are less than 70 kb away from SOCA9 on the genome, while the molecular markers and QTL sites reported in the aforementioned patents generally have unclear positional relationships (e.g., CN103667484A, CN103667485A, CN102766627A, CN101988118A, CN105505925A) or large distances (e.g., 181 kb in CN 114736986A). Therefore, using A9-M6 and A9-M7 for marker-assisted selection breeding will not result in site loss even in large populations of thousands of individual plants.

[0028] The SOCA9 gene with the nucleotide sequence shown in SEQ ID No. 1, and other DNA sequences containing this gene's nucleotide sequence, obtained by this invention, are particularly applicable to the breeding of Brassica napus. For example, this gene can be used in breeding to modify, supplement, knock out, or silence seeds in Brassica napus to improve seed oil content, lignocellulose content, or seed coat color, especially to increase seed oil content and achieve high oil content breeding of Brassica napus. Correspondingly, the SOCA9 protein with the amino acid sequence shown in SEQ ID No. 4, and other protein sequences containing this protein's amino acid sequence, can be used in commercial production. For example, codon optimization, point mutation, or sequence modification of this protein can be performed for commercial purposes. The complementary sequence of the nucleotide sequence shown in SEQ ID No. 1, or the derived nucleotide sequence with no less than 95% homology to the nucleotide sequence shown in SEQ ID No. 1 and the same function due to the addition, deletion, or substitution of one or more nucleotides, and the derived protein with the same activity as the amino acid sequence shown in SEQ ID No. 4 due to the addition, deletion, or substitution of one or more amino acids, also have considerable effects.

[0029] In genetic studies of rapeseed quality, the identification of functional genes often relies solely on big data mining. This involves using a reference genome, combined with the function of homologous genes in model crops such as Arabidopsis thaliana, or screening candidate genes based on relevant metabolic regulatory pathways in these model crops, and finally evaluating the function of these candidate genes (CN113564181A, CN113481213A, CN116515859A, etc.). However, this study relies more heavily on near-isogenic line phenotypic data, and does not depend on a reference genome, the function of homologous genes in model crops, or metabolic regulatory pathways. Compared to big data mining, this study places greater emphasis on the actual production effects of loci and genes. Furthermore, previous studies have shown that traits such as seed coat color, oil content, and lignocellulose content are regulated only by transcription factors or transcription factor complexes such as MYB and bHLH. In this invention, during the screening and identification of candidate genes for QTL fine mapping regions, the DNA sequences within the QTL fine mapping regions were first amplified by PCR and then sequenced by Sanger. The functions of each gene were re-annotated based on sequence variations, ultimately identifying a new gene, SOCA9, encoding a Skp1-like protein, as a candidate gene. This is the first time that the ubiquitin pathway has been found to simultaneously regulate traits such as seed coat color, oil content, and lignocellulose content. Therefore, this invention provides new materials and ideas for breeding high-oil-content rapeseed, and is of great significance. Attached Figure Description

[0030] Figure 1 This is a diagram showing the lignocellulose content and oil content of Ken-C8-SOCA9 and Ken-C8-WT.

[0031] Figure 2 This is a diagram showing the lignocellulose content and oil content of N53-2-soca9 and N53-2-WT.

[0032] Figure 3 These are seed coat color illustrations for Ken-C8-SOCA9, Ken-C8-WT, N53-2-soca9, and N53-2-WT; among them, Figure 3 A, B, and C in the text all correspond to Ken-C8-SOCA9. Figure 3 The D in the text corresponds to Ken-C8-WT. Figure 3 In this context, E and G both correspond to N53-2-soca9 (where G represents the magnified effect of the seed coat). Figure 3 F and H in the text both correspond to N53-2-WT (where H is the effect after the seed coat is magnified).

[0033] Figure 4 This is a diagram showing the lignocellulose content and oil content of different SOCA9 haplotypes in near-isogenic lines; Figure 4The first nine columnar shapes from left to right correspond to lignocellulose content (i.e., the three types of lignocellulose: ADL, ADF, and NDF), while the last three columnar shapes correspond to oil content.

[0034] Figure 5 This is a diagram showing the lignocellulose content and oil content of different SOCA9 haplotypes in the F2 segregating population.

[0035] Figure 6 This is a diagram showing the lignocellulose content and oil content of J9709-SOCA9 and J9709-WT.

[0036] Figure 7 These are seed coat color illustrations for J9709-SOCA9 and J9709-WT; among them, Figure 7 The A in J9709-SOCA9 corresponds to J9709-SOCA9. Figure 7 The B in the text corresponds to J9709-WT.

[0037] Figure 8 These are comparative charts of the seed fatty acid composition of J9709-SOCA9 and J9709-WT, and comparative charts of the seed fatty acid composition of Ken-C8-SOCA9 and Ken-C8-WT; among them, Figure 8 (a) in the figure corresponds to a comparison of the seed fatty acid composition of J9709-SOCA9 and J9709-WT. Figure 8 (b) in the figure corresponds to a comparison chart of the seed fatty acid composition of Ken-C8-SOCA9 and Ken-C8-WT, which compares the proportions of C16:0 (palmitic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (linolenic acid), C20:0 (eicosinate), and C20:1 (eicosinate).

[0038] Figure 9 This is a display of the results of fine mapping of cqOCA9-9 in near-isogenic lines.

[0039] Figure 10 This section shows the seed coat cross-sections of J9709-WT, J9709-SOCA9, Ken-C8-WT, Ken-C8-SOCA9, N53-2-WT, and N53-2-soca9, along with a comparison of their seed coat percentages; among them, Figure 10 The A in the text corresponds to J9709-WT (the lignin in the seed coat was stained with phloroglucinol, the same below). Figure 10 The B in the text corresponds to Ken-C8-WT. Figure 10 The C in the text corresponds to N53-2-WT. Figure 10The D in J9709-SOCA9 corresponds to J9709-SOCA9. Figure 10 The E in this context corresponds to Ken-C8-SOCA9. Figure 10 The F in the text corresponds to N53-2-soca9. Figure 10 G in the figure corresponds to the seed coat rate of the six materials mentioned above. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0041] Example 1

[0042] The QTL determination of oil content in Brassica napus includes the following steps:

[0043] a) Using the existing known yellow-seeded rapeseed variety N53-2 with high oil content as the female parent and the existing known black-seeded rapeseed variety Ken-C8 with low oil content as the male parent, N53-2 is a DH line derived from the quaternary hybrid combination '1721-1B'·'start'×'955'·'SpringShaanxi2B' via microspore culture, while Ken-C8 is derived from the hybridization of 'SE8'×'midas'. After hybridization, pollen from the F1 generation was collected and subjected to microspore culture and chromosome doubling to obtain a haploid-diploid population (KN DH population) containing 300 lines.

[0044] b) Extract DNA from each line of the N53-2, Ken-C8 and KN DH populations, and use the 60K rapeseed SNP chip developed by Illumina to genotype the DNA. Based on the genotyping results, use joinmap 4.0 software to construct the genetic linkage map of the KN DH population.

[0045] c) The oil content of mature seeds of each line in the N53-2, Ken-C8 and KN DH populations was investigated. QTL mapping was performed using Windows QTL Cartographer 2.5 software in conjunction with genetic linkage maps, with the sliding window set to 1 cM and the lod value threshold set to 3.0.

[0046] Through the above steps, the inventors obtained a major QTL for oil content at the end of the A9 chromosome of Brassica napus. Its effect value is stable over many years and in multiple environments, and the additive effect exceeds 20%. The dominant allele comes from N53-2.

[0047] Fine mapping of the major QTL cqOCA9-9 site for oil content in Brassica napus included the following steps:

[0048] a) N53-2 and Ken-C8 were resequencing, and their sequence differences within cqOCA9-9 were analyzed. Primers were designed based on these sequence differences, and 17 molecular markers (A9-M1 to A9-M17) were developed. The primer pairs for these 17 molecular markers are shown in the table below.

[0049]

[0050] b) Select the line containing cqOCA9-9 in the KN DH population, backcross it with Ken-C8 for 5 generations, and then self-cross it for 1 generation to obtain the BC4F1 population containing 2820 individual plants;

[0051] c) Extract DNA from each individual in the BC4F1 population, use 17 molecular markers (A9-M1 to A9-M17) to genotype them, and construct a genetic linkage map using joinmap 4.0 software based on the genotyping results.

[0052] d) The seed oil content and seed lignocellulose content of each individual plant in the BC4F1 population at maturity were investigated. Combined with the genetic linkage map, QTL location was performed using Windows QTL Cartographer 2.5 software and the composite interval mapping method was used. The sliding window was set to 1 cM and the lod value threshold was 3.0.

[0053] e) Select 15 lines containing cqOCA9-9 and 15 lines not containing cqOCA9-9 from the BC4F1 population, and take seeds from them 14 days, 28 days and 42 days after flowering for transcriptome sequencing.

[0054] Through the above steps, the inventors shortened the major QTL for oil content in Brassica napus, cqOCA9-9, to a 69.8 kbps interval between molecular markers A9-M6 and A9-M7. Within this interval, 17 genes were annotated, and molecular markers A9-M6 and A9-M7 were tightly linked to cqOCA9-9. Simultaneously, the 17 genes within the fine-mapping region of cqOCA9-9 and their expression levels were obtained. Based on differences in expression levels, SOCA9 was identified as a functional gene within cqOCA9-9.

[0055] like Figure 4 As shown, there were significant differences in lignocellulose content and oil content between near-isogenic lines, specifically between single plants with the cqOCA9-9 region derived from Ken-C8 (NIL-Ken-C8), single plants with the cqOCA9-9 region derived from N53-2 (NIL-N53-2), and heterozygous single plants (NIL-H).

[0056] like Figure 9 As shown, after fine mapping of cqOCA9-9 in near-isogenic lines, it was located within a 69.8 kbps interval between A9-M6 and A9-M7; that is, A9-M6 and A9-M7 are less than 70 kb away from SOCA9 on the genome.

[0057] Example 2

[0058] Functional validation of the SOCA9 gene in Ken-C8 and N53-2 includes the following steps:

[0059] a) The complete gene sequence and promoter sequence of SOCA9 were obtained by PCR amplification and Sanger sequencing. At the same time, its ORF sequence and SOCA9 amino acid sequence were obtained based on the gene sequence. The complete gene sequence of SOCA9 is shown in SEQ ID No. 1, the complete promoter sequence of SOCA9 is shown in SEQ ID No. 2, the nucleotide sequence of the open reading frame (ORF) is shown in SEQ ID No. 3, and the amino acid sequence of SOCA9 protein is shown in SEQ ID No. 4.

[0060] b) SOCA9 was ligated into the expression vector pCAMBIA1303 and transformed into Ken-C8 by Agrobacterium-mediated hypocotyl genetic transformation to obtain the SOCA9 gene complementation line in the Ken-C8 background (Ken-C8-SOCA9).

[0061] c) Design gRNA based on SOCA9 gene sequence, link it to knockout vector pHSN401, and transform it into N53-2 by Agrobacterium-mediated hypocotyl genetic transformation to obtain SOCA9 gene knockout line (N53-2-soca9) in N53-2 background.

[0062] d) The seed oil content, lignocellulose content, and seed coat color of Ken-C8, Ken-C8-SOCA9, N53-2, and N53-2-soca9 at maturity were examined and compared. The F-test was used to analyze whether the gene complementation of SOCA9 had a significant effect on the oil content and other traits of Ken-C8. At the same time, the F-test was used to analyze whether the gene knockout of SOCA9 had a significant effect on the oil content and other traits of N53-2.

[0063] Through the above steps, SOCA9 gene replacement lines under the Ken-C8 background and SOCA9 gene knockout lines under the N53-2 background were obtained. Phenotypic analysis results showed that in Ken-C8, SOCA9 gene replacement significantly increased seed oil content by 8.99% (from 35.06% to 38.22%). Figure 1As shown), the acid-washed lignin (ADL) content in the seeds decreased significantly by 13.38% (from 19.68% to 17.05%). Figure 1 As shown), acid detergent fiber (ADF) decreased significantly by 29.62% (from 10.68% to 7.51%). Figure 1 As shown), neutral detergent fiber (NDF) decreased significantly by 62.81% (from 4.73% to 1.76%). Figure 1 (As shown). Meanwhile, as... Figure 3 and Figure 8 As shown, SOCA9 gene replacement resulted in a lighter seed coat color in Ken-C8 seeds, while the seed fatty acid composition showed no significant difference. Furthermore, SOCA9 gene replacement thinned the Ken-C8 seed coat, reducing the seed coat percentage by 25.38% (from 19.35% to 14.44%). Figure 10 (As shown).

[0064] In N53-2, knockout of the SOCA9 gene resulted in a significant decrease in seed oil content of 3.75% (from 50.40% to 48.51%). Figure 2 As shown), the acid-washed lignin content in the seeds increased significantly by 11.75% (from 15.28% to 17.07%). Figure 2 As shown), the acid detergent content of the fibers increased significantly by 8.85% (from 6.26% to 6.82%). Figure 2 As shown), the percentage of neutral detergent fibers increased significantly by 85.07% (from 0.92% to 1.71%). Figure 2 (As shown). Meanwhile, as... Figure 3 As shown, SOCA9 gene knockout darkens the seed coat color of N53-2. Additionally, SOCA9 gene knockout thickens the seed coat of N53-2, increasing the seed coat percentage by 10.75% (from 13.12% to 14.53%). Figure 10 (As shown).

[0065] Example 3

[0066] Linkage testing of molecular markers in near-isogenic lines includes the following steps:

[0067] a) Select individual plants containing cqOCA9-9 from the KN DH population, backcross them with Ken-C8 for 5 generations, and then self-cross them for 1 generation to obtain the BC5F1 population containing 2348 individual plants.

[0068] b) Extract DNA from each individual plant of the BC5F1 population and the aforementioned BC4F1 population, and examine the seed oil content and seed lignocellulose content of each individual plant of the BC5F1 population.

[0069] c) Use molecular markers A9-M6 and A9-M7 to genotype each individual plant in the BC4F1 and BC5F1 populations, and detect the presence of SOCA9 in each individual plant in the BC4F1 and BC5F1 populations;

[0070] d) Measure the seed oil content and seed lignocellulose content of each individual plant in the BC4F1 and BC5F1 populations at maturity, and compare the traits of homozygous plants containing the SOCA9 gene (NIL-N53-2), heterozygous plants containing the SOCA9 gene (NIL-H), and plants without the SOCA9 gene (NIL-Ken-C8). Use the F test to detect the significance of differences.

[0071] The results showed that among a total of 5165 individual plants in the BC4F1 and BC5F1 populations, A9-M6, A9-M7, and SOCA9 were always closely linked. In the BC5F1 population, A9-M6, A9-M7, SOCA9 were closely linked to seed oil content and seed lignocellulose content.

[0072] Table: Linkage relationship between molecular markers A9-M6 and A9-M7 and SOCA9

[0073]

[0074] As shown in the table above, molecular markers A9-M6, A9-M7, and SOCA9 were always closely linked in the segregating population, and no segregation occurred in a total of 5178 individual plants.

[0075] Example 4

[0076] The commercially available rapeseed variety ZS11 was improved through assisted breeding using molecular markers A9-M6 and A9-M7, including the following steps:

[0077] a) Cross N53-2 with the commercially available rapeseed variety ZS11, and obtain F1 lines by sowing hybrid seeds;

[0078] b) Self-pollinate the F1 line, harvest the self-pollinated seeds and sow them to obtain an F2 population containing 300 individual plants;

[0079] c) Extract DNA from each individual in the F2 population and use A9-M6 and A9-M7 to label and genotype it;

[0080] d) Examine the oil content and lignocellulose content of mature seeds of each F2 plant, and classify the plants containing A9-M6 and A9-M7 (ZS11) as examples. SOCA9 (Superscript in uppercase) and single plants not included (ZS11) soca9 (The superscript is lowercase English) to compare traits, and the F test was used to detect the significance of differences.

[0081] The results show that ZS11 SOCA9 Compared to ZS11 soca9 The oil content increased significantly by 4.26% (from 40.16% to 41.87%). Figure 5 As shown), the lignin content of acid-washed lignin decreased significantly by 18.45% (from 19.72% to 16.08%). Figure 5 As shown), acid washing significantly reduced fiber content by 28.36% (from 8.31% to 5.95%). Figure 5 As shown), the percentage of neutral detergent fibers decreased significantly by 59.71% (from 2.66% to 1.07%). Figure 5 (As shown).

[0082] Example 5

[0083] The following steps were taken to modify the rapeseed variety J9709 by SOCA9 gene replacement:

[0084] a) SOCA9 was ligated into the expression vector pCAMBIA1303 and transformed into the conventional Brassica napus material J9709 using Agrobacterium-mediated hypocotyl genetic transformation to obtain the SOCA9 gene complementation line (J9709-SOCA9) with the J9709 background.

[0085] b) Examine the oil content, lignocellulose content, and seed coat color of mature seeds of J9709 and J9709-SOCA9, and compare them. Use the F test to detect the significance of differences.

[0086] The results showed that in J9709, gene replacement with SOCA9 significantly increased seed oil content by 11.06% (from 40.72% to 45.23%). Figure 6 As shown), the acid-washed lignin content in the seeds decreased significantly by 22.84% (from 17.99% to 13.88%). Figure 6 As shown), acid washing significantly reduced fiber content by 40.32% (from 12.05% to 7.19%). Figure 6 As shown), the amount of neutral detergent fibers decreased significantly by 68.24% (from 5.52% to 1.75%). Figure 6 (As shown). Meanwhile, as... Figure 7 and Figure 8 As shown, SOCA9 gene replacement resulted in a lighter seed coat color in J9709, while the seed fatty acid composition remained largely unchanged. Furthermore, SOCA9 gene replacement thinned the seed coat of J9709, reducing the seed coat percentage by 22.59% (from 20.49% to 15.86%). Figure 10 (As shown).

[0087] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. SOCA9 The application of genes in regulating oil content, lignocellulose content, and / or seed coat color in Brassica napus seeds is characterized by, SOCA9 The nucleotide sequence of the gene is shown in SEQ ID No. 1; SOCA9 Gene expression can increase the oil content of rapeseed seeds, reduce the lignocellulose content, and lighten the seed coat color.

2. SOCA9 The application of genes in regulating the oil content of rapeseed seeds is characterized by, SOCA9 The nucleotide sequence of the gene is shown in SEQ ID No.

1. SOCA9 Gene expression can increase the oil content of rapeseed seeds.

3. SOCA9 The application of genes in the breeding of Brassica napus is characterized by, SOCA9 The nucleotide sequence of the gene is shown in SEQ ID No. 1; By breeding SOCA9 Gene replacement in Brassica napus can: increase the oil content of Brassica napus seeds, reduce the lignocellulose content, and lighten the seed coat color; Alternatively, by in breeding SOCA9 Gene knockout or gene silencing in Brassica napus can reduce the oil content of Brassica napus seeds, increase the lignocellulose content, and darken the seed coat color.

4. The application as described in claim 3, characterized in that, The application is achieved through breeding... SOCA9 Gene replacement was performed in Brassica napus to increase seed oil content.

5. The application of an SOCA9 protein in regulating the oil content, lignocellulose content, and / or seed coat color of Brassica napus seeds, characterized in that, The SOCA9 protein has the amino acid sequence shown in SEQ ID No. 4; Expression of SOCA9 protein can increase the oil content of rapeseed seeds, reduce the lignocellulose content, and lighten the seed coat color.

6. The application of an SOCA9 protein in regulating the oil content of rapeseed seeds, characterized in that, SOCA9 protein has the amino acid sequence shown in SEQ ID No. 4; SOCA9 protein can increase the oil content of rapeseed seeds.

Images