Molecular marker combination remarkably related to effective branch number of rape, primer and application of molecular marker combination

Abstract

The invention provides a molecular marker combination remarkably related to the effective branch number of rape and application of the molecular marker combination, and belongs to the technical field of plant molecular breeding and gene editing. The molecular marker combination remarkably related to the effective branch number of the rape comprises one or more of 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 molecular marker combination is excavated based on whole genome association analysis of gene structure variation, and the number of effective branches of rape germplasm can be accurately judged so as to assist in breeding; a new method and a new gene sequence resource are provided for breeding of the brassica napus capable of increasing effective branches and increasing the yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding and gene editing, and in particular relates to a molecular marker combination, primers and applications thereof that are significantly correlated with the number of effective branches of a single oilseed plant. Background Art

[0002] Brassica napus (AACC, 2n=38) is a major oilseed crop in China and around the world, and the primary type of cultivated rapeseed. It is an allotetraploid crop in the genus Brassica, Brassica family, with two subgenomes, A and C. It originated approximately 7,500 years ago from a natural hybridization between Brassica rapa and Brassica oleracea, resulting in genome doubling. Consequently, Brassica napus (hereinafter referred to as rapeseed) possesses a rich genetic base of variation. Branching is a key characteristic of plant architecture; the number of branches determines the number of flowers and seeds, and thus the final yield. Optimizing branching structure facilitates adaptation to diverse cultivation systems, improves stress resistance, and thus ensures rapeseed yield and quality.

[0003] Effective branching in rapeseed refers to the number of branches bearing one or more valid siliques on a single plant. It is a key agronomic trait that determines yield. In particular, in multiple-cropping production systems, the appropriate number of branches influences the number of valid siliques, and thus the yield per unit area. The production of effective branches depends not only on early nutrient accumulation but also on later environmental adaptability. In recent years, with the continuous optimization of agricultural cropping patterns and the promotion of efficient planting techniques, effective branching in rapeseed has garnered increasing attention. In particular, in the "oil-rice-rice" three-cropping rotation system, the number of effective branches decreases due to the shortened growing season and adverse environmental conditions. Therefore, in practical production, rapeseed varieties with early and efficient branching are crucial. They can initiate branching at the appropriate growth stage, maintain branch quantity and quality, and maximize land resources and photosynthetic output, thereby increasing rapeseed yield. Therefore, breeding varieties and genetic material with a high number of effective branches is crucial.

[0004] Single nucleotide polymorphisms (SNPs) and small insertions or deletions (INDELs) are two common types of genomic variation in the genome, playing an important role in the formation and regulation of agronomic traits. With the development of high-throughput sequencing technology, SNP-based genome-wide association studies (GWAS) have been widely used to study the association between phenotype and genotype, particularly in crop variety improvement and the mapping of agronomic traits. Taking plant branch number as an example, previous studies have reported that the orthologous genes of LATERAL ORGANFUSION2 (LOF2) and CUPSHAPED COTYLEDON3 (CUC3) in Arabidopsis thaliana (At) contain multiple SNPs in Brassica napus. These SNPs may play a role in ecotype differentiation and the regulation of axillary meristem formation. Compared to the genetic variation caused by single-nucleotide polymorphisms (SNPs), chromosomal structural variations (SVs) have a much wider range of variation, often ranging from tens to thousands of base pairs (bp) depending on sequencing depth. These variations can cause changes in cis-regulatory regions (CREs) or even larger scales, thereby altering gene expression and the traits they determine. Structural variations can be categorized as either unbalanced or balanced. 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 structural variations play a key role in plant trait innovation and adaptive evolution. Genome-wide, structural variations can influence plant phenotype and, consequently, gene expression by generating large-scale sequence polymorphisms in cis-regulatory regions. Only a small number of SVs are found in coding sequence regions (CDS). Gene structural variations in exons usually lead to loss of gene function, while SVs in promoter and intron regions have more diverse effects on gene expression, and can promote or inhibit gene expression.In maize, a transposon (TE) insertion approximately 60 kb upstream of the tb1 gene played a significant role in altering plant architecture and tiller number during maize 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 branch number has yet to be systematically elucidated. Further research is needed to understand the genetic networks by which structural variation regulates branching under the context of polyploidy.

[0005] In Arabidopsis thaliana, BRANCHED1 (BRC1), encoding a TCP family transcription factor, is a key gene for integrating branching-controlling signals in axillary buds. In pea, auxin promotes the expression of BRC1 by inducing strigolactones and inhibiting cytokinins, thereby inhibiting bud growth (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 the expression of BRC1 and promote the release of buds (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 Acad Sci USA 118:e2004384118.); The bZIP transcription factor LONG HYPOCOTYL 5 (HY5) is a master regulator of light-regulated responses. In tomato (Solanum lycopersicum, Sl), HY5 promotes bud growth by directly repressing BRC1 transcripts and activating transcripts of brassinosteroid 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 Bud Outgrowth. Proc. Natl. Acad. Sci. USA 120:e2301879120.). BRC1 integrates multiple factors, including hormones, nutrients, and light, and is a core integrator of lateral bud growth. Therefore, the number of plant branches is affected by a complex interaction between the environment and genotype, and methods for determining 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 functional loss by designing sgRNA in the coding region of the gene, inducing frameshift mutations or premature translation termination, thereby changing the sequence of the protein product. However, in Brassica napus, how to systematically identify non-coding regulatory sites and use them to improve agronomic traits such as plant type and stress resistance still requires further exploration. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to analyze the gene structure variation of large fragments on the genome of the core germplasm resources of cabbage, explore new genetic loci that affect the formation of effective branches, develop molecular marker combinations, and verify the genetic and molecular functions of some genes through gene editing technology, create new genetic sequences, and directionally change the agronomic traits of the number of effective branches formed, providing a new method for the improvement of rapeseed traits.

[0008] The present invention provides a molecular marker combination significantly correlated with the number of effective branches of single oilseed oil strains, including one or more of the following: DEL00022485 gene structural variation, DEL00003485 gene structural variation, DEL00022483 gene structural variation, DEL00022460 gene structural variation, DEL00022459 gene structural variation and DEL00022484 gene structural variation;

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

[0010] Preferably, any one of the following is included:

[0011] 1) DEL00022485 gene structural variation and DEL00003485 gene structural variation;

[0012] 2) DEL00022485 gene structural variation and DEL00022483 gene structural variation;

[0013] 3) DEL00022485 gene structural variation and DEL00022460 gene structural variation;

[0014] 4) DEL00022485 gene structural variation and DEL00022459 gene structural variation;

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

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

[0017] Primers qOGB-1_DEL00003485_F and qOGB-1_DEL00003485_R for amplifying 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] The primers qOGB-5_DEL00022483_F and qOGB-5_DEL00022483_R for amplifying the structural variation of the DEL00022483 gene, the sequence of the qOGB-5_DEL00022483_F is shown in SEQ ID NO.5, and the sequence of the qOGB-5_DEL00022483_R is shown in SEQ ID NO.6.

[0019] The primers qOGB-6_DEL00022460_F and qOGB-6_DEL00022460_R for amplifying the structural variation of the DEL00022460 gene, the sequence of the qOGB-6_DEL00022460_F is shown in SEQ ID NO.7, and the sequence of the qOGB-6_DEL00022460_R is shown in SEQ ID NO.8.

[0020] The primers qOGB-7_DEL00022459_F and qOGB-7_DEL00022459_R for amplifying the structural variation of the DEL00022459 gene, the sequence of the qOGB-7_DEL00022459_F is shown in SEQ ID NO.9, and the sequence of the qOGB-7_DEL00022459_R is shown in SEQ ID NO.10.

[0021] The primers qOGB-8_DEL00022484_F and qOGB-8_DEL00022484_R for amplifying the structural variation of the DEL00022484 gene, the sequence of the qOGB-8_DEL00022484_F is shown in SEQ ID NO.11, and the sequence of the qOGB-8_DEL00022484_R is shown in SEQ ID NO.12.

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

[0023] 1) Extracting genomic DNA from the rapeseed germplasm sample to be screened;

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

[0025] 3) Analysis results: When the amplified 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 Plants; when the amplified product is 1545 bp, the rapeseed germplasm to be screened lacks the DEL00003485 gene structural variation sequence and is considered to be qOGB-1 单倍型2 plant;

[0026] When the amplified 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 Plants; when the amplified product is a 143 bp band, the rapeseed germplasm to be screened lacks the DEL00022485 gene structural variation and is considered to be qOGB-4 单倍型2 plant.

[0027] When the amplified 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 Plants; when the amplified product is 310 bp, the rapeseed germplasm to be screened lacks the DEL00022483 gene structural variation sequence and is considered to be qOGB-5 单倍型2 plant.

[0028] When the amplified 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1Plants; when the amplified product is 155 bp, the rapeseed germplasm to be screened lacks the DEL00022460 sequence and is considered to be qOGB-6 单倍型2 plant.

[0029] When the amplified 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 Plants; when no amplification product appears, the rapeseed germplasm to be screened lacks the DEL00022459 sequence and is considered to be qOGB-7 单倍型2 plant.

[0030] When the amplified 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 Plants; when the amplified product is 207 bp, the rapeseed germplasm to be screened lacks the DEL00022484 gene structural variation sequence and is considered to be qOGB-8 单倍型2 plant;

[0031] Screen any of the following germplasms for those with a large 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, any one of the following is selected as a germplasm with a large number of effective branches:

[0034] 4.1) At the same time, qOGB-4 单倍型1 and qOGB-1 单倍型2 ;

[0035] 4.2) At the same time, qOGB-4 单倍型1 and qOGB-5 单倍型1 ;

[0036] 4.3) At the same time, qOGB-4 单倍型1 and qOGB-6 单倍型1 ;

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

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

[0039] The present invention provides a guide RNA sequence for affecting the effective branch number of rapeseed, including sgRNA12 or sgRNA26; the sequence of the sgRNA12 is shown as SEQ ID NO.25, and the sequence of the sgRNA26 is shown as SEQ ID NO.26.

[0040] The present invention provides an application of the guide RNA sequence in increasing the number of effective branches of rapeseed, and utilizes CRISPR / Cas9 gene editing technology to transfer the guide RNA into rapeseed germplasm to perform genetic sequence editing on the BnBRC1 gene.

[0041] The present invention provides a genetic sequence for regulating the increase in the number of effective branches of rapeseed, wherein the genetic sequence is shown as 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] The present invention uses core field-grown Brassica napus germplasm resources to study the relationship between effective branch number and genetic structural variation. The natural population used has high genetic diversity and can represent various agronomic traits of rapeseed from different ecotypes. Based on genome-wide association analysis (GWAS) of genetic structural variation (SV), the present invention has discovered six significant genetic loci that affect effective branch number: qOGB-1, qOGB-4 to qOGB-8, corresponding to six genetic structural variations: DEL00003485, DEL00022485, DEL00022483, DEL00022460, DEL00022459, and DEL00022484. Using these genetic structural variations as molecular markers, the number of effective branches per plant in rapeseed is detected or screened to assist in rapeseed breeding.

[0044] Using qOGB-4 (i.e., the structural variant sequence DEL00022485) as the primary genetic locus, this method, combined with five other genetic loci to create molecular marker combinations, can accurately and rapidly identify rapeseed varieties with a high number of effective branches. This method uses a combination of multiple structural variants that influence the number of effective branches per plant, a trait that is influenced by both genotype and environment, to accurately detect or screen rapeseed germplasm with high effective branch numbers.

[0045] The present invention has developed five molecular marker combinations for qOGB-4, qOGB-5, qOGB-6, qOGB-7, qOGB-8 and qOGB-1. The molecular marker combinations can be used to distinguish the number of effective branches of a single Brassica napus strain. 单倍型1 and qOGB-5 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 qOGB-4 can identify the number of effective branches in the range of 9 to 11 with a high probability (25% to 75%). 单倍型1 With qOGB-1 单倍型2 The combination can identify the number of effective branches ranging from 10 to 13.5 with a high probability (25% to 75%).

[0046] The primers for amplifying the molecular marker combination provided by the present invention can be used for common PCR, and the size of the amplified product fragment can be used to more intuitively determine which haplotype of the gene structure the Brassica napus belongs to.

[0047] In the present invention, qOGB-1 is related to a TCP transcription factor BRANCHED 1 (BnaA1.BRC1) on chromosome A1, and has qOGB-1 单倍型2 The plant (i.e., lacking the 623bp-long DEL00003485 gene structural variation sequence) has a significantly reduced expression level of the BnaA1.BRC1 gene, and a significantly increased number of effective branches per plant. The present invention designed 46 different guide RNAs (sgRNAs) for the coding regions (CDS) of BnaA1.BRC1 and its other four homologous genes in the spring rapeseed Westar variety, selected two sgRNA12 and sgRNA26 sequences, and used CRISPR / Cas9 technology to perform genetic sequence editing on the five BnBRC1 genes. A T3 generation five-mutant bnbrc1 plant was obtained, which produced a new genetic sequence on the first exon of the five BnBRC1 genes, which significantly increased the number of effective branches of the main stem of each rapeseed plant. This method provides an identification method and gene resources for screening Brassica napus with many effective branches from the genetic locus and gene levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 These are representative germplasms of Brassica napus with more and fewer effective branches per plant, as well as haplotype combinations with different gene structure variations.

[0049] Figure 2Genome-wide analysis of gene structural variation revealed 12 significant qOGB loci. (a) Manhattan plot of the 12 significant qOGB loci. (b) Gene structural variation of qOGB-1 on chromosome A1 (left) and qOGB-4 to qOGB-9 on chromosome A4 (right), both located in regions of high linkage disequilibrium.

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

[0051] Figure 4 To investigate the effect of qOGB on the number of effective branches per plant, five molecular marker combinations were used to verify 23 typical germplasm resources, including (a) the molecular marker combination of qOGB-4 and qOGB-1; (b) the molecular marker combination of qOGB-4 and qOGB-5; (c) the molecular marker combination of qOGB-4 and qOGB-6; (d) the molecular marker combination of qOGB-4 and qOGB-7; (e) the molecular marker combination of qOGB-4 and qOGB-8; (f) the five molecular marker combinations effectively distinguished the number of effective branches in 16 germplasm resources. and 7 accessions with a large number of effective branches and a small number of effective branches; lane 1 (a) to (e) is the DNA molecular standard; lanes 2 to 24 are the accessions 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 locus.

[0053] Figure 6 To edit the five homologous genes of BnBRC1 in the Brassica napus variety Westar, (a) shows the genetic 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 homozygous for the five homologous genes, whose new genetic sequence characteristics are manifested as changes in small fragment deletions or insertions.

[0054] Figure 7Compared to Brassica napus (Brassica napus) Westar, a mutant homozygous for five homologous genes, bnbrc1 showed an increase in the number of effective branches per plant in the T3 generation. (a) Branching phenotypes of Westar and bnbrc1 mutants at maturity; (b) Compared to the wild type, the bnbrc1 mutant showed an increase in the number of effective branches on the main stem. DETAILED DESCRIPTION

[0055] The technical solutions provided by the present invention are 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 the same year, rapeseed seeds of Brassica napus were sown directly in the experimental field. The same variety of rapeseed germplasm materials were planted in a small plot of experimental field. The plot was 1.5 meters long and 1.5 meters wide. Before wintering, the seedlings were thinned out in rows with a spacing of 30 cm. The final planting density was 16 plants per plot.

[0058] (2) Phenotypic determination and analysis were carried out in the late growth period of the rapeseed population in the following year, generally in late May or early June. The number of primary branches of a single plant with one or more valid siliques in the late growth period was counted in the fields of Jiaxing. For each material, three representative samples were selected in the planting area as replicates.

[0059] (3) The statistical data of the number of primary branches of individual plants with one or more effective siliques in the late growth period of different germplasm materials were used for structural variation-based genome-wide association analysis (SV-GWAS). The GEMMAv0.98.1 software package was used to align the resequencing data of the core germplasm to the ZS11.v0 reference genome with a threshold of 4. Based on the mixed linear model, the genetic loci that affect the number of effective branches were screened and named quantitative O There are 12 outgrowingBranch sites, referred to as qOGB, which are named qOGB-1 to qOGB-12 ( Figure 2 ). These include 10 large deletion mutations (DEL) and 2 large insertion mutations (INS) (such as Figures 1 to 3 , Table 1).

[0060] (4) Through the analysis of (3), a total of 12 different qOGB sites with genomic structural variations were discovered. 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 the 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 and 3 ). BnaA01G0326500ZS encodes the BnaA1.BRC1 gene, which, together with four other homologous genes, is collectively referred to as BnBRC1.

[0061] Table 1 Genetic structural variation affecting the number of effective branches per plant

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

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

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

[0070] The gene structural variants DEL00022485, DEL00022483, and DEL00022484 are located at the qOGB-4, qOGB-5, and qOGB-8 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 gene structural variants are located in the intergenic region between the BnaA04G0189200ZS and BnaA04G0189300ZS genes ( Figures 2 and 3 , Table 1).

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

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

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

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

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

[0076] Among the 12 genetic loci mentioned above, qOGB-1, qOGB-4 to qOGB-8 can have a significant effect on the number of effective branches, while the other loci have a statistically weaker effect on effective branches; therefore, further research will be conducted on qOGB-1, qOGB-4 to qOGB-8.

[0077] (5) Haplotype analysis of the effective branch number per plant was performed on the six gene structural variants, qOGB-1, qOGB-4 to qOGB-8. The haplotype carrying the DEL00003485 gene structural variant sequence was named qOGB-1. 单倍型1 , the missing one is named qOGB-1 单倍型2 The haplotype carrying the DEL00022485 gene structural variation was 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 variation was 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 variation was 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 variation was 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 variation was named qOGB-8 单倍型1 , the missing one is named qOGB-8 单倍型2 Taking the structural variation of qOGB-4 gene as the main effect variation site, the qOGB-4 with more effective branches was used. 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 The germplasm is intersected ( Figure 4), screened haplotype combinations that could effectively distinguish the number of effective branches, a total of 5 groups (Table 2). Primers were designed for these six gene structural variations (Table 3) to verify the consistency of genotype and phenotype ( Figure 4 ).

[0078] Table 2 Statistics of different haplotypes and effective branch numbers of rapeseed

[0079]

[0080] (6) On the BnaA01G0326500ZS / BnaA1.BRC1 gene, the haplotype carrying the DEL00003485 gene structural variation sequence is BnaA1.BRC1 单倍型1 , the missing one is BnaA1.BRC1 单倍型2 ( Figure 5 (a) BnaA1.BRC1 with DEL00003485 gene structural variant deleted 单倍型2 The Brassica napus germplasm showed an agronomic phenotype of increased effective branch number, and the expression level of BnaA1.BRC1 during bud development was significantly lower than that of BnaA1.BRC1 containing the structural variation of this gene. 单倍型1 ( Figure 5 b). This locus significantly affects effective branch formation and provides an important genetic material basis for breeding multi-branched and high-yield rapeseed.

[0081] (7) Molecular markers as shown in SEQ ID NO.3 and NO.4 were designed for the structural variation qOGB-4 / DEL00022485. Molecular markers as shown in SEQ ID NO.1 and NO.2 were designed for qOGB-1 / DEL00003485. Molecular markers as shown in SEQ ID NO.5 and NO.6 were designed for qOGB-5 / DEL00022483. Molecular markers as shown in SEQ ID NO.7 and NO.8 were designed for qOGB-6 / DEL00022460. Molecular markers as shown in SEQ ID NO.9 and NO.10 were designed for qOGB-7 / DEL00022459. Molecular markers as shown in SEQ ID NO.11 and NO.12 were designed for qOGB-8 / DEL00022484. The sequence information is shown in Table 3. Use 5 μl of KOD enzyme (Toyobo, KMM-201), 0.3 μl of 10 μM forward primer, 0.3 μl of 10 μM reverse primer, 0.5 μl of extracted genomic DNA, and 3.9 μl of water for a total of 10 μl. Use a commonly used PCR instrument on the market and perform the experiment according to the reaction conditions in Table 3. Perform a total of 35 cycles from denaturation to extension. After the experiment is completed, take 2 μl and run it on a 1% agarose gel to detect band size.

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

[0083]

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

[0085]

[0086]

[0087]

[0088]

[0089] The above germplasms are core rapeseed germplasms from different regions around the world, as described in the following literature:

[0090] The Intronic Structure Variation of Rapeseed BnaC3.LEAFY Regulatesthe Timing of Inflorescence Formation and Flowering.Mengjie Gong,Guangyu Wu,Xiuchen Weng,Huiqi Zhang,Tao Zhou,Wenbin Guo,Shuijin Hua,Weizhen Hu,ShanLiang,YifengXu,Shuijin Hua,Lixi Jiang andYangZhu*2025Plant Communicationshttps: / / 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 BnaA1.BRC1 with qOGB-1 / DEL00003485 单倍型1 Using the CRISPR / Cas9 gene editing system, a total of 46 sgRNA sequences were designed targeting the first exon sequences of the five BnBRC1 homologous genes in Westar (Table 5). Through analysis and experiments, sgRNA12, i.e., sequence SEQ ID NO.25, and sgRNA26, i.e., sequence SEQ ID NO.26, were able to 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 loss of gene function.

[0093] Table 5 sgRNA sequence design for the first exon of five BnBRC1 homologous genes in Brassica napus Westar

[0094]

[0095]

[0096]

[0097] (9) Using the sgRNA design and CRIPSPR / Cas9 technology in (8), a new material with homozygous mutations of five homologous genes was created in Westar and named bnbrc1. In the T3 generation plants of this strain, it was found that the coding regions of 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 the sgRNA26 position and an 11-base deletion at the sgRNA12 position. The BnaC3.BRC1 gene has a 4-base deletion at the sgRNA26 position and a 1-base insertion at the sgRNA12 position (Table 6); In a plant incubator, a potted experiment was conducted using white LED light, 20,000 lux light intensity, 16-hour day length and 24 degrees Celsius. It was found that compared with the wild-type Westar, the bnbrc1 plant showed a phenotype of increased effective branch number ( Figure 7 ), demonstrating the negative effect of BnBRC1 on effective branch formation in rapeseed, and providing important genetic resources for molecular breeding of Brassica napus.

[0098] Table 6. New sequences generated from five homologous genes of BnBRC1 in Brassica napus Westar.

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Note: CRISPR / Cas9-mediated base deletions are marked with strikethroughs, base insertions are marked with underlines and italics, the sgRNA recognition sequence is underlined in black, and the PAM recognition position NGG is bolded.

[0105] As can be seen from the above examples, the present invention mainly analyzes the structural variation of large fragments of genes on the genome of the core germplasm resources of Brassica napus, and explores the 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 formed. Primers are developed based on these structural variation sequences for screening different germplasm resources of Brassica napus for germplasms with multiple effective branching traits, which is conducive to accelerating rapeseed breeding. The gene structure variation DEL00003485 belongs to the qOGB-1 locus and is located in the upstream region of the BnaA01G0326500ZS / BnaA1.BRC1 gene. The present invention also verified the genetic and molecular functions of BnBRC1 through gene editing technology, created a new genetic sequence, and directionally changed the agronomic trait of the number of effective branches formed.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A molecular marker combination significantly associated with the number of effective branches in a single oil plant, characterized in that: Including one or more of the DEL00022485 gene structural variation, DEL00003485 gene structural variation, DEL00022483 gene structural variation, DEL00022460 gene structural variation, DEL00022459 gene structural variation and DEL00022484 gene structural variation; The sequences of the DEL00022485 gene structural variation, DEL00003485 gene structural variation, DEL00022483 gene structural variation, DEL00022460 gene structural variation, DEL00022459 gene structural variation and DEL00022484 gene structural variation are shown in SEQ ID NO.16, SEQ ID NO.13, and SEQ ID NO.17 to SEQ ID NO.20, respectively.

2. The molecular marker combination according to claim 1, characterized in that Includes any one of the following 1) to 5): 1) DEL00022485 gene structural variation and DEL00003485 gene structural variation; 2) DEL00022485 gene structural variation and DEL00022483 gene structural variation; 3) DEL00022485 gene structural variation and DEL00022460 gene structural variation; 4) DEL00022485 gene structural variation and DEL00022459 gene structural variation; 5) DEL00022485 gene structural variation and DEL00022484 gene structural variation.

3. A primer for amplifying the molecular marker combination according to claim 1 or 2, characterized in that: The method comprises primers qOGB-4_DEL00022485_F and qOGB-4_DEL00022485_R for amplifying the structural variation of the DEL00022485 gene, wherein 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; Primers qOGB-1_DEL00003485_F and qOGB-1_DEL00003485_R for amplifying 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; Primers qOGB-5_DEL00022483_F and qOGB-5_DEL00022483_R for amplifying 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; Primers qOGB-6_DEL00022460_F and qOGB-6_DEL00022460_R for amplifying 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; Primers qOGB-7_DEL00022459_F and qOGB-7_DEL00022459_R for amplifying 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; The primers qOGB-8_DEL00022484_F and qOGB-8_DEL00022484_R for amplifying the structural variation of the DEL00022484 gene, the sequence of the qOGB-8_DEL00022484_F is shown in SEQ ID NO.11, and the sequence of the qOGB-8_DEL00022484_R is shown in SEQ ID NO.

12.

4. A method for screening rapeseed germplasm using the molecular marker combination according to claim 1 or 2 and the primer according to claim 3, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the rapeseed germplasm sample to be screened; 2) using the genomic DNA as a template and performing PCR amplification using the primers described in claim 3 to obtain amplified 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 a qOGB-1 haplotype 1 plant; when the amplification product is 1545 bp, the rapeseed germplasm to be screened lacks the DEL00003485 gene structural variation sequence and is considered a qOGB-1 haplotype 2 plant; When the amplified 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 a qOGB-4 haplotype 1 plant; when the amplified product is a 143 bp band, the rapeseed germplasm to be screened lacks the DEL00022485 gene structural variation and is considered to be a qOGB-4 haplotype 2 plant. When the amplified 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 a qOGB-5 haplotype 1 plant; when the amplified product is 310 bp, the rapeseed germplasm to be screened lacks the DEL00022483 gene structural variation sequence and is considered to be a qOGB-5 haplotype 2 plant; When the amplified 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 as a qOGB-6 haplotype 1 plant; when the amplified product is 155 bp, the rapeseed germplasm to be screened lacks the DEL00022460 sequence and is considered as a qOGB-6 haplotype 2 plant; When the amplified 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 a qOGB-7 haplotype 1 plant; when no amplified product appears, the rapeseed germplasm to be screened lacks the DEL00022459 sequence and is considered to be a qOGB-7 haplotype 2 plant; When the amplified 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 a qOGB-8 haplotype 1 plant; when the amplified product is 207 bp, the rapeseed germplasm to be screened lacks the DEL00022484 gene structural variation sequence and is considered to be a qOGB-8 haplotype 2 plant. Screen any of the following germplasms for those with a large number of effective branches: qOGB-1 haplotype 2, qOGB-4 haplotype 1, qOGB-5 haplotype 1, qOGB-6 haplotype 1, qOGB-7 haplotype 1, and qOGB-8 haplotype 1.

5. The method according to claim 4, characterized in that Screen any of the following 5.1) to 5.5) for germplasms with a large number of effective branches: 5.1) Both qOGB-4 haplotype 1 and qOGB-1 haplotype 2; 5.2) Both qOGB-4 haplotype 1 and qOGB-5 haplotype 1; 5.3) Both qOGB-4 haplotype 1 and qOGB-6 haplotype 1; 5.4) Both qOGB-4 haplotype 1 and qOGB-7 haplotype 1; 5.5) Both qOGB-4 haplotype 1 and qOGB-8 haplotype 1.

6. A guide RNA sequence that affects the number of effective branches in rapeseed, characterized in that: Including sgRNA12 or sgRNA26; the sequence of the sgRNA12 is shown as SEQ ID NO.25, and the sequence of the sgRNA26 is shown as SEQ ID NO.

26.

7. Use of the guide RNA sequence according to claim 6 in increasing the number of effective branches of rapeseed, characterized in that The CRISPR / Cas9 gene editing technology is used to transfer the guide RNA into rapeseed germplasm to perform genetic sequence editing on the BnBRC1 gene.

8. A genetic sequence for regulating the increase in the number of effective branches of rapeseed, characterized in that: The genetic sequences include SEQ ID NO.72, SEQ ID NO.74, SEQ ID NO.76, SEQ ID NO.78 and SEQ ID NO.80.

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