Application of BnaA07.MKK9 gene or its SNAP molecular marker in regulating plant resistance to Sclerotinia sclerotiorum

Abstract

The present invention discloses the application of the BnaA07.MKK9 gene or its SNAP molecular marker in regulating plant sclerotinia resistance. The present invention locates and clones a gene BnaA07.MKK9 for regulating rapeseed sclerotinia resistance for the first time. Overexpression of the gene can enhance rapeseed sclerotinia resistance; the encoded protein of the gene and the SNAP molecular marker can be used for genetic improvement of sclerotinia resistance.

Description

Technical Field

[0001] The invention relates to application of BnaA07.MKK9 gene or its SNAP molecular marker in regulating plant sclerotinia resistance, and belongs to the fields of molecular biology and genetic breeding. Background Art

[0002] As a major consumer of vegetable oil, my country has a very low supply rate of domestic vegetable oil and needs to import to meet consumer demand. Increasing the supply rate of domestic vegetable oil is the key to ensuring the security of my country's edible oil supply. As my country's largest oil crop, Brassica napus (Brassicanapus.L) contributes about 50% of my country's domestic vegetable oil. Rapeseed sclerotinia was listed as a national Class I crop disease in 2023. Rapeseed sclerotinia caused by Sclerotinia not only seriously affects the yield of rapeseed, but also affects the quality of rapeseed oil. Rapeseed sclerotinia can cause an average annual yield loss of 20%, and the loss in severely affected areas is as high as 80%. Therefore, cultivating sclerotinia resistant (tolerant) rapeseed germplasm is the most economical and effective way to reduce the occurrence of sclerotinia and reduce yield losses.

[0003] At present, no rapeseed germplasm that is completely immune to Sclerotinia sclerotinia has been found in rapeseed and its closely related species. A large number of genetic experiments have shown that rapeseed's resistance to Sclerotinia sclerotinia is mainly controlled by quantitative traits (QTLs). Although hundreds of Sclerotinia sclerotinia resistance QTLs / associated sites have been located through research methods such as whole-genome association analysis, most of them are minor QTLs / associated sites, and the major QTLs have not yet been cloned.Through reverse genetics, several genes involved in regulating rapeseed resistance to Sclerotinia sclerotinia were discovered, such as: BnaWRKY33 (Liu et al., Interactions of WRKY15 and WRKY33 transcription factors and their roles in the resistance of oilseed rape to Sclerotinia infection. Plant Biotechnol J, 2018, 16, 911-925), BnaMPK4 (Wang et al., Overexpression of Brassica napusMPK4 enhances resistance to Sclerotinia sclerotiorum in oilseed rape. Mol Plant-microbe In, 2009, 22, 235-244), BnaMPK3 (Wang et al., BnaMPK3 is a key regulator of defense responses to the devastating plant pathogen Sclerotiniasclerotiorum in oilseed rape. Front Plant Sci, 2019, 10: 91), BnaMPK6 (Wang et al., BnaMPK6 is a determinant of quantitative disease resistance against Sclerotinia sclerotiorum in oilseed rape. Plant Sci, 2020, 291, 110362), BnaWRKY28 (Zhang et al., Transcription factor WRKY28 curbs WRKY33-mediated resistance to Sclerotinia sclerotiorum in Brassica napus. Plant Physiol, 2022, 190, 2757-2774), etc. However, it is still unclear whether these genes have excellent allelic variation, and there is a lack of effective functional molecular markers, so it is difficult to directly apply them to actual production practice. In summary, isolating and cloning rapeseed anti-sclerotial genes and developing corresponding functional molecular markers play an important role in breeding and improving sclerotinia resistance. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide for the first time the application of the BnaA07.MKK9 gene or its SNAP molecular marker in regulating plant resistance to Sclerotinia sclerotiorum.

[0005] Technical solution: To solve the above technical problems, the present invention provides the application of BnaA07.MKK9 gene or its SNAP molecular marker in regulating plant resistance to sclerotinia sclerotinia.

[0006] The present invention also provides application of the BnaA07.MKK9 gene or its SNAP molecular marker in genetic improvement of plant sclerotinia resistance.

[0007] Wherein, the SNAP molecular marker includes one or more of Hap0, Hap1 or Hap2; the nucleotide sequence of Hap0 is shown in SEQ ID NO.4; the nucleotide sequence of Hap1 is shown in SEQ ID NO.5; and the nucleotide sequence of Hap2 is shown in SEQ ID NO.6.

[0008] The present invention also provides a method for improving plant resistance to sclerotinia sclerotinia, characterized in that it comprises the following steps: DD The gene overexpression vector can be transformed into plants.

[0009] The present invention also provides a method for reducing plant resistance to sclerotinia sclerotinia, characterized in that it comprises the following steps: transforming a recombinant vector in which the BnaA07.MKK9 gene is knocked out into a plant.

[0010] The present invention also provides the use of the SNAP molecular marker of the BnaA07.MKK9 gene or a primer combination thereof in identifying plant resistance to sclerotinia sclerotinia, wherein the SNAP molecular marker includes one or more of Hap0, Hap1 or Hap2; the nucleotide sequence of Hap0 is shown in SEQ ID NO.4; the nucleotide sequence of Hap1 is shown in SEQ ID NO.5; and the nucleotide sequence of Hap2 is shown in SEQ ID NO.6.

[0011] Wherein, the primer combination includes one or more of the primer combination of Hap0, the primer combination of Hap1 or the primer combination of Hap2.

[0012] The sequences of the primer combinations of Hap0 are shown in SEQ ID NO.35 to SEQ ID NO.36.

[0013] The sequences of the primer combinations of Hap1 are shown in SEQ ID NO.37 to SEQ ID NO.38.

[0014] The sequences of the primer combinations of Hap2 are shown in SEQ ID NO.39 to SEQ ID NO.40.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention locates and clones a gene BnaA07.MKK9 that regulates rapeseed's resistance to sclerotinia sclerotinia for the first time. Overexpression of this gene can enhance rapeseed's resistance to sclerotinia sclerotinia; 2. The encoded protein of this gene and the SNAP molecular marker can be used for genetic improvement of sclerotinia sclerotinia resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a genome-wide association analysis of rapeseed sclerotinia resistance: A: Statistics of stem sclerotinia resistance of 322 rapeseed germplasm resources; BD: Manhattan plot, QQ plot and linkage disequilibrium analysis heat map of rapeseed sclerotinia resistance association analysis; E: Expression heat map of candidate genes 48 hours after inoculation with Sclerotinia and uninoculated control; The expression abundance of each gene is expressed in FPKM (fragments per kilobase of transcript per million fragments mapped);

[0017] Figure 2 BnaA07.MKK9 DD Creation of overexpression plants and analysis of sclerotinia resistance: A: BnaA07.MKK9 D D Schematic diagram of overexpression vector construction; B: RT-PCR analysis of BnaA07.MKK9 DD The expression level of BnaA07.MKK9 in the T0 generation overexpression and transgenic recipient J9712; CD: BnaA07.MKK9 DD Statistical diagram of leaf lesions and lesion area of ​​T1 overexpression line and transgenic recipient J9712 48 hours after inoculation with Sclerotinia sclerotiorum, the scale bar represents 1 cm; EF: BnaA07.MKK9 DD Statistics of stem lesions and lesion length of T1 generation overexpression transgenic recipient J9712 inoculated with Sclerotinia sclerotiorum 168 h later, the scale bar represents 5 cm; n represents the number of inoculated plants;

[0018] Figure 3Creation of Bnamkk9 knockout mutants (#5 and #7) and analysis of resistance to sclerotinia: A: mutation of Bnamkk9 knockout mutants (#5 and #7); base insertions or deletions are marked in red, target sites are underlined, and PAM (protospacer adjacent motif) is marked in blue; BC: leaf lesions and lesion area statistics of Bnamkk9 knockout mutants (#5 and #7) and transgenic recipient J9712 48 hours after inoculation with Sclerotinia, the scale bar represents 1 cm; DE: statistics of stem lesions and lesion length of Bnamkk9 knockout mutants (#5 and #7) and transgenic recipient J9712 168 hours after inoculation with Sclerotinia, the scale bar represents 5 cm; * and ** indicate P < 0.05 and P < 0.01 in Student's t-test, respectively; n represents the number of inoculated plants;

[0019] Figure 4 The gene structure of BnaA07.MKK9 and the DNA polymorphisms of three haplotypes;

[0020] Figure 5 The figure is a statistical diagram of the stem lesion length of the three haplotypes of BnaA07.MKK9; different letters indicate significant differences in Tukey's honestly significant difference (HSD) in variance analysis;

[0021] Figure 6 Analysis of the in vivo kinase activity of the three haplotypes of BnaA07.MKK9 after inoculation with Sclerotinia sclerotiorum; A: In vivo kinase activity of the three haplotypes of BnaA07.MKK9 12 hours after simulated inoculation with potato medium (PDA); B: In vivo kinase activity of the three haplotypes of BnaA07.MKK9 12 hours after inoculation with Sclerotinia sclerotiorum;

[0022] Figure 7 SNAP molecular marker detection of three haplotypes of BnaA07.MKK9: A: Hap1; B: Hap0; C: Hap2; the DNA size indicated by the DNA marker is shown on the left side of the gel image. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1 Genome-wide association analysis of rapeseed sclerotinia resistance

[0025] The 322 rapeseed germplasm resources used in this experiment were donated by the National Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University. After years of breeding in this laboratory, they were planted in the experimental fields of Yangzhou University. The 322 rapeseed resources can be queried in the BnIR (https: / / yanglab.hzau.edu.cn / ) database.

[0026] Table 1322 Rapeseed Germplasm Resources Information Table

[0027]

[0028]

[0029]

[0030]

[0031] The original strain SS-1 of Sclerotinia sclerotiorum was provided by Li Guoqing, a teacher at the State Key Laboratory of Microbiology, Huazhong Agricultural University. The original strain was stored in a 4°C refrigerator in the dark and inoculated on potato culture medium (PDA, Becton, Dickinson and Company, USA) before use, cultured at 23°C in the dark for 2 days, and activated twice before inoculation. The original strain SS-1 of Sclerotinia sclerotiorum was derived from a published paper (Wu et al., Identification of QTLs for resistance to Sclerotinia stem rot and BnaC.IGMT5.a as a candidate gene of the majorresistant QTL SRC6 in Brassica napus, PloS one, 2013, 8, e67740.).

[0032] Inoculation of live stems of rapeseed at the end of flowering with Sclerotinia: Use a 7mm puncher to punch out the mycelium of Sclerotinia growing on the edge of the potato culture medium and spread it in the center of the plastic wrap for inoculation. 322 rapeseed germplasm resources were inoculated with stems, and two biological replicates were designed. In each replicate, 6 plants of each line were used for inoculation. Each plant was inoculated with Sclerotinia at 50 cm above the ground on the main stem and fixed with plastic wrap to keep it moist. Measure the length of the lesion with a ruler 7 days after inoculation and record it.

[0033] The transcriptome data of rapeseed after stem inoculation with Sclerotinia sclerotiorum for 48 hours were referred to the published paper (Wu et al., Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorumin Brassicanapus, Sci Rep, 2016, 6, 19007).

[0034] Genome-wide association analyses (GWAS): The resequencing results of 322 rapeseed samples were aligned with the reference genome in the BnIR database (https: / / yanglab.hzau.edu.cn / ) to obtain associated SNP markers. These SNP markers were quality controlled and filtered, and SNP markers with a minimum allele frequency of less than 0.05% and a missing rate of more than 30% were eliminated. Finally, 4,984,924 SNP markers were obtained. The mixed linear model of GEMMA (v0.98.1) was used to perform genome-wide association analysis (GWAS), and -log10 (1 / n) (where n is all SNP markers) was used as the threshold to define SNP markers significantly associated with the target trait. The GWAS results were presented using Manhattan plots and QQ plots. Manhattan plots and QQ plots were drawn using the CMplot package (v.4.0) of R language (v.x644.1.1). PLINK (v.1.90b4.4) was used to calculate the linkage disequilibrium (LD) between SNP loci, and LDheatmap software was used to display the results.

[0035] Through the investigation of stem resistance of 322 rapeseed varieties, it was found that none of the 322 rapeseed varieties were completely immune to Sclerotinia sclerotiorum, and different rapeseed varieties had different resistance to Sclerotinia sclerotiorum ( Figure 1 A). GWAS results showed that 120 SNPs associated with sclerotinia resistance were located between 24.439-24.590Mb on the A07 chromosome of rapeseed ( Figure 1 BD). Subsequently, combined with the published transcriptome sequencing data, it was found that BnaA07.MKK9 (BnaA07G0255500ZS) was significantly induced after the stems were inoculated with Sclerotinia sclerotiorum, and the degree of induced expression was the most intense ( Figure 1 E), therefore BnaA07.MKK9 was selected as a candidate gene.

[0036] Example 2 Cloning of BnaA07.MKK9

[0037] Cloning of BnaA07.MKK9: The coding region (CDS) sequence of BnaA07.MKK9 (BnaA07G0255500ZS) was retrieved using the BnIR database (https: / / yanglab.hzau.edu.cn / ), and specific primers were designed. The primer sequences were: SEQ ID NO.7: BnaA07.MKK9-F1: 5'-ATGGCTTTGGTAAGAGAACG-3'; SEQ ID NO.8: BnaA07.MKK9-R1: 5'-CTACAAAACGAATCGAATTAAC-3'. The primers were synthesized by Qingke Biotechnology Co., Ltd.

[0038] High-quality DNA from Brassica napus strain J9712 (laboratory-preserved strain) was extracted using the CTAB method and used as a template. PCR amplification was performed using the high-fidelity enzyme Max Super-Fidelity DNA Polymerase, P505 (Vazyme, Nanjing, China). The reaction system was configured according to the product manual, and the PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, and full extension at 72°C for 10 min after 33 cycles of reaction. After the PCR reaction, 1% agarose gel electrophoresis was performed, and a gel block of about 1000 bp was cut out. The DNA fragment was purified and recovered using a DNA recovery kit from Kangwei Century Company. The recovered DNA fragment was incubated with pCE2-TA / Blunt-Zero vector, C601-01 (Vazyme, Nanjing, China) at 25°C for 1 hour, transformed into DH5α Escherichia coli competent cells, and positive clones were selected to extract plasmids. The sequencing was completed by Beijing Qingke Biotechnology Co., Ltd., and the plasmid was named BnaA07.MKK9-CDS. The sequencing results showed that a DNA fragment with a length of 927 bp was obtained, as shown in the nucleotide sequence of SEQ ID NO.1; the protein sequence encoded by it is shown in SEQ ID NO.2.

[0039] SEQ ID NO.1:ATGGCTTTGGTAAGAGAACGTCGTCAGCTCAACCTCCGTCTT CCTCTTCCTCCGATCTCCGACCGCCGCTTCTCCCTCCCATCTGTCACCACCACAGTCGCTACCTCCGGTGACATCTCCGCCGCTGATCTCGAGAAACTCAACGTTCTCGGATGCGGAAACGGCGGGATTGTTTACAGAGTCCGTCACAAGAACACGGCGGACCTCTACGCTTTGAAAACTGTCAACGGCGACATGGATCCGATCTTAACACGGCAGCTGATGCGAGAGATGGAGATCCTCCGCCGTACGGACTCTCCCTACGTCGTCCGGTGTCACGGAATCTTCGAGAAACAAGTCGTCGGCGAAGTGTCGATTCTGATGGAGTACATGGACGGCGGAACTCTAGAGTCTCTCCGCGGCGCCGTGACGGAGCAACGCCTCGCCGGGTTCGCGAGACAGATCTTGAAAGGTCTTAGTTACTTGCACGCTCTCAAGATCGTGCACAGGGACATCAAACCGGCGAACCTCCTCCTCAACTCGAAGGACGAGGTCAAGATCGCCGACTTCGGAGTCAGCAAGATCTTGGTCCGGTCGTTGGATTCCTGCAA CTCTTACGTCGGGACTTGTGCTTACATGAGTCCGGAGAGGTTTGATTCCGAATCTTCTTCCGGTGGTGGCTCCGACGTGTACGCCGGAGATATCTGGAGCTTCGGGTTGATGATGCTGGAGCTTCTCGTTGGTCACTTTCCGTTATTACCGCCGGGACAGAGACCGGACTGGGCGACTTTGATGTGCGCGGTGTGTTTTGGGGAGCCGCCGCGGGCGCCGGAGGGGTGCTCGGAGGAGTTTAGGAGCTTTGTTGAGTGTTGTCTACGTAAAGATTCGAGTAAGAGATGGACGGCGTCGCAGCTTCTCGCCCATCCTTTTCTCCGGGAGGATCTTTAG

[0040] SEQ ID NO.2:

[0041] MALVRERRQLNLRLPLPPISDRRFSLPSVTTTVATSGDISAADLEKLNVLGCGNGGIVYRVRHKNTADLYALKTVNGDMDPILTRQLMREMEILRRTDSPYVVRCHGIFEKQVVGEVSILMEYMDGGTLESLRGAVTEQRLAGFARQILKGLSY LHALKIVHRDIKPANLLNSKDEVKIADFGVSKILVRSLDSCNSYVGTCAYMSPERFDSESSSGGGSDVYAGDIWSFGLMMLELLVGHFPLLPPGQRPDWATLMCAVCFGEPPRAPEGCSEEFRSFVECCLRKDSSKRWTASQLLAHPFLREDL

[0042] Example 3 BnaA07.MKK9 DD Creation of overexpression strains and analysis of resistance to sclerotinia sclerotinia

[0043] The BnaA07.MKK9 gene encodes a mitogen-activated protein kinase. To further study the function of the gene, the plasmid BnaA07.MKK9-CDS was used as a template to mutate the serine at positions 192 and 198 to aspartic acid by splicing by the overlap extension PCR technique, thereby converting BnaA07.MKK9 into a kinase-activated state. DD , the recovered BnaA07.MKK9 DD The DNA fragment was incubated with pCE2-TA / Blunt-Zerovector, C601-01 (Vazyme, Nanjing, China) at 25°C for 1 hour, transformed into DH5α Escherichia coli competent cells, and the positive clones were selected to extract plasmids, which were sequenced by Qingke Company and named BnaA07.MKK9. DD -CDS. The primer sequence of overlap extension shear PCR technology is: SEQ ID NO.9: BnaA07.MKK9 DD -F1:5'-ATGGCTTTGGTAAGAGAACG-3'; SEQ ID NO.10: BnaA07.MKK9 DD -R1:5'-GTTGCAGGAATCCAAGTCCCGGACC-3'; SEQ ID NO.11: BnaA07.MKK9 DD-F2:5'-TTGGATTCCTGCAACGACTACGTCG-3'; SEQ ID NO.12: BnaA07.MKK9 DD -R2:5'-CTAAAGATCCTCCCGGAGAA-3'. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, and full extension at 72°C for 10 min after 33 cycles. The overlap extension shearing PCR system was carried out according to the instructions of Max Super-Fidelity DNA Polymerase, P505 (Vazyme, Nanjing, China), and the system was 50 μL. The sequencing result is the nucleotide sequence shown in SEQ ID NO.3.

[0044] SEQ ID NO.3: ATGGCTTTGGTAAGAGAACGTCGTCAGCTCAACCTCCGTCTT CCTCTTCCTCCGATCTCCGACCGCCGCTTCTCCCTCCCATCTGTCACCACCACAGTCGCTACCTCCGGTGACATCTCCGCCGCTGATCTCGAGAAACTCAACGTTCTCGGATGCGGAAACGGCGGGATTGTTTACAGAGTCCGTCACAAGAACACGGCGGACCTCTACGCTTTGAAAACTGTCAACGGCGACATGGATCCGATCTTAACACGGCAGCTGATGCGAGAGATGGAGATCCTCCGCCGTACGGACTCTCCCTACGTCGTCCGGTGTCACGGAATCTTCGAGAAACAAGTCGTCGGCGAAGTGTCGATTCTGATGGAGTACATGGACGGCGGAACTCTAGAGTCTCTCCGCGGCGCCGTGACGGAGCAACGCCTCGCCGGGTTCGCGAGACAGATCTTGAAAGGTCTTAGTTACTTGCACGCTCTCAAGATCGTGCACAGGGACATCAAACCGGCGAACCTCCTCCTCAACTCGAAGGACGAGGTCAAGATCGCCGACTTCGGAGTCAGCAAGATCTTGGTCCGGGACTTGGATTCCTGCAACGACTACGTCGGGACTTGTGCTTACATGAGTCCGGAGAGGTTTGATTCCGAATCTTCTTCCGGTGGTGGCTCCGACGTGTACGCCGGAGATATCTGGAGCTTCGGGTTGATGATGCTGGAGCTTCTCGTTGGTCACTTTCCGTTATTACCGCCGGGACAGAGACCGGACTGGGCGACTTTGATGTGCGCGGTGTGTTTTGGGGAGCCGCCGCGGGCGCCGGAGGGGTGCTCGGAGGAGTTTAGGAGCTTTGTTGAGTGTTGTCTACGTAAAGATTCGAGTAAGAGATGGACGGCGTCGCAGCTTCTCGCCCATCCTTTTCTCCGGGAGGATCTTTAG

[0045] Using plasmid BnaA07.MKK9 DD-CDS was used as a template, and the forward and reverse amplification primers of the insert fragment were designed with reference to the instructions of ClonExpress II One Step Cloning Kit, C112-01 (Vazyme, Nanjing, China). The high-fidelity enzyme Max Super-Fidelity DNA Polymerase was used to perform PCR on BnaA07.MK K9 DD -Add restriction endonuclease AscI and PacI recognition sites and corresponding upstream / downstream vector end homologous sequences at both ends of CDS. At the same time, pMDC83 stored in the laboratory was double-digested with 5U AscI and 2.5U PacI. The AscI digestion system was referred to the NEB official website (www.neb-china.com). After 2h of digestion at 37℃, heat inactivation was performed at 80℃ for 20min. The PCR product and the digestion product were detected by 1% agarose gel electrophoresis, and the corresponding fragments were recovered. The recombination reaction was carried out according to the instructions of ClonExpress IIOne Step Cloning Kit, C112-01 (Vazyme, Nanjing, China). After the recombination reaction was completed, it was transformed into Escherichia coli DH5α competent cells, and the positive clones were selected to extract the plasmid. The above-mentioned vector is BnaA07.MKK9 DD The overexpression vector was named BnaA07.MKK9 DD -OE. BnaA07.MKK9 was transformed by electroporation DD -OE transforms Agrobacterium GV3101. The primer sequence is: SEQ ID NO.13: BnaA07.MKK9 DD -F:5'-ACTAGTTCTAGAGTCGAGGTCATGGCTTTGGTAAGAGAAC G-3'; SEQ ID NO.14: BnaA07.MKK9 DD -R:5'-GGCGCGCCTCGAGGGGGGGCCTAA GATCCTCCCGGAGA-3'. The reaction procedure for PCR positive identification was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, and full extension at 72°C for 10 min after 28 cycles of reaction.

[0046] BnaA07.MKK9 was transformed into hypocotyls by Agrobacterium-mediated transformation. DD-OE recombinant vector was transformed into rapeseed line J9712 recipient, 25mg / L hygromycin was used as a screening marker, and the selected T0 generation tissue culture seedlings resistant to hygromycin were positively identified by PCR using vector universal primers and gene specific primers, and the primer sequences were SEQ ID NO.15: 35s: 5'-TCCCACTATCCTTCGCAAG-3'; SEQ ID NO.16: BnaA07.MKK9-R2: 5'-TAGGCGCGCCTAAGATCCTCCCGGAGA-3'. The expression level of BnaA07.MKK9 in the T0 generation positive tissue culture seedlings was detected by RT-PCR, and the positive T0 generation tissue culture seedlings were transplanted to the field for continued growth, and self-pollination and bagging were performed after flowering to harvest the corresponding T1 generation transgenic lines. Among them, the reaction procedure for PCR positive identification is as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, and full extension at 72°C for 10 min after 33 cycles of reaction. The transformation method refers to the article (Dai et al. An efficient Agrobacterium-mediated transformation method using hypocotyl asexplants for Brassica napus. Mol. Breed, 2020, 40, 1-13).

[0047] RNA isolater Total RNA Extraction Reagent (Vazyme, China) was used to isolate BnaA07.MKK9. DDRNA was extracted from positive tissue culture seedlings of generation T0, and the experimental procedure was referred to the instructions of the kit. RNA was reverse transcribed using HiScript III1st Strand cDNA Synthesis Kit (+gDNA wiper) reverse transcription kit (Vazyme, China), and the reverse transcription system and reaction procedure were referred to the instructions of the kit. The reverse transcribed cDNA was diluted 50 times as a template for RT-PCR. In the RT-PCR experiment, the internal reference semi-quantitative primer sequence was SEQ ID NO.17: BnActinF2: 5'-AGCTGGAGACGGCTAAGAG-3'; SEQ ID NO.18: BnActinR2: 5'-GTTGGAAAGTGCTGAGGGA-3'. The sequences of the semi-quantitative primers for BnaA07.MKK9 are SEQ ID NO.19: BnaA07.MKK9-F2: 5'-ACAGTCGCTACCTCCGGTGA-3'; SEQ ID NO.20: BnaA07.MKK9-R2: 5'-CCTAAACTCCTCCGAGCACC-3'. The RT-PCR reaction procedure is as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, and full extension at 72°C for 10 min after 28 cycles of reaction. The RT-PCR reaction system was carried out according to the instructions of 2×Taq Master Mix (Dye Plus) C112-01 (Vazyme, Nanjing, China), and the system was 20 μL.

[0048] BnaA07.MKK9 was isolated from leaves and inoculated into living stems by two methods. DD Analysis of the resistance of the T1 generation of the overexpression lines of Sclerotinia sclerotiorum. Inoculation of live stems: Use a 7mm puncher to punch out the mycelium of Sclerotinia sclerotiorum growing on the edge of the potato culture medium, inoculate Sclerotinia sclerotiorum on the main stems of the T1 generation of the overexpression lines of the rapeseed lines J9712 and BnaA07.MKK9 at the end of flowering at 50 cm above the ground and fix them with plastic wrap to keep them moist. Select at least 10 strains of each line for live stem inoculation of Sclerotinia sclerotiorum. Measure the length of the lesions with a ruler 7 days after inoculation and record them. Inoculation of detached leaves: Take the fully expanded true leaves of the rapeseed line J9712 and the overexpression line of BnaA07.MKK9 that have grown in the field for about 8 weeks and place them in a plastic pot with a wet gauze on the bottom. Use a 7mm puncher to punch the mycelium of Sclerotinia sclerotiorum growing on the edge of the potato culture medium and inoculate it on the 1 / 3 of the leaf away from the main vein. Spray the plastic film with a spray bottle, cover the plastic film on the plastic pot, and culture in a dark room at 23℃. Take pictures 48 hours after inoculation to investigate the area of ​​lesions. Select at least 10 strains of each strain for inoculation of Sclerotinia sclerotiorum on detached leaves. The specific inoculation number for each strain is shown in Figure 2 C.

[0049] Results: BnaA07.MKK9 was transformed into hypocotyls by Agrobacterium-mediated genetic transformation. DD -OE vector was transformed into rapeseed recipient J9712 ( Figure 2 A). In the T0 generation, 15 positive seedlings were obtained. RT-PCR results showed that the expression of BnaMKK9 was significantly increased in 10 of the 15 positive seedlings ( Figure 2 B), and these positive seedlings were propagated to obtain T1 generation. The BnaA07.MKK9 T1 generation overexpression lines (#12, #35, #51) were inoculated with bacteria in vitro on leaves and in vivo on stems according to the above method. It was found that the lesion area of ​​these three overexpression lines was significantly smaller than that of the transgenic receptor J9712 ( Figure 2 CD), and the lesion lengths of the three overexpression lines were significantly shorter than those of the transgenic receptor J9712 ( Figure 2 EF). The above results indicate that overexpression of BnaA07.MKK9 enhances the resistance of rapeseed to Sclerotinia sclerotiorum.

[0050] Example 4 Creation of Bnamkk9 knockout mutant and analysis of sclerotinia resistance

[0051] There are four copies of BnaMKK9 in Brassica napus, namely BnaA07.MKK9 (BnaA07G0255500Z S), BnaC06.MKK9 (BnaC06G0283400ZS), BnaA02.MKK9 (BnaA02G0206900ZS) and Bna C02.MKK9 (BnaC02G0277100ZS). Four target sites were designed by CRISPRP2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / C RISPR2 / CRISPR), and the gene editing vector of BnaMKK9 was constructed using the CRISPR / Cas9 system provided by Professor Liu Yaoguang of South China Agricultural University. The specific method of vector construction is referenced to (Ma et al. A robust CRIS PR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015, 8, 1274-1284.). The gene editing vector of BnaMKK9 was named B naMKK9-CRI. The BnaMKK9-CRI vector was transformed into Agrobacterium GV3101. The target site sequences of BnaMKK9 are: SEQ ID NO.21: Tgt1: 5'-CAAGAACACTCTCTACGCTCT-3'; SEQ ID NO.22: Tgt2: 5'-CCGCGAGCGCCCGAAGGATG-3'; SEQ ID NO.23: Tgt3: 5'-ACGGCAGCTG ATGCGAGAGA-3'; SEQ ID NO.24: Tgt4: 5'-CCTCCTCAACTCGAAGGACG-3'.

[0052] The BnaA07.MKK9-CRI recombinant vector was transformed into the rapeseed line J9712 recipient by Agrobacterium-mediated hypocotyl transformation. 25 mg / L hygromycin was used as a selection marker. The T0 generation tissue culture seedlings resistant to hygromycin were screened out and positively identified by PCR using universal primers of the vector. The primer sequences were: SEQ ID NO.25: SP-DL: 5'-GTCGTGCTCCACATGTTGACCG-3'; SEQ ID NO.26: SP-R: 5'-CCCGACATAGATGCA ATAACTTC-3'. The PCR reaction procedure was as follows: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 59℃ for 30s, extension at 72℃ for 2min, and full extension at 72℃ for 10min after 33 cycles of reaction. The PCR positive identification reaction system was carried out according to the instructions of 2×Taq Master Mix (Dye Plus) C112-01 (Vazyme, Nanjing, China), and the system was 20μL.

[0053] Copy-specific primers were designed for BnaA07.MKK9, BnaC06.MKK9, BnaA02.MKK9 and BnaC02.MKK9, and then PCR was performed using these copy-specific primers, and the gene editing of the four copies was analyzed by Sanger sequencing. The edited single plants of the T0 generation were self-pollinated by bagging and propagated until a strain with mutations in all four copies and homozygous was obtained. Among them, the copy-specific primer sequences are: SEQ ID NO.27: BnaA07.MKK9-F3: 5'-CACGGCGGACCTCTACGCTT-3'; SEQ ID NO.28: BnaA07.MKK9-R3: 5'-CGT CGGAGCCACCACCGGAA-3'; SEQ ID NO.29: BnaA02.MKK9-F: 5'-AACCGTCCTCT CTAACTC-3'; SEQ ID NO.30: BnaA02.MKK9-R: 5'-CTGGAGAAAAGGGTGGCCG-3'; SEQ ID NO.31: BnaC02.MKK9-F: 5'-TCCATCACCACCGCCGCCT-3'; SEQ ID NO.32: BnaC02.MKK9-R: 5'-TTCCCGGAGAAAAGGATGGCCA-3'; SEQ ID NO.33: BnaA02.MKK9-F: 5'-AACCGTCCTCT CTAACTC-3'; SEQ ID NO.34: BnaA02.MKK9-R: 5'-CTGGAGAAAAGGGTGGCCG-3'; aC06.MKK9-F: 5'-CCACAGTCGCTACCTCCGGT-3'; SEQ ID NO.34: BnaC06.MKK9-R: 5'-GCGTACACGTCGGAGCCT-3'. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 30 s, and full extension at 72°C for 10 min after 33 cycles of reaction. The PCR reaction system was carried out according to the instructions of 2×Taq Master Mix (Dye Plus) C112-01 (Vazyme, Nanjing, China), and the system was 50 μL.

[0054] The resistance of Bnamkk9 knockout mutant and transgenic recipient J9712 to sclerotinia was analyzed by in vitro leaf inoculation and in vivo stem inoculation. The in vitro leaf inoculation was referred to Example 3.

[0055] BnaA07.MKK9 was transformed into hypocotyls by Agrobacterium-mediated genetic transformation. DD-CRI vector was transferred into rapeseed recipient J9712, and 11 positive seedlings were obtained in the T0 generation. Two gene-edited plants (#5 and #7) were obtained in the T0 generation by PCR amplification with copy-specific primers and Sanger sequencing. They were self-crossed to the T4 generation to obtain a line with four copies mutated and homozygous ( Figure 3 A). The #5 and #7 strains were inoculated with bacteria in vitro on leaves and in vivo on stems. It was found that the lesion area and lesion length of the two mutants of Bnamkk9 were significantly larger than those of the transgenic receptor J9712 ( Figure 3 BE). By investigating BnaA07.MKK9 DD The leaf resistance and stem resistance of the overexpression strain and the Bnamkk9 deletion mutant further demonstrated that BnaA07.MKK9 positively regulates the resistance of rapeseed to sclerotinia. Example 5 Natural variation analysis of BnaA07.MKK9

[0056] Using the data of 322 rapeseed germplasm resources provided by the BnIR database (https: / / yanglab.hzau.edu.cn / ), BnaA07.MKK9 was divided into three haplotypes: Hap0, Hap1 and Hap2. The CDS sequences of Hap0, Hap1 and Hap2 are shown in SEQ ID NOs. 4 to 6.

[0057] The nucleotide sequence of the coding region of Hap0 (SEQ ID NO.4):

[0058] ATGGCTTTGGTAAGAGAACGTCGTCAGCTCAACCTCCGTCTTCCTCTTCCTCCGATCTCCGACCGCCGCTTCTCCCTCCCATCTGTCACCACCACAGTCGCTACCTCCGGTGACATCTCCGCCGCTGATCTCGAGAAACTCAACGTTCTCGGATGCGGAAACGGCGGGATTGTTTACAGAGTCCGTCACAAGAACACGGCGGACCTCTACGCTTTGAAAACTGTCAACGGCGACATGGATCCGATCTTACCACGGCAGCTGATGCGAGAGATGGAGATCCTCCGCCGTACGGACTCTCCCTACGTCGTCCGGTGTCACGGAATCTTCGAGAAACAAGTCGTCGGCGAAGTGTCGATTCTGATGGAGTACATGGACGGCGGAACTCTAGAGTCTCTCCGCGGCGCCGTGACGGAGCAACGCCTCGCCGGGTTCGCGAGACAGATCTTGAAAGGTCTTAGTTACTTGCACGCTCTCAAGATCGTGCACAGGGACATCAAACCGGCGAACCTCCTCCTCAACTCGAAGGACGAGGTCAAGATCGCCGACTTCGGAGTCAGCAAGATCTTGGTCCGGGACTTGGATTCCTGCAACGACTACGTCGGGACTTGTGCTTACATGAGTCCGGAGAGGTTTGATTCCGAATCTTCTTCCGGTGGTGGCTCCGACGTGTACGCCGGAGATATCTGGAGCTTCGGGTTGATGATGCTGGAGCTTCTCGTTGGTCACTTTCCGTTATTACCGCCGGGACAGAGACCGGACTGGGCGACTTTGATGTGCGCGGTGTGTTTTGGGGAGCCGCCGCGGGCGCCGGAGGGGTGCTCGGAGGAGTTTAGGAGCTTTGTTGAGTGTTGTCTACGTAAAGATTCGAGTAAGAGATGGACGGCGTCGCAGCTTCTCGCCCATCCTTTTCTCCGGGAGGATCTTTAG

[0059] Nucleotide sequence of the coding region of Hap1 (SEQ ID NO.5):

[0060] ATGGCTTTGGTAAGAGAACGTCGTCAGCTCAACCTCCGTCTTCCTCTTCCTCCGATCTCCGACCGCCGCTTCTCCCTCCCATCTGTCACCACCACAGTCGCTACCTCCGGTGGCATCTCCGCCGCTGATCTCGAGAAACTCAACGTTCTTGGATGCGGAAACGGCGGGGTTGTTTACAGAGTCCGTCACAAGAACACGGCGGACCTCTACGCTTTGAAAACTGTCAACGGCGACACGGATCCGATCTTAACACGGCAGCTGATGCGCGAGATGGAGATCCTCCGCCGTACGGACTCTCCCTACGTAGTCCGGTGTCACGGGATCTTCGAGAAACAAGTCGTCGGCGAAGTATCGATTCTGATGGAGTACATGGACGGCGGAACTCTAGAGTCTCTCCGCGGCGCCGTGACGGAGCAACGCCTCGCCGGGTTCGCGAGACAGATCTTGAAAGGTCTTAGTTACTTGCACGCTCTCAAGATCGTGCACAGGGACATCAAACCGGCGAACCTCCTCCTCAACTCGAAGGACGAGGTCAAGATCGCCGACTTCGGAGTCAGCAAGATCTTGGTCCGGGACTTGGATTCCTGCAACGACTACGTCGGGACTTGTGCTTACATGAGTCCGGAGAGGTTTGATTCCGAATCTTCTTCCGGTGGTGGCTCCGACGTGTACGCCGGAGATATCTGGAGCTTCGGGTTGATGATGCTGGAGCTTCTCGTTGGTCACTTTCCGTTATTACCGCCGGGACAGAGACCGGACTGGGCGACTTTGATGTGCGCGGTGTGTTTTGGGGAGCCGCCGCGGGCGCCGGAGGGGTGCTCGGAGGAGTTTAGGAGCTTTGTTGAGTGTTGCCTACGTAAAGATTCGAGTAAGAGATGGACGGCGTCGCAGCTTCTCGCCCATCCTTTTCTCCGGGAGGATCTTTAG

[0061] Nucleotide sequence of the coding region of Hap2 (SEQ ID NO.6): ATGGCTTTGGTAAGAGAACGT

[0062] CGTCAGCTCAACCTCCGTCTTCCTCTTCCTCCGATCTCCGACCGCCGCTTCTCCCTCCCATCTGTCACCACCACAGTCGCTACCTCCGGTGACATCTCCGCCGCTGATCTCGAGAAACTCAACGTTCTCGGATGCGGAAACGGCGGGATTGTTTACAGAGTCCGTCACAAGAACACGGCGGACCTCTACGCTTTGAAAACTGTCAACGGCGACATGGATCCGATCTTACCACGGCAGCTGATGCGCGAGATGGAGATCCTCCGCCGTACGGACTCTCCCTACGTAGTCCGGTGTCACGGAATATTCGAGAAACAAGTCGTCGGCGAAGTATCGATTCTGATGGAGTACATGGACGGCGGAACTCTAGAGTCTCTCCGCGGCGCCGTGACGGAGCAGCGCCTCGCCGGGTTCGCGAGACAGATCTTGAAAGGTCTTAGTTACTTGCACGCTCTCAAGATCGTGCACAGGGACATCAAACCGGCGAACCTCCTCCTCAACTCGAAGGACGAGGTCAAGATCGCCGACTTCGGAGTCAGCAAGATCTTGGTCCGGGACTTGGATTCCTGCAACGACTACGTCGGGACTTGTGCTTACATGAGTCCGGAGAGGTTTGATTCCGAATCTTCTTCCGGTGGTAGCTCCGACGTGTACGCCGGAGATATCTGGAGCTTCGGGTTGATGATGCTGGAGCTTCTCGTTGGTCACTTTCCGTTATTACCGCCGGGACAGAGACCGGACTGGGCGACGTTGATGTGCGCGGTGTGTTTTGGGGAGCCGCCGCGGGCGCCGGAGGGATGCTCGGAGGAGTTTAGGAGCTTTGTTGAGTGTTGTCTACGTAAAGATTCGAGTAAGAGATGGACGGCGTCGCAGCTTCTCGCCCATCCTTTTCTCCGGGAGGATCTTTAG

[0063] In vivo kinase activity analysis: 30 rapeseed varieties of Hap0, Hap1 and Hap2 were selected (Table 2) and inoculated with detached leaves and simulated PDA inoculation. The in vitro leaf inoculation was referred to Example 3, and the PDA simulated inoculation was performed by punching 7 mm potato culture medium with a 7 mm puncher. The remaining inoculation steps were consistent with the in vitro leaf inoculation in Example 3. Samples were taken 12 hours after the above samples were inoculated and stored at -80°C. The leaves of Hap0, Hap1 and Hap2 were ground into powder, and the three powders were added in equal amounts to the extraction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 5 mM ethylene glycol bis-α-aminoethyl ether tetraacetic acid EGTA, 5 mM Na 3 VO 4 , 5mM NaF, 50mM sodium β-glycerophosphate, 10mM dithiothreitol DTT, 1mM phenylmethylsulfonyl fluoride, 5g mL -1 Leupeptin, 5 g mL -1 Aprotinin, 10% glycerol, 0.1% Tween 20), mixed at 4°C for 30 minutes, and centrifuged at 12000g for 30 minutes. The supernatant was taken as the total protein of Hap0, Hap1 and Hap2. 200μg of Hap0, Hap1 and Hap2 total protein were incubated with anti-BnaA07.MKK9 antibody at 4°C overnight, followed by adding 30μl protein A+G magnetic beads (Beyotime, Jiangsu, China) and incubated at 4°C for 3h and then gently washed three times with extraction buffer. The protein A+G magnetic beads were mixed with 10μg myelin basic protein (MBP, SigmaAldrich, St.Louis, MO, USA) in kinase buffer (25mMTris–HCl [pH 7.5], 5mM MgCl 2 , 1 mM EGTA, 0.1 mM Na 3 VO 4 , 1mM DTT and 100μM ATP) at 30℃ for 30min, phosphorylated MBP was obtained, and then protein immunoblotting was performed. The protein immunoblotting process was referenced to (Kurien et al. Western blotting. Methods, 2006, 38, 283-293.), and phosphorylated MBP reflected the kinase activity of Hap0, Hap1 and Hap2. Anti-pMBP (Millipore, Eschborn, Germany) was used to detect phosphorylated MBP, anti-MBP (SigmaAldrich, St. Louis, MO, USA) was used to detect MBP, and anti-BnaA07.MKK9 antibody was synthesized by Shanghai Kaijing Biotechnology Co., Ltd.

[0064] Table 2 Rapeseed varieties used in in vivo kinase activity analysis

[0065]

[0066]

[0067] The results showed that BnaA07.MKK9 had three major haplotypes (Hap0, Hap1 and Hap2), and these three haplotypes had four missense SNPs and 11 synonymous SNPs in the coding region, and two missense SNPs were located in the ATP binding site of BnaA07.MKK9 ( Figure 4 ). Analysis of stem lesion length showed that the three haplotypes of BnaA07.MKK9 had significant differences in resistance to sclerotinia, with hap0 having the strongest resistance and hap1 having the weakest resistance ( Figure 5 ). In vivo kinase activity analysis revealed that the kinase activity of the disease-resistant haplotype hap0 increased most dramatically after inoculation with Sclerotinia sclerotiorum, while the kinase activity of the susceptible haplotype hap1 increased most slowly after inoculation with Sclerotinia sclerotiorum ( Figure 6 ), further demonstrating that the differences in resistance of different haplotypes to Sclerotinia sclerotiorum were positively correlated with their kinase activities.

[0068] Example 6 Design of SNAP Molecular Markers for BnaA07.MKK9 Haplotype

[0069] The CDS sequences of the three haplotypes of BnaA07.MKK9 were used as references and sequence alignment was performed using the Clustal W (https: / / www.genome.jp / tools-bin / clustalw) online website. SNAP molecular markers for Hap0, Hap1 and Hap2 were designed at the SNPs of the three haplotypes. High-quality DNA of Brassica napus lines SWU94, Zhongshuang No. 11, Niklas, Monty, WH-88 and SWU47 were extracted by CTAB method. SWU94 and Zhongshuang No. 11 correspond to Hap0, Niklas and Monty correspond to Hap1, and WH-88 and SWU47 correspond to Hap2. The DNA concentration of the above samples was diluted to 10 ng / μL for PCR. The PCR reaction program was pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, and full extension at 72℃ for 10 min after 30 cycles of reaction. The PCR reaction system was carried out according to the instructions of 2×Taq Master Mix (Dye Plus) C112-01 (Vazyme, Nanjing, China), and the PCR reaction system was 20 μL. The sequences of the SNAP molecular marker primers are as follows: SEQ ID NO.35: Hap0-F: 5'-AACACGGCAGCTGATGTTA-3'; SEQ ID NO.36: Hap0-R: 5'-CGTCGGAGCCACCACCGGAA-3'; SEQ ID NO.37: Hap1-F: 5'-CTCGAGAAACTCAA CGTTCCT-3'; SEQ ID NO.38: Hap1-R: 5'-CGTCGGAGCCACCACCGGAA-3'; SEQ ID NO.39: Hap2-F: 5'-CGTAGTCCGGTGTCACGGAATA-3'; SEQ ID NO.40: Hap2-R: 5'-CGTCGGAGCCACCACCGGAA-3'.

[0070] 1% agarose gel electrophoresis revealed that Hap1-F and Hap1-R can specifically detect Hap1( Figure 7 A), Hap0-F and Hap0-R can be used together to specifically detect Hap0 ( Figure 7 B), while Hap2-F and Hap2-R can specifically detect Hap2( Figure 7 C).

[0071] In summary, the above experiments prove that BnaA07.MKK9 is a gene that regulates the resistance of rapeseed to Sclerotinia sclerotinia. Overexpression of this gene in rapeseed can enhance the resistance of rapeseed to Sclerotinia sclerotinia. After knocking out this gene in rapeseed, the resistance of Bnamkk9 deletion mutant to Sclerotinia sclerotinia was significantly weakened. In addition, there are three main haplotypes of this gene, among which Hap0 has the strongest resistance to Sclerotinia sclerotinia, while Hap1 has the weakest resistance to Sclerotinia sclerotinia. The corresponding SNAP molecular markers are designed according to the SNP differences of haplotypes. The above research results provide useful target genes for breeding of Sclerotinia sclerotinia resistance.

Claims

1. BnaA07.MKK9 DD The application of gene overexpression in improving the resistance of rapeseed to sclerotinia sclerotinia is characterized in that: BnaA07.MKK9 DD The nucleotide sequence of the overexpressed gene is shown in SEQ ID NO.

3.

2. Application of a deletion mutant of the BnaA07.MKK9 gene in reducing resistance to sclerotinia sclerotiorum in rapeseed, characterized in that: The deletion mutant of the BnaA07.MKK9 gene is a mutant in which four copies of BnaA07.MKK9 with accession number BnaA07G0255500ZS, BnaC06.MKK9 with accession number BnaC06G0283400ZS, BnaA02.MKK9 with accession number BnaA02G0206900ZS and BnaC02.MKK9 with accession number BnaC02G0277100ZS are knocked out simultaneously.

3. A method for improving the resistance of rapeseed to sclerotinia sclerotinia, characterized in that: The following steps are involved: BnaA07.MKK9 DD The gene overexpression vector can be transformed into rapeseed; the BnaA07.MKK9 DD The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. A method for reducing the resistance of rapeseed to Sclerotinia sclerotiorum, characterized in that: The following steps are involved: The recombinant vector with four copies of the BnaA07.MKK9 gene knocked out at the same time can be transformed into rapeseed; the four copies are BnaA07.MKK9 with accession number BnaA07G0255500ZS, BnaC06.MKK9 with accession number BnaC06G0283400ZS, BnaA02.MKK9 with accession number BnaA02G0206900ZS and BnaC02.MKK9 with accession number BnaC02G0277100ZS.

5. Application of the SNAP molecular marker of the BnaA07.MKK9 gene or its primer combination in identifying the resistance of rapeseed to Sclerotinia sclerotiorum, characterized in that: The SNAP molecular marker is one or more of Hap0, Hap1 or Hap2; the nucleotide sequence of Hap0 is shown in SEQ ID NO.4; the nucleotide sequence of Hap1 is shown in SEQ ID NO.5; the nucleotide sequence of Hap2 is shown in SEQ ID NO.

6.

6. The use according to claim 5, characterized in that: The primer combination is one or more of a primer combination of Hap0, a primer combination of Hap1 or a primer combination of Hap2.

7. The use according to claim 6, characterized in that: The sequences of the primer combination of Hap0 are shown in SEQ ID NO.35 to SEQ ID NO.

36.

8. The use according to claim 6, characterized in that: The sequences of the primer combination of Hap1 are shown in SEQ ID NO.37 to SEQ ID NO.

38.

9. The use according to claim 6, characterized in that: The sequences of the primer combination of Hap2 are shown in SEQ ID NO.39 to SEQ ID NO.40.

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