Molecular marker combination significantly related to effective branch number of rape, primer and application thereof

Abstract

The application provides a molecular marker combination significantly related to the effective branch number of oilseed rape and an application thereof, and belongs to the technical field of plant molecular breeding and gene editing. The molecular marker combination significantly related to the effective branch number of oilseed rape comprises one or more of the following: a DEL00022485 gene structure variation, a DEL00003485 gene structure variation, a DEL00022483 gene structure variation, a DEL00022460 gene structure variation, a DEL00022459 gene structure variation and a DEL00022484 gene structure variation. The application mines the above-mentioned molecular marker combination based on whole genome association analysis of gene structure variations, can accurately determine the number of effective branch numbers of oilseed rape germplasm, and further assists breeding. The application provides a new method and a new gene sequence resource for increasing effective branches and improving yield of Brassica napus breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding and gene editing technology, and in particular relates to molecular marker combinations, primers and their applications that are significantly related to the number of effective branches of oilseed plants. Background Technology

[0002] Brassica napus (AACC, 2n=38) is one of the major oilseed crops in China and the world, and is the main type of cultivated rapeseed. Brassica napus is an allotetraploid crop of the genus Brassica in the family Brassicaceae, possessing two subgenomes, A and C. It evolved approximately 7500 years ago from a natural hybridization of Chinese cabbage (Brassica rapa) and cabbage (Brassica oleracea) followed by genome doubling. Therefore, Brassica napus (hereinafter referred to as rapeseed) has a very rich genetic variation base. Branching is an important characteristic of plant architecture; the number of branches determines the number of flowers and seeds, thus affecting the final yield. Optimizing the branching structure helps adapt to different cultivation systems, improves stress resistance, and thus ensures the yield and quality of the rapeseed.

[0003] Effective branching in rapeseed refers to the primary branching of a single plant with one or more effective siliques, a crucial agronomical trait determining yield. Especially in multi-cropping systems, the optimal number of branches influences the number of effective siliques, thus determining yield per unit area. The development of effective branching depends not only on early nutrient accumulation but also on later environmental adaptability. In recent years, with the continuous optimization of agricultural planting patterns and the promotion of efficient planting technologies, the effective branching trait in rapeseed has received increasing attention. Particularly in the "oilseed-rice-rice" three-crop rotation system, the shortened growth period and adverse environmental conditions reduce the number of effective branches. Therefore, in production practice, rapeseed varieties with early and efficient branching can initiate branching growth at appropriate growth stages, ensuring both the quantity and quality of branches, thereby maximizing the utilization of land resources and photosynthetic products and increasing rapeseed yield. Therefore, breeding varieties and genetic materials with a high number of effective branches is crucial.

[0004] In the genome, single nucleotide polymorphisms (SNPs) and small insertions or deletions (INDELs) are two common types of genomic variation, playing important roles in the formation and regulation of agronomic traits. With the development of high-throughput sequencing technology, genome-wide association studies (GWAS) based on SNPs have been widely used in phenotypic and genotypic association studies, particularly in crop variety improvement and agronomic trait localization research. Taking the number of branches in plants as an example, previous studies have reported that homologous genes of *Arabidopsis thaliana* (At.) *Lateral ORGANFUSION2* (LOF2) and *CUPSHAPED COTYLEDON3* (CUC3) contain multiple SNPs in *Brassica napus*. These SNPs may play a role in the regulation of ecotype differentiation and axillary meristem formation. Compared to the genetic variations of SNPs, chromosomal structural variations (SVs) have a much wider range of variation, often ranging from tens to thousands of base pairs depending on the sequencing depth. They can cause changes in cis-regulatory regions (CREs) or even larger-scale variations, thereby altering gene expression levels and the traits they determine. Gene structural variations can be classified into unbalanced and balanced types. Unbalanced variations include presence and absence variations (PAVs) and copy number variants (CNVs), while balanced variations include inversions (INVs) and inter- and intrachromosomal translocations. These gene structural variations play a crucial role in plant trait innovation and adaptive evolution; across the entire genome, gene structural variations can influence plant phenotypes and, consequently, gene expression by generating large-scale sequence polymorphisms in cis-regulatory regions. Only a small percentage of SVs exist in coding sequence (CDS) regions. Gene structural variations in exons usually lead to gene loss of function, while SVs located in promoter and intron regions have more diverse effects on gene expression, and can promote or inhibit gene expression.In maize, the transposon (TE) insertion approximately 60 kb upstream of the tb1 gene plays a crucial role in altering maize plant structure and tiller number during domestication (Studer, A., Zhao, Q., Ross-Ibarra, J., and Doebley, J. (2011). Identification of a Functional Transposon Insertion in the Maize Domestication Gene tb1. Nat Genet 43:1160-1163.). Due to the high redundancy and complexity of the Brassica napus genome, the contribution of SVs to complex traits such as branching number has not yet been systematically analyzed. Future research should delve deeper into the genetic network regulating branching formation through structural variations in the context of polyploidization.

[0005] In Arabidopsis thaliana, BRANCHED1 (BRC1) encodes a TCP family transcription factor and is a key gene controlling the integration of branching signals into axillary buds. In peas, auxin promotes BRC1 expression and inhibits shoot growth by inducing strigolactone and inhibiting cytokinin (Dun, EA, de Saint Germain, A., Rameau, C., and Beveridge, CA (2012). Antagonistic Action of Strigolactone and Cytokinin in Bud Outgrowth Control. Plant Physiol 158:487-498.). Exogenous application of sucrose can also inhibit BRC1 expression and promote shoot release (Xia, XJ, Dong, H., Yin, YL, Song, XW, Gu, XH, Sang, KQ, Zhou, J., Shi, K., Zhou, Y., Foyer, CH, and Yu, JQ (2021). Brassinosteroid Signaling Integrates Multiple Pathways to Release Apical Dominance in Tomato. Proc Natl. AcadSci.USA118:e2004384118.); bZIP transcription factor LONG HYPOCOTYL 5 (HY5) is a major regulator of light-regulated responses. In tomato (Solanum lycopersicum, Sl), HY5 promotes shoot growth by directly inhibiting BRC1 transcripts and activating transcripts of brassinolide biosynthesis genes in tomato lateral buds (Dong, H., Wang, J., Song, X., Hu, C., Zhu, C., Sun, T., Zhou, Z., Hu, Z., Xia, X., Zhou, J., Shi, K., Zhou, Y., Foyer, CH., and Yu, JQ (2023). HY5 Functions as a Systemic Signal by Integrating BRC1-Dependent Hormone Signaling in Tomato Buds (Outgrowth.Proc.Natl.Acad.Sci.USA120:e2301879120.). BRC1 integrates multiple factors such as hormones, nutrients, and light, and is a core integrative factor for lateral bud growth. Therefore, the number of branches in a plant is influenced by the complex interaction between the environment and genotype, and methods for judging and regulating this trait have certain limitations.

[0006] With the development of CRISPR / Cas9 technology, targeted editing of candidate genes to knock out their function has become an important means of verifying the association between genes and specific traits. Currently, this technology mainly achieves loss of function by designing sgRNA in the coding region of a gene to induce frameshift mutations or premature translation termination, thereby altering the protein product sequence. However, in Brassica napus, how to systematically explore non-coding regulatory sites and utilize them to directionally improve agronomic traits such as plant architecture and stress resistance still requires further in-depth exploration. Summary of the Invention

[0007] In view of this, the purpose of this invention is to analyze the gene structure variation of large segments in the genome of core germplasm resources of Brassica napus, to discover new genetic loci that affect the formation of effective branches, to develop molecular marker combinations, and to verify the heredity and molecular function of some genes through gene editing technology, thereby creating new genetic sequences and directionally altering the agronomic trait of the number of effective branches, thus providing a new method for improving rapeseed traits.

[0008] This invention provides a combination of molecular markers that are significantly associated with the number of effective branches of an oilseed plant, including one or more of the following gene structural variations: DEL00022485, DEL00003485, DEL00022483, DEL00022460, DEL00022459, and DEL00022484.

[0009] The sequences of the DEL00022485 gene structural variant, DEL00003485 gene structural variant, DEL00022483 gene structural variant, DEL00022460 gene structural variant, DEL00022459 gene structural variant and DEL00022484 gene structural variant are shown in SEQ ID NO.16, SEQ ID NO.13, SEQ ID NO.17 to SEQ ID NO.20, respectively.

[0010] Preferably, it includes any one of the following:

[0011] 1) Structural variations in the DEL00022485 and DEL00003485 genes;

[0012] 2) Structural variations in the DEL00022485 and DEL00022483 genes;

[0013] 3) Structural variations in the DEL00022485 and DEL00022460 genes;

[0014] 4) Structural variations in the DEL00022485 and DEL00022459 genes;

[0015] 5) DEL00022485 gene structural variation and DEL00022484 gene structural variation.

[0016] This invention provides primers for amplifying the aforementioned molecular marker combination, including primers qOGB-4_DEL00022485_F and qOGB-4_DEL00022485_R for amplifying structural variations in the DEL00022485 gene. The sequence of qOGB-4_DEL00022485_F is shown in SEQ ID NO.3, and the sequence of qOGB-4_DEL00022485_R is shown in SEQ ID NO.4.

[0017] Primers qOGB-1_DEL00003485_F and qOGB-1_DEL00003485_R were used to amplify the structural variation of the DEL00003485 gene. The sequence of qOGB-1_DEL00003485_F is shown in SEQ ID NO.1, and the sequence of qOGB-1_DEL00003485_R is shown in SEQ ID NO.2.

[0018] Primers qOGB-5_DEL00022483_F and qOGB-5_DEL00022483_R were used to amplify the structural variation of the DEL00022483 gene. The sequence of qOGB-5_DEL00022483_F is shown in SEQ ID NO.5, and the sequence of qOGB-5_DEL00022483_R is shown in SEQ ID NO.6.

[0019] Primers qOGB-6_DEL00022460_F and qOGB-6_DEL00022460_R were used to amplify the structural variation of the DEL00022460 gene. The sequence of qOGB-6_DEL00022460_F is shown in SEQ ID NO.7, and the sequence of qOGB-6_DEL00022460_R is shown in SEQ ID NO.8.

[0020] Primers qOGB-7_DEL00022459_F and qOGB-7_DEL00022459_R were used to amplify the structural variation of the DEL00022459 gene. The sequence of qOGB-7_DEL00022459_F is shown in SEQ ID NO.9, and the sequence of qOGB-7_DEL00022459_R is shown in SEQ ID NO.10.

[0021] Primers qOGB-8_DEL00022484_F and qOGB-8_DEL00022484_R were used to amplify the structural variation of the DEL00022484 gene. The sequence of qOGB-8_DEL00022484_F is shown in SEQ ID NO.11, and the sequence of qOGB-8_DEL00022484_R is shown in SEQ ID NO.12.

[0022] This invention provides a method for screening rapeseed germplasm using the aforementioned molecular marker combination or primers, comprising the following steps:

[0023] 1) Extract genomic DNA from the rapeseed germplasm samples to be screened;

[0024] 2) Using the genomic DNA as a template, perform PCR amplification using the primers to obtain amplification products;

[0025] 3) Analysis results: When the amplification product of the DEL00003485 gene structural variation is 2168 bp, the rapeseed germplasm to be screened carries the DEL00003485 gene structural variation sequence and is considered as qOGB-1. 单倍型1 For plants; when the amplification product is 1545bp, the structural variation sequence of the DEL00003485 gene deletion in the rapeseed germplasm to be screened is considered as qOGB-1. 单倍型2 plant;

[0026] When the amplification product of the DEL00022485 gene structural variation is a 2040bp band, the rapeseed germplasm to be screened carries the DEL00022485 gene structural variation sequence and is considered as qOGB-4. 单倍型1 For plants, when the amplification product is a 143bp band, the rapeseed germplasm to be screened has a structural variation in the gene DEL00022485 deletion, which is considered as qOGB-4. 单倍型2 plant.

[0027] When the amplification product of the DEL00022483 gene structural variation is 890 bp, the rapeseed germplasm to be screened carrying the DEL00022483 gene structural variation sequence is considered as qOGB-5. 单倍型1 Plants; when the amplification product is 310 bp, the rapeseed germplasm to be screened has a gene structural variation sequence that deletes the DEL00022483 gene, which is considered as qOGB-5. 单倍型2 plant.

[0028] When the amplification product of the DEL00022460 gene structural variation is an 820bp band, the rapeseed germplasm to be screened carries the DEL00022460 gene structural variation sequence and is considered as qOGB-6. 单倍型1For plants; when the amplification product is 155bp, the rapeseed germplasm to be screened lacks the DEL00022460 sequence and is considered as qOGB-6. 单倍型2 plant.

[0029] When the amplification product of the DEL00022459 gene structural variation is a 254bp band, the rapeseed germplasm to be screened carries the DEL00022459 gene structural variation sequence and is considered as qOGB-7. 单倍型1 For plants, if no amplification product is found, the rapeseed germplasm to be screened should have the DEL00022459 sequence deleted, and should be considered as qOGB-7. 单倍型2 plant.

[0030] When the amplification product of the DEL00022484 gene structural variation is a 1363bp band, the rapeseed germplasm to be screened carries the DEL00022484 gene structural variation sequence and is considered as qOGB-8. 单倍型1 For plants; when the amplification product is 207 bp, the structural variation sequence of the DEL00022484 gene deletion in the rapeseed germplasm to be screened is considered as qOGB-8. 单倍型2 plant;

[0031] Select any of the following germplasm types that have a high number of effective branches:

[0032] qOGB-1 单倍型2 qOGB-4 单倍型1 qOGB-5 单倍型1 qOGB-6 单倍型1 qOGB-7 单倍型1 qOGB-8 单倍型1 .

[0033] Preferably, the following germplasm is selected based on the number of effective branches:

[0034] 4.1) Simultaneously qOGB-4 单倍型1 and qOGB-1 单倍型2 ;

[0035] 4.2) Simultaneously qOGB-4 单倍型1 and qOGB-5 单倍型1 ;

[0036] 4.3) Simultaneously qOGB-4 单倍型1 and qOGB-6 单倍型1 ;

[0037] 4.4) Simultaneously qOGB-4 单倍型1 and qOGB-7 单倍型1 ;

[0038] 4.5) Simultaneously qOGB-4单倍型1 and qOGB-8 单倍型1 .

[0039] This invention provides a guide RNA sequence that affects the effective number of branches in rapeseed, including sgRNA12 or sgRNA26; the sequence of sgRNA12 is shown in SEQ ID NO.25, and the sequence of sgRNA26 is shown in SEQ ID NO.26.

[0040] This invention provides the application of the guide RNA sequence in increasing the number of effective branches in rapeseed. The guide RNA is transferred into rapeseed germplasm using CRISPR / Cas9 gene editing technology to edit the genetic sequence of the BnBRC1 gene.

[0041] This invention provides genetic sequences for regulating the increase of the number of effective branches in rapeseed, as shown in SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.78 and SEQ ID NO.80.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention uses core germplasm resources of Brassica napus grown in the field to study the correspondence between the number of effective branches and gene structural variations. The natural population used has good genetic diversity and can represent various agronomic traits of rapeseed of different ecotypes. Based on genome-wide association analysis (GWAS) of gene structural variations (SVs), this invention identified six significant genetic loci affecting the number of effective branches, namely qOGB-1, qOGB-4 to qOGB-8, corresponding to six gene structural variations, DEL00003485, DEL00022485, DEL00022483, DEL00022460, DEL00022459, and DEL00022484. These gene structural variations are used as molecular markers to detect or screen the number of effective branches per rapeseed plant, assisting in rapeseed breeding.

[0044] Using qOGB-4 (i.e., the gene structural variation sequence DEL00022485) as the major genetic locus, and combining it with five other genetic loci to form molecular marker combinations, it is possible to accurately and rapidly identify rapeseed varieties with a high number of effective branches. For the number of effective branches per plant, an agronomic trait influenced by both genotype and environment, this invention can accurately detect or screen rapeseed germplasm with a high number of effective branches by combining multiple gene structural variations that affect the number of effective branches per plant.

[0045] This invention develops five molecular marker combinations for qOGB-4, qOGB-5, qOGB-6, qOGB-7, qOGB-8, and qOGB-1. Using these molecular marker combinations, the effective branch number of Brassica napus oleifera plants can be distinguished. qOGB-4 单倍型1 and qOGB-5 respectively 单倍型1 qOGB-6 单倍型1 qOGB-7 单倍型1 and qOGB-8 单倍型1 The combination of these methods can identify 9 to 11 effective branches with a high probability (25%–75%); qOGB-4 单倍型1 With qOGB-1 单倍型2 The combination can identify the effective number of branches in the range of 10 to 13.5 with a high probability (25% to 75%).

[0046] The primers provided by this invention for amplifying the molecular marker combination can be used for conventional PCR. By measuring the size of the amplified product fragment, it is possible to intuitively determine which haplotype of gene structure the rapeseed belongs to.

[0047] In this invention, qOGB-1 is associated with a TCP transcription factor BRANCHED 1 (BnaA1.BRC1) on chromosome A1, and possesses qOGB-1. 单倍型2 Plants lacking the 623bp DEL00003485 gene structural variation sequence exhibited significantly reduced expression of the BnaA1.BRC1 gene and a significantly increased number of effective branches per plant. This invention designed 46 different guide RNAs (sgRNAs) targeting the coding sequences (CDS) of BnaA1.BRC1 and four other homologous genes in the spring rapeseed variety Westar. Two sgRNA sequences, sgRNA12 and sgRNA26, were selected, and genetic sequences of five BnBRC1 genes were edited using CRISPR / Cas9 technology. A T3 generation five-mutant bnbrc1 plant was obtained, generating new genetic sequences in the first exons of the five BnBRC1 genes, resulting in a significant increase in the number of effective branches on the main stem of each rapeseed plant. This method provides an identification method and gene resource for screening rapeseed varieties with a high number of effective branches at both the genetic locus and gene level. Attached Figure Description

[0048] Figure 1 These are representative germplasms of Brassica napus with a high or low number of effective branches, and haplotype combinations of different gene structural variations.

[0049] Figure 2A genome-wide analysis of gene structural variations yielded 12 significant qOGB genetic loci, where (a) is a Manhattan plot of the 12 significant qOGB genetic loci; and (b) shows the gene structural variations of qOGB-1 on chromosome A1 (left) and qOGB-4 to qOGB-9 on chromosome A4 (right), all located in highly linked disequilibrium regions.

[0050] Figure 3 The 12 qOGB gene structural variations are shown alongside their adjacent genes; the gene structural variations from qOGB-1 to qOGB-12 are all located in the non-coding regions of the genes; green areas represent exons, blue areas represent deletion SVs, blank areas represent non-coding regions, and dashed lines represent insertion SVs.

[0051] Figure 4 To investigate the effect of qOGB on the number of effective branches per plant and to validate the effect of five molecular marker combinations on 23 typical germplasm resources, the following molecular marker combinations were used: (a) qOGB-4 and qOGB-1; (b) qOGB-4 and qOGB-5; (c) qOGB-4 and qOGB-6; (d) qOGB-4 and qOGB-7; (e) qOGB-4 and qOGB-8; and (f) the five molecular marker combinations effectively distinguished the number of effective branches in 16 accessions. More than 7 germplasms with fewer effective branches; lanes (a) to (e) 1 are DNA molecular standard quantities; lanes 2 to 24 are germplasms of R4268, R4718, R4728, R4731, R4894, R5018, R5032, R5033, R5038, R5041, R5043, R5044, R5059, R5067, R5073, R5156, R4181, R4182, R4213, R4272, R4283, R4314 and R4342, respectively.

[0052] Figure 5 The expression levels of BnaA1.BRC1 in different haplotypes of rapeseed at the qOGB-1 site.

[0053] Figure 6 To edit five homologous genes of BnBRC1 in the Brassica napus variety Westar, (a) shows the gene structure of the five homologous genes of BnBRC1 in Brassica napus Westar, and the positions of sgRNA12 and sgRNA26; (b) shows a T3 generation mutant bnbrc1 plant with homozygous five homologous genes, whose new genetic sequence features are characterized by small deletions or insertions.

[0054] Figure 7Compared with Brassica napus Westar, the T3 generation homozygous mutant bnbrc1 had an increased number of effective branches per plant. (a) Branching phenotype of Westar and bnbrc1 mutants at maturity; (b) Compared with wild type, bnbrc1 mutant had an increased number of effective branches on the main stem. Detailed Implementation

[0055] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] (1) In mid-October of that year, the seeds of Brassica napus were sown directly in the experimental field. The same variety of rapeseed germplasm was planted in one plot of experimental field. The plot was 1.5 meters long and 1.5 meters wide. Before overwintering, the seedlings were fixed at a spacing of 30 centimeters between rows and columns. The final planting density was 16 plants per plot.

[0058] (2) In the later stage of rapeseed population growth the following year, usually at the end of May or the beginning of June, phenotypic determination and analysis were carried out; in the field in Jiaxing, the number of primary branches of a single plant with one or more effective siliques in the later stage of growth was counted, and 3 representative samples were selected from each planting plot as replicates for each material.

[0059] (3) Statistical data on the number of primary branches of a single plant bearing one or more effective siliques in the later stages of growth of different germplasm materials were analyzed using a genome-wide association study (SV-GWAS) based on structural variation. Using the GEMMAv0.98.1 software package, the resequencing data of the core germplasm were aligned to the ZS11.v0 reference genome, with a threshold set to 4. Based on a mixed linear model, genetic loci affecting the number of effective branches were screened out and named quantitative loci. O The utgrowingBranch sites, abbreviated as qOGB, consist of 12 sites, named qOGB-1 to qOGB-12. Figure 2 This includes 10 large deletion mutations (DEL) and 2 large insertion mutations (INS) (e.g. Figures 1-3 (Table 1).

[0060] (4) Through the analysis in (3), a total of 12 different qOGB sites of genomic structural variations were identified, as shown in Table 1. The gene structural variation DEL00003485 belongs to the qOGB-1 site and is located in the upstream region of the BnaA01G0326500ZS gene. The gene structural variations DEL00022485, DEL00022483, DEL00022460, DEL00022459, and DEL00022484 belong to qOGB-4 to qOGB-8 sites, respectively (Table 1). They all belong to genomic structural variations in non-coding regions and are in high linkage disequilibrium. Figures 2-3 BnaA01G0326500ZS encodes the BnaA1.BRC1 gene, which, along with four other homologous genes, is collectively referred to as BnBRC1.

[0061] Table 1 Gene structural variations affecting the number of effective branches per plant

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] The gene structural variant DEL00004495, located at the qOGB-2 genetic locus, is 160 bp in length and is shown in sequence SEQ ID NO. 14. It is situated in the intergenic region between the two genes BnaA02G0004100ZS and BnaA02G0004200ZS. Figures 2-3 (Table 1).

[0069] The gene structural variant DEL00011547, located at the qOGB-3 genetic locus, is 2542 bp in length and, as shown in SEQ ID NO. 15, lies in the intergenic region between the two genes BnaA03G0098900ZS and BnaA03G0099000ZS. Figures 2-3 (Table 1).

[0070] The structural variants DEL00022485, DEL00022483, and DEL00022484 are located at the qOGB-4, qOGB-5, and qOGB-8 genetic loci, respectively, with lengths of 1897 bp, 580 bp, and 1156 bp, as shown in SEQ ID NO.16, NO.17, and NO.20. These three linked structural variants are located in the intergenic region between the genes BnaA04G0189200ZS and BnaA04G0189300ZS. Figures 2-3 (Table 1).

[0071] The gene structural variant DEL00022460, located at the qOGB-6 genetic locus, is 665 bp in length and, as shown in SEQ ID NO. 18, lies in the intergenic region between the genes BnaA04G0188300ZS and BnaA04G0188400ZS. The gene structural variant DEL00022459, located at the qOGB-7 genetic locus, is 225 bp in length and, as shown in SEQ ID NO. 19, lies in the intergenic region between the genes BnaA04G0188200ZS and BnaA04G0188300ZS. These three genes are linked to the two gene structural variants. Figures 2-3 (Table 1).

[0072] The gene structural variant INS00022504 is located at the qOGB-9 genetic locus, with a length of 32 bp, as shown in the sequence SEQ ID NO.21, in the intergenic region between the two genes BnaA04G0190000ZS and BnaA04G0190100ZS.

[0073] The structural variant DEL00053080, located at the qOGB-10 genetic locus, is 100 bp in length and is shown in sequence SEQ ID NO. 22. It is situated in the intergenic region between the two genes BnaA09G0051600ZS and BnaA09G0051700ZS. Figures 2-3 (Table 1).

[0074] The structural variant DEL00105694, located at the qOGB-11 genetic locus, is 505 bp in length and, as shown in SEQ ID NO. 23, lies in the intergenic region between the genes BnaC03G0345100ZS and BnaC03G0345200ZS. Figures 2-3 (Table 1).

[0075] The structural variant INS00177470, located at the qOGB-12 genetic locus, is 30 bp in length and, as shown in sequence SEQ ID NO. 24, lies in the intergenic region between the two genes BnaC08G0485100ZS and BnaC08G0485200ZS. Figures 2-3 (Table 1).

[0076] Of the 12 genetic loci mentioned above, qOGB-1, qOGB-4 to qOGB-8 have a significant effect on the number of effective branches. Other loci have a statistical effect on the number of effective branches, but the effect is weak. Therefore, further research should be conducted on qOGB-1, qOGB-4 to qOGB-8.

[0077] (5) Haplotype analysis of the number of effective branches per plant was performed on the six gene structural variations of qOGB-1, qOGB-4 to qOGB-8. The haplotype carrying the DEL00003485 gene structural variation sequence was named qOGB-1. 单倍型1 The missing one is named qOGB-1 单倍型2 The haplotype carrying the structural variant of the DEL00022485 gene is named qOGB-4. 单倍型1 The missing one is named qOGB-4 单倍型2 The haplotype carrying the DEL00022483 gene structural variant is named qOGB-5. 单倍型1 The missing one is named qOGB-5 单倍型2 The haplotype carrying the DEL00022460 gene structural variant is named qOGB-6. 单倍型1 The missing one is named qOGB-6 单倍型2 The haplotype carrying the DEL00022459 gene structural variant is named qOGB-7. 单倍型1 The missing one is named qOGB-7 单倍型2 The haplotype carrying the DEL00022484 gene structural variant is named qOGB-8. 单倍型1 The missing one is named qOGB-8 单倍型2 Using structural variations in the qOGB-4 gene as the major variable sites, the qOGB-4 variant with the most effective branches was selected. 单倍型1 Germplasm, respectively with qOGB-1 单倍型2 qOGB-5 单倍型1 qOGB-6 单倍型1 qOGB-7 单倍型1 or qOGB-8 单倍型1 Intersection of germplasm ( Figure 4Five haplotype combinations with a high number of effective branches were screened (Table 2). Primers were designed for these six gene structural variations (Table 3) to verify the consistency between genotype and phenotype. Figure 4 ).

[0078] Table 2. Statistical table of different haplotypes and effective branch number of rapeseed.

[0079]

[0080] (6) The haplotype carrying the DEL00003485 gene structural variant sequence on the BnaA01G0326500ZS / BnaA1.BRC1 gene is BnaA1.BRC1. 单倍型1 The missing value is BnaA1.BRC1. 单倍型2 ( Figure 5 (a) BnaA1.BRC1 with deletion of the DEL00003485 gene structural variant. 单倍型2 The Brassica napus germplasm exhibited an agronomical trait phenotype with increased effective branching, and the expression level of BnaA1.BRC1 during bud development was significantly lower than that of BnaA1.BRC1 containing this gene structural variation. 单倍型1 ( Figure 5 (b) This locus significantly affects effective branching, providing an important genetic basis for cultivating multi-branched, high-yielding rapeseed.

[0081] (7) Molecular markers were designed for the structural variant qOGB-4 / DEL00022485 as shown in SEQ ID NO.3 and NO.4. Molecular markers were designed for qOGB-1 / DEL00003485 as shown in SEQ ID NO.1 and NO.2. Molecular markers were designed for qOGB-5 / DEL00022483 as shown in SEQ ID NO.5 and NO.6. Molecular markers were designed for qOGB-6 / DEL00022460 as shown in SEQ ID NO.7 and NO.8. Molecular markers were designed for qOGB-7 / DEL00022459 as shown in SEQ ID NO.9 and NO.10. Molecular markers were designed for qOGB-8 / DEL00022484 as shown in SEQ ID NO.11 and NO.12. Sequence information is shown in Table 3. Using 5 μl of KOD enzyme (Toyobo, KMM-201), 0.3 μl of 10 μM forward primer, 0.3 μl of 10 μM reverse primer, and 0.5 μl of extracted genomic DNA, 3.9 μl of water were added, for a total system of 10 μl. The experiment was conducted using a commercially available PCR instrument according to the reaction conditions in Table 3. A total of 35 cycles were performed from denaturation to extension. After the experiment, 2 μl of the sample was run on a 1% agarose gel for electrophoresis to determine the band size.

[0082] Table 3 Primer design and PCR reaction conditions for gene structural variation sites.

[0083]

[0084] Table 4 Germplasm names of 278 accessions of Brassica napus

[0085]

[0086]

[0087]

[0088]

[0089] The above-mentioned germplasm is core rapeseed germplasm from different regions around the world, as documented in the following literature:

[0090] The Intronic Structure Variation of Rapeseed BnaC3.LEAFY Regulates the Timing of Inflorescence Formation and Flowering. Mengjie Gong, Guangyu Wu, Xiuchen Weng, Huiqi Zhang, Tao Zhou, Wenbin Guo, Shuijin Hua, Weizhen Hu, Shan Liang, Yifeng Xu, Shuijin Hua, Lixi Jiang and Yang Zhu* 2025 Plant Communications https: / / doi.org / 10.1016 / j.xplc.2025.101318.

[0091] Wu D,Liang Z,Yan T,XuY,Xuan L,Tang J,Zhou G,Lohwasser U,Hua S,Wang H,Wang Q,Zhu L,MaodzekaA,Hussain N,Li Z,Li X,Shamsi IH,Jilani G,Wu L,Zheng H,Zhang G,Chalhoub B,Shen L,Yu H,Jiang LX(2019).Whole-genome resequencing of aworld-wide collection of rapeseed accessions reveals genetic basis oftheirecotype divergence.MolecularPlant 12-36-43.

[0092] (8) The Brassica napus variety Westar is a BnaA1.BRC1 with the seed code qOGB-1 / DEL00003485. 单倍型1 Based on the germplasm, targeting the first exon sequences of five BnBRC1 homologous genes in Westar, a total of 46 sgRNA sequences were designed using the CRISPR / Cas9 gene editing system (Table 5). Analysis and experiments showed that sgRNA12 (SEQ ID NO. 25) and sgRNA26 (SEQ ID NO. 26) can target the five BnBRC1 homologous genes and generate new genetic sequences such as single-base insertions or multi-base deletions within the editing window, resulting in the loss of gene function.

[0093] Table 5. Design of sgRNA sequences for the first exons of five BnBRC1 homologous genes in Westar rapeseed.

[0094]

[0095]

[0096]

[0097] (9) Using the sgRNA design and CRIPSPR / Cas9 technology from (8), a new material with five homozygous mutant homologous genes was created in Westar and named bnbrc1. In this T3 generation plant, it was found that the coding regions on the first exon (exon 1 or e1) of the five homologous genes produced new gene sequences. Figure 6 Among them, the BnaA1.BRC1, BnaC1.BRC1, and BnaC5.BRC1 genes have a 1-base insertion in the first exon. The BnaA3.BRC1 gene has a 2-base deletion at sgRNA26 and an 11-base deletion at sgRNA12. The BnaC3.BRC1 gene has a 4-base deletion at sgRNA26 and a 1-base insertion at sgRNA12 (Table 6). In a plant culture chamber, using white LED lights, a light intensity of 20,000 lux, a 16-hour day length, and a temperature of 24 degrees Celsius, pot experiments were conducted. It was found that compared with the wild-type Westar, the bnbrc1 plants exhibited a phenotype with an increased number of effective branches. Figure 7 This study demonstrates the negative effect of BnBRC1 on the effective branching formation of rapeseed, providing important genetic resources for molecular breeding of Brassica napus.

[0098] Table 6 shows the new sequences generated by the five homologous genes of BnBRC1 in Westar rapeseed.

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Note: CRISPR / Cas9-mediated base deletions are indicated with strikethroughs, base insertions are indicated with underlines and italics, sgRNA recognition sequences are marked with black underlines, and PAM recognition sites are bolded with NGG.

[0105] As can be seen from the above embodiments, this invention mainly focuses on the analysis of gene structural variations in large segments of the genome of core germplasm resources of Brassica napus. It has identified structural variation sequences qOGB-4 / DEL00022485, qOGB-1 / DEL00003485, qOGB-5 / DEL00022483, qOGB-6 / DEL00022460, qOGB-7 / DEL00022459, and qOGB-8 / DEL00022484 that may affect the number of effective branches. Primers based on these structural variation sequences are developed to screen germplasm resources of Brassica napus that have multiple effective branching traits, which is beneficial to accelerating rapeseed breeding. The gene structural variant DEL00003485 belongs to the qOGB-1 site and is located in the upstream region of the BnaA01G0326500ZS / BnaA1.BRC1 gene. This invention also verified the genetic and molecular function of BnBRC1 through gene editing technology, created a new genetic sequence, and directionally changed the agronomic trait of effective branching number.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker combination significantly correlated with the number of effective branches of an oilseed plant, characterized in that, Includes any one of the following: 1) to 5) 1) Structural variations in the DEL00022485 and DEL00003485 genes; 2) Structural variations in the DEL00022485 and DEL00022483 genes; 3) Structural variations in the DEL00022485 and DEL00022460 genes; 4) Structural variations in the DEL00022485 and DEL00022459 genes; 5) Structural variations in the DEL00022485 and DEL00022484 genes; The sequences of the DEL00022485 gene structural variant, DEL00003485 gene structural variant, DEL00022483 gene structural variant, DEL00022460 gene structural variant, DEL00022459 gene structural variant and DEL00022484 gene structural variant are shown in SEQ ID NO.16, SEQ ID NO.13 and SEQ ID NO.17~SEQ ID NO.20, respectively.

2. Primers for amplifying the molecular marker combination of claim 1, characterized in that, Primers for amplifying structural variations in the DEL00022485 gene. qOGB-4 _DEL00022485_F and qOGB-4 _DEL00022485_R, the qOGB-4 The sequence _DEL00022485_F is shown in SEQ ID NO.

3. qOGB-4 The sequence of _DEL00022485_R is shown in SEQ ID NO.4; Primers for amplifying the structural variation of the DEL00003485 gene qOGB-1 _DEL00003485_F and qOGB-1 _DEL00003485_R, the qOGB-1 The sequence _DEL00003485_F is shown in SEQ ID NO.

1. qOGB-1 The sequence of _DEL00003485_R is shown in SEQ ID NO.2; Primers for amplifying the structural variation of the DEL00022483 gene qOGB-5 _DEL00022483_F and qOGB-5 _DEL00022483_R, the qOGB-5 The sequence _DEL00022483_F is shown in SEQ ID NO.

5. qOGB-5 The sequence of _DEL00022483_R is shown in SEQ ID NO.6; Primers for amplifying the structural variation of the DEL00022460 gene qOGB-6 _DEL00022460_F and qOGB-6 _DEL00022460_R, the qOGB-6 The sequence _DEL00022460_F is shown in SEQ ID NO.

7. qOGB-6 The sequence of _DEL00022460_R is shown in SEQ ID NO.8; Primers for amplifying the structural variation of the DEL00022459 gene qOGB-7 _DEL00022459_F and qOGB-7 _DEL00022459_R, the qOGB-7 The sequence _DEL00022459_F is shown in SEQ ID NO.

9. qOGB-7 The sequence of _DEL00022459_R is shown in SEQ ID NO.10; Primers for amplifying the structural variation of the DEL00022484 gene qOGB-8 _DEL00022484_F and qOGB-8 _DEL00022484_R, the qOGB-8 The sequence _DEL00022484_F is shown in SEQ ID NO.

11. qOGB-8 The sequence of _DEL00022484_R is shown in SEQ ID NO.

12.

3. A method for screening rapeseed germplasm using the molecular marker combination of claim 1 or the primers of claim 2, characterized in that, Includes the following steps: 1) Extract genomic DNA from the rapeseed germplasm samples to be screened; 2) Using the genomic DNA as a template, perform PCR amplification using the primers described in claim 2 to obtain amplification products; 3) Analysis results: When the amplification product of the DEL00003485 gene structural variation is 2168 bp, the rapeseed germplasm to be screened carries the DEL00003485 gene structural variation sequence and is considered to be... qOGB-1 单倍型1 For plants; when the amplification product is 1545 bp, the structural variation sequence of the DEL00003485 gene deletion in the rapeseed germplasm to be screened is considered as... qOGB-1 单倍型2 plant; When the amplification product of the DEL00022485 gene structural variation is a 2040 bp band, the rapeseed germplasm to be screened carries the DEL00022485 gene structural variation sequence and is considered to be... qOGB-4 单倍型1 In plants, when the amplification product is a 143 bp band, the rapeseed germplasm to be screened has a gene deletion of DEL00022485 structural variation, which is considered as... qOGB-4 单倍型2 plant; When the amplification product of the DEL00022483 gene structural variation is 890 bp, the rapeseed germplasm to be screened carries the DEL00022483 gene structural variation sequence and is considered to be... qOGB-5 单倍型1 In plants, when the amplification product is 310 bp, the DEL00022483 gene structural variation sequence in the rapeseed germplasm to be screened is considered as... qOGB-5 单倍型2 plant; When the amplification product of the DEL00022460 gene structural variation is an 820 bp band, the rapeseed germplasm to be screened carries the DEL00022460 gene structural variation sequence and is considered to be... qOGB-6 单倍型1 For plants; when the amplification product is 155 bp, the rapeseed germplasm to be screened is considered to have a deletion of the DEL00022460 sequence. qOGB-6 单倍型2 plant; When the amplification product of the DEL00022459 gene structural variation is a 254 bp band, the rapeseed germplasm to be screened carries the DEL00022459 gene structural variation sequence and is considered to be... qOGB-7 单倍型1 In plants, if no amplification product appears, the rapeseed germplasm to be screened is considered to have the DEL00022459 sequence missing. qOGB-7 单倍型2 plant; When the amplification product of the DEL00022484 gene structural variation is a 1363 bp band, the rapeseed germplasm to be screened carries the DEL00022484 gene structural variation sequence and is considered to be... qOGB-8 单倍型1 For plants, when the amplification product is 207 bp, the DEL00022484 gene structural variation sequence in the rapeseed germplasm to be screened is considered as... qOGB-8 单倍型2 plant; Select any of the following germplasm types that have a high number of effective branches: 1) At the same time qOGB-4 单倍型1 and qOGB-1 单倍型2 ; 2) At the same time qOGB-4 单倍型1 and qOGB-5 单倍型1 ; 3) At the same time qOGB-4 单倍型1 and qOGB-6 单倍型1 ; 4) At the same time qOGB-4 单倍型1 and qOGB-7 单倍型1 ; 5) At the same time qOGB-4 单倍型1 and qOGB-8 单倍型1 .

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