A gene BnaLACS related to oil content and nitrogen efficiency in rapeseed grains and its superior haplotype and application

Abstract

The present invention relates to the field of agricultural molecular biology, and in particular to a gene BnaLACS related to high nitrogen efficiency in oil content in rapeseed grains, and its excellent haplotype and application. The present invention found that there are 8 base mutations on the BnaLACS gene of rapeseed A09 chromosome that are closely related to high nitrogen efficiency in oil content in rapeseed grains. By detecting its genotype, it is possible to effectively distinguish the germplasm of rapeseed grain high nitrogen efficiency in oil content / low nitrogen efficiency in oil content. The present invention provides favorable gene resources and excellent haplotype germplasm for screening and breeding rapeseed varieties with high nitrogen efficiency in oil content, and significantly shortens the breeding cycle, reducing the time and workload in the breeding process. In addition, the present invention integrates OC LN ‑OC HN Diversified comprehensive methods such as phenotypic analysis, whole genome association analysis, LD analysis, and haplotype analysis not only discovered the gene BnaLACS related to rapeseed grain oil content and nitrogen efficiency and identified its excellent haplotype, but also provided new ideas for the discovery and application of other related genes, laying a theoretical foundation and technical framework for subsequent related research.

Description

Technical Field

[0001] The present invention relates to the field of agricultural molecular biology, and in particular to a gene BnaLACS related to the oil content and nitrogen efficiency of rapeseed grains, and its excellent haplotype and application. Background Art

[0002] Rapeseed is an annual plant of the genus Brassica in the family Brassicaceae. It is one of the major oil crops in the world and the largest oil crop in China. Improving rapeseed oil production is of great significance to ensuring the security of edible oil supply in my country. Rapeseed oil production is determined by two factors: the yield per unit area of ​​rapeseed and the oil content of the grain. Nitrogen fertilizer is crucial to the growth and development of plants. Increasing nitrogen fertilizer application to rapeseed can increase yield, but it will reduce the oil content of the grain. Unlike gramineous crops such as corn and rice, which focus on yield, the ultimate goal of rapeseed production is oil. In production, both yield and oil content of the grain must be increased. Therefore, it is of great significance to analyze the genetic basis of rapeseed oil nitrogen efficiency, explore oil-nitrogen efficient germplasm resources with no or insignificant decrease in oil content after nitrogen application, and cultivate new oil-nitrogen efficient varieties.

[0003] At present, relevant research reports are still at the stage of selecting and breeding rapeseed germplasm resources with high oil content and nitrogen efficiency based on traditional breeding methods. However, breeding using traditional methods has disadvantages such as low selection efficiency, great breeding difficulty, and long breeding cycle. Summary of the invention

[0004] In view of this, the present invention proposes a gene BnaLACS related to the oil content and nitrogen efficiency of rapeseed grains, its excellent haplotype and its application. Only by genetic typing detection, the oil content and nitrogen efficiency phenotype of rapeseed samples can be quickly identified, and then rapeseed samples with oil content and nitrogen efficiency can be selected, and the rapid breeding of oil content and nitrogen efficiency germplasm resources can be realized.

[0005] The technical solution of the present invention is achieved in this way:

[0006] In the first aspect, the present invention provides an application of gene BnaLACS in identifying excellent haplotypes with high oil content and high nitrogen efficiency in rapeseed grains, including: gene BnaLACS contains 8 mutation sites, and the mutation sites are respectively located at 8416147bp C / T base mutation, 8416322bp A / G base mutation, 8416337bp G / A base mutation, 8416403bp A / G base mutation, 8416529bp G / A base mutation, 8416532bp T / C base mutation, 8416544bp A / G base mutation and 8416625bp T / C base mutation on rapeseed A09 chromosome; when the haplotype is C+A+G+A+G+T+A+T, it is identified as an excellent haplotype with high oil content and high nitrogen efficiency in rapeseed grains.

[0007] In a second aspect, the present invention provides a method for detecting a rapeseed grain oil content nitrogen high efficiency / oil content nitrogen low efficiency phenotype, comprising: detecting the genotypes of eight mutation sites on rapeseed A09 chromosome: 8416147bp, 8416322bp, 8416337bp, 8416403bp, 8416529bp, 8416532bp, 8416544bp and 8416625bp; when the genotypes are CC, AA, GG, AA, GG, TT, AA and TT respectively, the rapeseed grain oil content nitrogen high efficiency phenotype is identified; when the genotypes are TT, GG, AA, GG, AA, CC, GG and CC respectively, the rapeseed grain oil content nitrogen low efficiency phenotype is identified.

[0008] In some preferred embodiments, the detection method includes sequencing, PCR amplification, specific probe hybridization method, specific primer extension method or gene chip method.

[0009] In a third aspect, the present invention provides a kit for detecting the oil content and nitrogen efficiency of rapeseed grains, the kit comprising reagents for detecting the genotypes of eight mutation sites on rapeseed A09 chromosome: 8416147bp, 8416322bp, 8416337bp, 8416403bp, 8416529bp, 8416532bp, 8416544bp and 8416625bp.

[0010] In a fourth aspect, the present invention also provides the use of the kit in the identification of high-efficiency oil content and low-efficiency oil content and nitrogen phenotype in rapeseed grains.

[0011] In a fifth aspect, the present invention also provides an application of the kit in rapeseed grain oil content and nitrogen-efficient breeding, comprising: based on the detection results of the kit on the genotypes of the eight mutation sites, selecting homozygous samples with genotypes of CC, AA, GG, AA, GG, TT, AA and TT for breeding.

[0012] In a sixth aspect, the present invention provides an application of the kit in any one of the following (A1)-(A3): (A1) preparing products for rapeseed breeding or assisted breeding; (A2) preparing products for identifying or assisted identifying the oil content and nitrogen efficiency phenotype of rapeseed grains; (A3) preparing products for breeding or assisted breeding of rapeseed grains with oil content and nitrogen efficiency phenotypes.

[0013] In a seventh aspect, the present invention provides a method for identifying an excellent haplotype with high nitrogen efficiency in rapeseed kernel oil content, comprising the following steps:

[0014] S1. Obtaining whole genome SNP data of a natural population of rapeseed, and phenotypic data of oil content and nitrogen efficiency of the grains of the natural population;

[0015] S2. Combining the whole genome SNP data with the natural population grain oil content and nitrogen high efficiency phenotype data, performing whole genome association analysis to obtain a significantly associated peak SNP, and selecting 200 kb segments upstream and downstream of the peak SNP for LD analysis;

[0016] S3 identifies excellent haplotypes with high grain oil content and high nitrogen efficiency in natural populations of rapeseed based on the SNP information that is significantly associated within the LD block linked to the peak SNP.

[0017] In some preferred embodiments, the natural population grain oil content and nitrogen high efficiency phenotypic data include: the difference in oil content of rapeseed grains at maturity under different nitrogen levels.

[0018] In some preferred embodiments, the different nitrogen levels specifically include: dividing the samples into a high nitrogen treatment group and a low nitrogen treatment group, applying nitrogen fertilizer with a high nitrogen content to the samples in the high nitrogen treatment group, and applying nitrogen fertilizer with a low nitrogen content to the samples in the low nitrogen treatment group.

[0019] In some preferred embodiments, the mass ratio of nitrogen application in the high nitrogen treatment group to the culture matrix is ​​0.3 g·kg-1, and the mass ratio of nitrogen application in the low nitrogen treatment group to the culture matrix is ​​0.1 g·kg-1. -1 .

[0020] The beneficial effects of the present invention include at least the following:

[0021] (1) The identification method of the gene BnaLACS and its excellent haplotype related to the high nitrogen efficiency of rapeseed grain oil content provided by the present invention not only provides favorable gene resources and excellent haplotype germplasm for screening and breeding rapeseed varieties with high nitrogen efficiency of oil content, but also significantly shortens the breeding cycle and reduces the time and workload in the breeding process. The application of the BnaLACS gene and its excellent haplotype enables the bred rapeseed varieties to show higher nitrogen utilization efficiency when the application of nitrogen fertilizer increases, effectively avoiding the potential negative impact of nitrogen fertilizer on oil content. Therefore, while ensuring or increasing rapeseed yield, it also further optimizes the quality of the grain, meeting the urgent needs of modern agriculture for efficient, environmentally friendly, and high-quality agricultural products.

[0022] (2) The present invention proposes a method for identifying an excellent haplotype with high nitrogen efficiency in oil content in rapeseed grains. First, a whole-genome association analysis is performed to accurately locate the SNP site that is significantly correlated with high nitrogen efficiency in oil content in rapeseed grains. Subsequently, linkage disequilibrium (LD) analysis is used to identify the potential candidate gene BnaLACS3 for high nitrogen efficiency in oil content in rapeseed in the segment near the peak SNP. Furthermore, by comparing with known homologous genes and verifying the expression level in grains under different nitrogen levels, it is confirmed that the BnaLACS3 gene is involved in nitrogen fertilizer regulation of rapeseed oil synthesis and metabolism. Based on the haplotype analysis of the BnaLACS3 gene, 8 base mutations were found in its CDS sequence, and an excellent haplotype with high nitrogen efficiency in oil content in rapeseed grains was successfully identified. The above analysis method also provides important theoretical and technical support for the mining and application of other related genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 A Manhattan diagram showing the chromosome segments associated with rapeseed grain oil content and nitrogen efficiency identified by genome-wide association analysis in an embodiment of the present invention;

[0025] Figure 2 This is a diagram showing the LD analysis results within a segment of about 0.005Mb of peakSNP of rapeseed A09 chromosome in an embodiment of the present invention;

[0026] Figure 3 It is a comparison diagram of the amino acid sequences encoded by the rapeseed BnaLACS and Arabidopsis thaliana AtLACS6 genes in the embodiments of the present invention;

[0027] Figure 4This is a graph showing changes in fatty acid content in grains at different developmental stages under different nitrogen treatments in an embodiment of the present invention;

[0028] Figure 5 This is a graph showing the relative expression of BnaLACS genes in grains under different nitrogen treatments in the embodiment of the present invention, wherein: LN represents a low nitrogen treatment group, and HN represents a high nitrogen treatment group;

[0029] Figure 6 The BnaLACS gene structure and non-synonymous mutation site polymorphism diagram in the embodiment of the present invention;

[0030] Figure 7 This is a diagram showing the analysis results of the nitrogen efficiency of rapeseed grain oil content of the three haplotypes of the BnaLACS gene in the example of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in this application are all conventional reagents and can be obtained from commercial sources; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by technicians in the technical field of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

[0032] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in the present invention refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the previously associated objects are in an "or" relationship.

[0033] Abbreviations and terms

[0034] Low nitrogen: Low nitrogen, LN;

[0035] High nitrogen: High nitrogen, HN;

[0036] Oil content: Oil content, OC;

[0037] Linkage disequilibrium analysis: Linkage disequilibrium, LD analysis;

[0038] Peak SNP: peakSNP;

[0039] Haplotype analysis: Haplotype analysis is a method of using genetic markers to conduct linkage analysis on family data and sequence a certain chromatid, as well as using mathematical models and computing software to estimate the frequencies of various haplotypes in a population.

[0040] Haplotype: also known as haploid genotype, refers to the combination of alleles at multiple loci inherited together on the same chromosome, and also refers to a type of single nucleotide polymorphism (SNP) with statistical association in a chromatid.

[0041] OC LN -OC HN : represents the difference in oil content (of rapeseed grains at maturity) under different nitrogen levels. That is, the oil content of rapeseed grains at maturity (represented as OC LN and OC HN ), therefore, the difference is recorded as OC LN -OC HN ;

[0042] Oil content nitrogen efficiency (OCNE) is evaluated based on the changes in oil content in rapeseed grains under different nitrogen levels. It aims to quantify the sensitivity and adaptability of rapeseed to changes in oil content in grains after the application of nitrogen fertilizer, so as to more effectively utilize nitrogen when nitrogen fertilizer increases, avoid or reduce the negative impact on oil content, and optimize the quality of rapeseed grains while ensuring or increasing rapeseed yield.

[0043] Oil content nitrogen high efficiency: the phenotype is that after increasing the nitrogen fertilizer supply, the oil content of rapeseed grains does not decrease or decreases insignificantly; in some preferred embodiments, specifically OC LN -OC HN Values ​​below the mean for a group;

[0044] Oil content nitrogen inefficiency: the phenotype is that after increasing the nitrogen fertilizer supply, the oil content of rapeseed grains decreases significantly; in some preferred embodiments, specifically OC LN -OC HN A value that is higher than the average for a group.

[0045] Example 1 Mining of genes related to rapeseed oil content and nitrogen efficiency

[0046] 1. Material selection and test treatment

[0047] The rapeseed materials used in the present invention are 70 Brassica napus diversity germplasm resources, which can be divided into: Asian resources (40 copies), European resources (23 copies), North American resources (3 copies), and Oceania resources (4 copies) according to the germplasm source. The materials are all provided by the National Oil Crop Germplasm Resources Medium-term Bank (Wuhan).

[0048] 70 Brassica napus germplasms were planted in pots on October 9, 2022 at the Yangluo Experimental Base in Wuhan, Hubei Province. The pot test was conducted in a drought shed, and the pots were arranged completely randomly. The materials were evenly sown in the pots on October 10, 2022. Plastic film was laid on the bottom of the pot to prevent nutrient and water loss. The potting soil was mixed in a ratio of yellow-brown soil: river sand = 2:1, and the soil alkaline nitrogen content was 32.38 mg kg -1 , organic matter content 9.23g·kg -1 , total nitrogen content 0.39g·kg -1 , available phosphorus content 8.58 mg kg -1 , available potassium content 77.80 mg·kg-1, soil pH 8.0.

[0049] The pot experiment was set up with low nitrogen (LN: N 0.1g kg -1 ) and high nitrogen (High nitrogen, HN: N 0.3g·kg -1 ) Two nitrogen treatments were used. The nitrogen fertilizer was urea (chemical formula: CH4N2O); N 0.1 g kg -1 It means that in every 1kg of culture medium (the culture medium in this embodiment is sandy soil), the amount of pure nitrogen contained in the nitrogen fertilizer applied is 0.1g. Three replicates were set for each nitrogen treatment, and 15 rapeseed seeds were sown in each replicate. Each pot was filled with 8kg of sandy soil, and the nitrogen fertilizer was mixed with the sandy soil and applied at one time. Other fertilizer dosage: KH2PO4 0.38 g·kg -1 , KCl 0.26 g kg -1 , H3BO35.75 mg·kg -1 During the winter, samples were taken, seedlings were thinned, and two plants were planted. Zinc and iron fertilizers were supplemented by applying nitrogen-deficient Hoagland solution. The nitrogen fertilizer pot experiment used gravimetric irrigation to maintain a soil moisture content of 60% to 75%.

[0050] 2. Phenotypic analysis

[0051] In order to screen and obtain nitrogen-efficient genes for rapeseed grain oil content, the inventors first detected the rapeseed grain oil content under different nitrogen treatments to preliminarily determine whether there are differences in the oil content phenotype between different germplasms under different nitrogen levels. The specific method is as follows:

[0052] When the plants were harvested, 3 replicates of 70 materials under different nitrogen treatments were selected, and 100 seeds from each plant were taken for oil content determination. The average value was taken as the oil content value of each germplasm material under this treatment. The oil content (OC) of rapeseed was determined by FossNIR Systems 5000 near infrared analyzer.

[0053] The difference in oil content under different nitrogen levels at maturity (OC LN -OC HN ) as a phenotypic indicator to analyze the oil content nitrogen efficiency of rapeseed, and it was divided into two types: (1) Oil content nitrogen efficiency type: OC LN -OC HN Lower than the average; (2) Oil content nitrogen inefficient type: OC LN -OC HN Above average.

[0054] 3. Mining candidate genes related to rapeseed grain oil content and nitrogen efficiency

[0055] (1) Genotype data collection and analysis

[0056] The genotype data of all germplasms were obtained by whole genome sequencing platform (Illumina HiSeq 4000 platform, sequencing depth was 17X), using Zhongshuang 11 (ZS11.v0) as the reference genome, and SNP genotype data were obtained.

[0057] (2) Genome-wide association analysis and gene mining

[0058] The genome-wide efficient mixed model software (GEMMA v.0.94.1) was used to convert OC LN -OC HN The data were combined with genotype data for genome-wide association analysis, and a mixed linear model (MLM): P+K model (controlling PCA and Kinship) was used to improve the accuracy of the association analysis.

[0059] The Manhattan plot of the association results was drawn using the R software qqman package. The threshold of the SNP site where the phenotypic data and the resequencing data were significantly associated was set to -log10(p)=5.0, and the candidate segment was located 200 kb before and after the significantly associated SNP site. Figure 1As shown, OC was found LN -OC HN There was a significant association signal on chromosome A09.

[0060] Linkage disequilibrium (LD) analysis was performed on the region near the peak SNP (e.g. Figure 2 As shown in Figure 3 ), gene BnaA09G0139900ZS (BnaLACS3) was found in the LD block linked to peakSNP.

[0061] The CDS sequence of the BnaLACS3 gene is 1983 bp in length, as shown in SEQ ID NO.1.

[0062] (3) Homologous gene comparison and verification

[0063] Referring to the annotation information of homologous genes in the Arabidopsis genome, it was found that the Arabidopsis AtLACS6 gene regulates fatty acid synthesis and metabolism, lipid biosynthesis, etc. in Arabidopsis.

[0064] The CrLACS3 gene in Chlamydomonas has been reported to be involved in lipid metabolism and synthesis under abiotic stresses such as nitrogen deficiency, affecting its total fatty acid content and triglyceride content. The Arabidopsis AtLACS6 and Chlamydomonas CrLACS3 genes have the highest similarity (BAIF, YU LH, ShI JN, LI-BEISSON YH, LIU J, 2022. Long-chain acyl-CoA synthetases activate fatty acids for lipid synthesis, remodeling and energy production in Chlamydomonas. New Phytologist.).

[0065] Comparison of the rapeseed BnaLACS3 and Arabidopsis thaliana AtLACS6 genes revealed that the amino acid sequences encoded by the BnaLACS3 and AtLACS6 genes were 35.11% similar ( Figure 3 ), indicating that the rapeseed BnaLACS3 gene located in the present invention has a lower sequence similarity with the Chlamydomonas oil content nitrogen efficiency-related gene CrLACS3.

[0066] Example 2 Verification of the expression level of gene BnaLACS3 in rapeseed grains under different nitrogen treatments

[0067] In order to further verify the expression of BnaLACS3 gene in grains under different nitrogen fertilizers, this example performed transcriptome sequencing on grains under different nitrogen fertilizer treatments. The specific method is as follows:

[0068] Plants were bagged when they were in full bloom and self-pollinated. One day before self-pollination, the flowers and pods that had already bloomed on the main inflorescence were removed and bagged. The flowers that were self-pollinated on the second day on the main inflorescence were marked as day 0 (day after pollination, DAP), and the bags were removed after 7 days. Siliques at 21DAP, 28DAP, 35DAP, 42DAP, 49DAP and 56DAP after flowering were taken, and the seeds were quickly separated from the silique skin, quickly frozen in liquid nitrogen, and then stored in a -80℃ refrigerator.

[0069] The internal standard method was used to determine the fatty acid content of samples at different developmental stages, and 42 DAP was the period with the greatest difference in oil content (e.g. Figure 4 As shown in Figure 2, samples of grains at 42 DAP under different nitrogen levels were selected and sent to Beijing Novogene Technology Co., Ltd. for sequencing. The transcriptome sequencing results showed that the relative expression level (FPKM value) of the BnaLACS3 gene under LN was significantly higher than that under HN, indicating that the BnaLACS3 gene is involved in the regulation of rapeseed oil synthesis and metabolism by nitrogen fertilizer (as shown in Figure 2). Figure 5 shown).

[0070] Example 3 Haplotype analysis of BnaLACS3

[0071] In order to further analyze whether the candidate gene BnaLACS3 is related to oil content and nitrogen efficiency, this example uses the genotype of the peak SNP associated with the above to perform genotyping on 70 potted materials, and then analyzes the phenotypic changes between different genotypes. The results are as follows: Figure 6 and Figure 7 As shown in Table 1, there are 8 base mutations in the CDS sequence of BnaLACS3 gene. According to the mutation type, it is divided into 3 haplotypes Hap1, 2 and 3. As shown in Table 1, there are 9, 21 and 40 haplotypes in 70 germplasms, respectively. LN -OC HN That is, after nitrogen application, the degree of oil content decrease in Hap3 individuals was significantly lower than that in Hap1, indicating that Hap3 is a haplotype with excellent oil content and nitrogen efficiency compared with Hap1.

[0072] Table 1 Haplotypes of BnaLACS3

[0073] Number of samples 8416147 8416322 8416337 8416403 8416529 8416532 8416544 8416625 9 TT GG AA GG AA CC GG CC Hap1 21 CT AG GA AG GA TC AG TC Hap2 40 CC AA GG AA GG TT AA TT Hap3

[0074] Note: 8416147, 8416322, 8416337, 8416403, 8416529, 8416532, 8416544 and 8416625 represent the base loci of rapeseed A09 chromosome.

[0075] like Figure 6As shown, the CDS of gene BnaLACS3 contains 8 mutation sites, which are located at 8416147 bp C / T base mutation, 8416322 bp A / G base mutation, 8416337 bp G / A base mutation, 8416403 bp A / G base mutation, 8416529 bp G / A base mutation, 8416532 bp T / C base mutation, 8416544 bp A / G base mutation and 8416625 bp T / C base mutation on rapeseed A09 chromosome. When the haplotype is C+A+G+A+G+T+A+T, it can be identified as an excellent haplotype with high nitrogen efficiency in rapeseed grain oil content.

[0076] The results showed that the genotypes of eight mutation sites on rapeseed A09 chromosome, namely 8416147 bp, 8416322 bp, 8416337 bp, 8416403bp, 8416529 bp, 8416532 bp, 8416544 bp and 8416625 bp, could accurately identify whether rapeseed was of high-efficiency nitrogen-oil content phenotype; when the sites were CC, AA, GG, AA, GG, TT, AA and TT respectively, it was identified as a high-efficiency nitrogen-oil content phenotype in rapeseed grains; when the sites were TT, GG, AA, GG, AA, CC, GG and CC respectively, it was identified as a low-efficiency nitrogen-oil content phenotype in rapeseed grains.

[0077] In summary, the present invention utilizes genes related to rapeseed grain oil content and nitrogen efficiency, and by detecting the genotypes of eight mutation sites on chromosome A09, namely 8416147bp, 8416322bp, 8416337bp, 8416403bp, 8416529bp, 8416532bp, 8416544bp and 8416625bp, the oil content and nitrogen efficiency phenotype of rapeseed samples can be quickly identified, rapeseed samples with oil content and nitrogen efficiency can be selected, and rapid breeding of oil content and nitrogen efficiency varieties can be achieved.

[0078] In addition, the present invention incorporates OC LN -OC HN Diversified comprehensive methods such as phenotypic analysis, whole genome association analysis, LD analysis, and haplotype analysis not only discovered the gene BnaLACS3 related to rapeseed grain oil content and nitrogen efficiency, and successfully identified the excellent haplotype of rapeseed grain oil content and nitrogen efficiency, but also provided new ideas for the discovery and application of other related genes, laying an important theoretical foundation and technical framework for subsequent related research.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of rapeseed gene BnaLACS3 in identification of excellent haplotypes with high oil content and high nitrogen efficiency in rapeseed grains, characterized in that: include: With reference to the double 11 rapeseed ZS11.v0, the rapeseed gene BnaLACS3 contains 8 mutation sites; the mutation sites are respectively located at 8416147bp C / T base mutation, 8416322bp A / G base mutation, 8416337bp G / A base mutation, 8416403bp A / G base mutation, 8416529bp G / A base mutation, 8416532bp T / C base mutation, 8416544bp A / G base mutation and 8416625bp T / C base mutation on the rapeseed A09 chromosome; when the haplotype is C+A+G+A+G+T+A+T, it is identified as an excellent haplotype with high oil content and nitrogen efficiency in rapeseed grains.

2. A method for detecting the high-efficiency / low-efficiency phenotype of oil-nitrogen content in rapeseed grains, characterized in that: include: With reference to Zhongshuang 11 rapeseed ZS11.v0, the genotypes of the eight mutation sites of 8416147bp, 8416322bp, 8416337bp, 8416403bp, 8416529bp, 8416532bp, 8416544bp and 8416625bp on the rapeseed A09 chromosome were detected; when they were CC, AA, GG, AA, GG, TT, AA and TT respectively, they were identified as rapeseed grain oil nitrogen high-efficiency phenotype; when they were TT, GG, AA, GG, AA, CC, GG and CC respectively, they were identified as rapeseed grain oil nitrogen low-efficiency phenotype.

3. The detection method according to claim 2, characterized in that: The detection method includes sequencing, PCR amplification, specific probe hybridization method, specific primer extension method or gene chip method.

4. Application of the detection method according to claim 2 in the identification of rapeseed grain oil content high nitrogen efficiency / oil content low nitrogen efficiency phenotype.

5. Application of the detection method of claim 2 in rapeseed seed oil content nitrogen efficient breeding, characterized in that: include: Based on the detection results of the genotypes of the eight mutation sites by the detection method, homozygous samples with genotypes of CC, AA, GG, AA, GG, TT, AA and TT were selected for breeding.

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