SNP (Single Nucleotide Polymorphism) molecular marker linked with sclerotinia rot resistance QTL-C04 site of brassica napus and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker linked with a sclerotinia rot resistance QTL-C04 site of brassica napus and application of the SNP molecular marker. The SNP molecular marker is located between the 31813481 basic group and the 33945641 basic group of a C04 chromosome of brassica napus. The molecular marker developed by the invention can accurately predict the sclerotiniose resistance of plants in the early stage, is independent of the limitation of field disease attack conditions or natural conditions, can realize early, efficient and accurate breeding, and greatly improves the disease-resistant breeding efficiency.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular biology and rapeseed breeding, and in particular to a SNP molecular marker linked to a QTL-C04 locus for sclerotinia rot resistance in Brassica napus and applications thereof. Background Art

[0002] Rapeseed (Brassica napus) is an important oilseed crop in the genus Brassica in the family Cruciferae and is widely cultivated worldwide. Increasing rapeseed production mainly relies on three approaches: increasing rapeseed yield per unit area, increasing rapeseed oil content, and expanding the cultivated area. The main goal of current rapeseed breeding is to strive to improve oil content, yield, and quality. Because rapeseed traits such as oil content, yield, and quality are complex quantitative traits and are easily affected by the environment, traditional breeding methods and technologies have difficulty achieving significant breakthroughs. Therefore, the combination of quantitative genetics and molecular marker technology provides a new opportunity for the development of rapeseed genetic breeding.

[0003] SNP-based molecular marker technology is considered the third generation of molecular markers, following RFLP and SSR. SNPs refer to variations such as single nucleotide differences between alleles at the same locus, or small deletions, insertions, and mutations. This technology enables automated, batch testing through sequencing or PCR combined with DNA microarrays, offering significant advantages and broad application prospects in genetic mapping studies.

[0004] Sclerotinia sclerotiorum is a plant disease caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) deBary. Sclerotinia sclerotiorum has a wide host range, infecting over 600 plant species from more than 70 families. To date, no rapeseed material has been found that is completely immune or highly resistant to sclerotinia sclerotiorum, but resistance variation within its germplasm is rich, manifesting as a complex quantitative trait controlled by multiple genes. In recent years, with the completion of whole-genome sequencing, the widespread application of molecular marker technology, and the continuous development of marker-assisted selection (MAS) technology, a significant amount of progress has been made in the mapping of molecular markers and QTLs associated with sclerotinia sclerotiorum resistance in rapeseed, although most are still in the preliminary positioning stage.

[0005] Therefore, further studying the resistance of Brassica napus to sclerotinia sclerotiorum at the molecular level with the help of molecular markers and quantitative trait loci (QTL) mapping technology will not only help improve the disease resistance of rapeseed, but also lay the foundation for revealing its genetic and molecular mechanisms of resistance. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention proposes a SNP molecular marker linked to the QTL-C04 locus for Sclerotinia sclerotiorum resistance in Brassica napus and its application.

[0007] The present invention provides a SNP molecular marker for a QTL locus for sclerotinia resistance in Brassica napus. The SNP molecular marker is located between bases 31813481 and 33945641 on chromosome C04 of Brassica napus. The SNP molecular marker has a high contribution rate to sclerotinia resistance in Brassica napus leaves and simultaneously regulates the content of 2',6-dihydroxyflavone in Brassica napus leaves. It is speculated that the site may contribute to sclerotinia resistance by regulating the content of 2',6-dihydroxyflavone. The SNP molecular marker can be used for map-based cloning and molecular marker-assisted selection, and is suitable for large-scale promotion and application.

[0008] In some embodiments, the SNP molecular marker is located at base 31813481 of chromosome C04 of Brassica napus, and the base 31813481 exhibits polymorphism of two alleles, C and T.

[0009] In some embodiments, the SNP molecular marker is located at base 33945641 of chromosome C04 of Brassica napus, and the base 33945641 exhibits polymorphism of two alleles, G and A.

[0010] In some embodiments, the SNP molecular marker is located at base 31890428 of chromosome C04 of Brassica napus, and the base 31890428 exhibits polymorphism of two alleles, G and A.

[0011] The present invention also provides the use of the SNP molecular marker in any of the following:

[0012] (1) Detect and / or predict resistance to sclerotinia sclerotiorum in Brassica napus;

[0013] (2) Selecting Brassica napus for its resistance to Sclerotinia sclerotiorum;

[0014] (3) Molecular marker-assisted breeding of Brassica napus;

[0015] (4) Accelerate the breeding process of Brassica napus varieties for resistance to Sclerotinia sclerotiorum.

[0016] The present invention also provides a primer or probe, comprising a primer or probe for amplifying or detecting the SNP molecular marker.

[0017] In some embodiments, the primers are designed based on any one of the following DNA fragments:

[0018] (1) DNA fragments containing 400 bp of sequence before and after bases 31813481 on the C04 chromosome of Brassica napus;

[0019] (2) DNA fragments containing 400 bp before and after base 33945641 of the C04 chromosome of Brassica napus;

[0020] (3) DNA fragments with a sequence of 400 bp before and after base 31890428 of the C04 chromosome of Brassica napus.

[0021] In some embodiments, (1) the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1;

[0022] (2) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 2;

[0023] (3) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO.3.

[0024] In some embodiments, the primers are any one of the following groups:

[0025] (1) The nucleotide sequences shown in SEQ ID NOs. 4 to 5;

[0026] (2) the nucleotide sequences shown in SEQ ID NOs. 6 to 7;

[0027] (3) The nucleotide sequences shown in SEQ ID NOs. 8 to 9.

[0028] Preferably, the primer or probe is labeled with a fluorescent group, and the fluorescent group includes FAM, HEX, VIC, and ROX.

[0029] The present invention also provides a reagent or a kit comprising the primer or the probe.

[0030] The present invention also provides a rapeseed breeding method, which is characterized by comprising the following steps: extracting genomic DNA of the rapeseed to be tested, testing the SNP molecular markers, and screening plants with high resistance to sclerotinia sclerotinia according to the test results for breeding.

[0031] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0032] 1. Through genome-wide association analysis, this study mapped a QTL region (31813481–33945641 bp) significantly associated with sclerotinia resistance on chromosome C04 of Brassica napus. The peak SNP (chrC04_31890428) and two boundary SNPs were precisely identified. This QTL contributes 11.65% to the resistance phenotype, demonstrating high genetic contribution and stability.

[0033] 2. The SNP molecular markers constructed in the present invention can accurately predict resistance to sclerotinia sclerotinia in the early stages of plant development, overcoming the limitations of traditional field inoculation that relies on environmental conditions. They have the advantages of high throughput, rapidity, and stability, and can significantly improve the efficiency of molecular-assisted selection (MAS) and accelerate the breeding process of new disease-resistant varieties.

[0034] 3. The method for identifying resistance to Sclerotinia sclerotiorum in Brassica napus of the present invention is simple and easy to operate, the markers are stable and reliable, and the selection efficiency is high. It is suitable for resistance screening of large-scale breeding materials and breeding of new varieties. It has broad prospects for promotion and application, and can provide reliable technical support for molecular breeding of Brassica napus resistance to Sclerotinia sclerotiorum. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is a schematic diagram of the distribution results of the Sclerotinia sclerotiorum resistance phenotype of Brassica napus in Example 1 of the present invention.

[0037] Figure 2 This is the Manhattan plot of the association analysis of the Sclerotinia disease resistance phenotype in the associated population of Example 3 of the present invention.

[0038] Figure 3 This is the molecular marker allelic analysis map of the peak SNP (chrC04_31890428) of the resistance QTL site in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] To help those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available.

[0041] Example 1 Evaluation of resistance to sclerotinia sclerotiorum in Brassica napus

[0042] Evaluation of Sclerotinia resistance in associated populations

[0043] (1) A natural population of pure lines of Brassica napus (Brassica napus) consisted of 220 accessions, 171 of which were from China and 49 from abroad (published in Zhang, Y., Structural variation reshapes population gene expression and trait variation in 2,105 Brassica napus accessions. Nature Genetics 2024, 56(11), 2538-2550.). A survey on leaf sclerotinia resistance was conducted at the Yangluo Experimental Base in Wuhan and the Yangzhou Academy of Agricultural Sciences Experimental Base.

[0044] (2) Direct seeding was used, with a row spacing of 33 cm and a plant spacing of 15 cm. Each plot had 4 rows and three replicates were randomly designed. Protective rows were planted around the test material plots.

[0045] (3) Evaluation of resistance to sclerotinia sclerotiorum: Mature leaves of 10 plants in each plot were infected with Sclerotinia sclerotiorum. The lesion area after inoculation was recorded, and the lesion expansion area between each two time points was calculated.

[0046] The disease resistance phenotype results of 220 materials are summarized as shown in Table 1:

[0047] Table 1 Sclerotinia disease resistance phenotypes of 220 accessions

[0048]

[0049]

[0050]

[0051] The results of the leaf lesion expansion area distribution of the associated population showed that the phenotypic distribution of the lesion expansion area was normally distributed, such as Figure 1 shown.

[0052] Example 2 Acquisition of high-quality SNP datasets from associated populations

[0053] The CTAB method was used to extract total DNA from leaves of each material of the associated population. The specific method was as follows:

[0054] The young leaves were rinsed in 10% ethanol; 0.1-0.2 g of leaves were cut and placed in a mortar, quickly ground with liquid nitrogen to powder, and placed in a 2 mL centrifuge tube; 700 μL of preheated DNA extraction solution was added; after mixing, the tube was placed in a 65°C water bath for 1 hour, mixing once every 10-15 minutes; 700 μL of a mixture (phenol: chloroform: isoamyl alcohol = 25:24:1) was added and gently inverted to mix for 10 minutes; centrifuged at 10,000 × g for 15 minutes at room temperature; the supernatant was transferred to a new 2 mL centrifuge tube; an equal volume of a mixture (chloroform: isoamyl alcohol = 24:1) was added and inverted to mix, allowed to stand for 5 minutes, and centrifuged at 10,000 × g for 15 minutes. The supernatant was pipetted into a new centrifuge tube; 2 volumes of anhydrous ethanol were added, mixed, and allowed to stand at -20°C for 1 hour, 10 The pellet was centrifuged at 10,000 × g for 10 min, and the supernatant discarded. The pellet was then washed with 500 μL of pre-cooled 75% ethanol, and the supernatant discarded. After two consecutive washes, the pellet was air-dried. 100 μL of a 2% RNase A solution was added, and the pellet was incubated at 37°C for 1 h before incubating at 4°C overnight. DNA was extracted again with an equal volume of a mixture (chloroform:isoamyl alcohol = 24:1), mixed by inversion, and incubated for 10 min. The pellet was centrifuged at 10,000 × g for 15 or 20 min to remove RNase A. The supernatant (approximately 60 μL) was aspirated and centrifuged again for 1 min. DNA concentration, quality, and integrity were assessed by agarose gel electrophoresis (0.8%) and UV spectrophotometry. The absorbance 260 / 280 ratio for all DNA samples was determined to be between 1.8 and 2.0. DNA samples were then shipped on dry ice to a sequencing company (BGI Genomics Co., Ltd.), with each sample sequenced at a depth of approximately 7×.

[0055] After obtaining high-quality DNA as described above, the sequencing company (BGI Genomics) performed 7× coverage depth sequencing and returned data. FastQC software was used to assess sequencing quality, followed by adapter and low-quality read filtering. Clean data from paired-end sequencing of each material was obtained, followed by mapping using bwa software and variant detection using GATK software. After obtaining the total SNP dataset for the associated population, the SNP dataset was quality-filtered based on a minimum allele frequency ≥ 0.05, a missingness rate ≤ 0.1, and a heterozygosity rate ≤ 0.1. Ultimately, a high-quality population SNP dataset was obtained for subsequent analysis.

[0056] Example 3 Genome-wide association analysis

[0057] Based on the VCF file of the high-quality SNP dataset generated in step 2 of Example 2, genome-wide association analysis of sclerotinia disease-related phenotypes was performed using the rMVP package of R language using the GLM model to obtain the P value of each SNP site. When the P value was less than 2.98×10-7 The SNP with the smallest P value is the peak SNP. The materials with different allele types of the peak SNP in the population are grouped and variance analysis is performed. The percentage of the ratio of the variance between groups to the total variance is the contribution rate of the peak SNP. Manhattan plot of association analysis of Sclerotinia disease resistance phenotype in the associated population is shown below. Figure 2 shown.

[0058] Through analysis, the interval of the QTL locus for Sclerotinia sclerotiorum resistance in Brassica napus was limited to bases 31813481 to 33945641 on chromosome C04 of Brassica napus. The corresponding SNPs were chrC04_31813481 (C / T) and chrC04_33945641 (G / A). The peak SNP was chrC04_31890428 (G / A). The contribution rate of this QTL to Sclerotinia sclerotiorum resistance in Brassica napus was 11.65% (the materials were grouped according to the different allele types of the peak SNP, and a one-way analysis of variance was performed. The percentage of the variance between groups divided by the total variance was the contribution rate).

[0059] The peak SNP for Sclerotinia sclerotiorum resistance is: chrC04_31890428 (G / A), and the corresponding Sclerotinia sclerotiorum resistance phenotype grouping is: when the SNP at the chrC04_31890428 position is G, the average lesion expansion area of ​​the material is 7.12; when it is A, the average lesion expansion area of ​​the material is 6.58; the contribution rate of this peak SNP is 11.65%; the molecular marker allelic analysis diagram of the peak SNP (chrC04_31890428) of the resistance QTL site is shown in Figure 3 shown.

[0060] A boundary SNP marker for the Sclerotinia sclerotinia phenotype is chrC04_31813481 (C / T). The corresponding Sclerotinia sclerotinia resistance phenotype grouping is as follows: when the SNP at the chrC04_31813481 position is C, the average lesion expansion area of ​​the material is 7.13; when it is T, the average lesion expansion area of ​​the material is 6.63; the contribution rate of this boundary SNP is 8.01%;

[0061] Another boundary SNP marker of the sclerotinia phenotype is: chrC04_33945641 (G / A), and the corresponding sclerotinia phenotype grouping is: when the SNP at the chrC04_33945641 position is G, the average lesion expansion area of ​​the material is 7.09; when it is A, the average lesion expansion area of ​​the material is 6.64; the contribution rate of this boundary SNP is 7.91%.

[0062] The complete genome sequence of Brassica napus has been published, including the 400 bp sequence before and after chrC04_31813481 (C / T) (801 bp in total) as shown in SEQ ID NO.1, the 400 bp sequence before and after chrC04_33945641 (G / A) (801 bp in total) as shown in SEQ ID NO.2, and the 400 bp sequence before and after chrC04_31890428 (G / A) (801 bp in total) as shown in SEQ ID NO.3.

[0063] Those skilled in the art can use conventional methods to design specific primers or probes for detecting SNP sites based on known sequences. The primers or probes can also be labeled with fluorescent groups such as FAM, HEX, VIC, ROX, etc., and quenching groups such as BHQ1 or TAMRA through conventional techniques in the art, so that the genotype of the above-mentioned SNP site can be detected by conventional methods in the art such as sequencing or PCR, thereby detecting and predicting the level of resistance of Brassica napus to Sclerotinia sclerotinia, and then effectively selecting the level of resistance of Brassica napus to Sclerotinia sclerotinia, which can be used for molecular marker-assisted breeding of Brassica napus with Sclerotinia sclerotinia resistance, thereby accelerating the process of breeding Brassica napus for Sclerotinia sclerotinia resistance.

[0064] Therefore, the present invention detected a QTL site for Sclerotinia sclerotinia resistance in Brassica napus on chromosome C04 through phenotypic analysis of leaf Sclerotinia sclerotinia resistance and whole-genome resequencing, followed by whole-genome association analysis, with a contribution rate of 11.65% to Sclerotinia sclerotinia resistance in Brassica napus. The QTL locus for sclerotinia sclerotinia resistance in Brassica napus is located between bases 31813481 and 33945641 on chromosome C04 of Brassica napus. The boundary significant SNPs are chrC04_31813481 (C / T) and chrC04_33945641 (G / A), and the peak SNP is chrC04_31890428 (G / A). The SNP molecular marker tightly linked to the QTL locus can be used to detect the level of sclerotinia sclerotinia resistance in Brassica napus leaves, predict the level of sclerotinia sclerotinia resistance in Brassica napus, and effectively select Brassica napus for the level of sclerotinia sclerotinia resistance, thereby being used for molecular marker-assisted breeding of Brassica napus for sclerotinia sclerotinia resistance, thereby accelerating the process of breeding Brassica napus for sclerotinia sclerotinia resistance.

[0065] The molecular marker-assisted selection is carried out by the SNP molecular markers disclosed by the present invention. The identification method is simple, the selection efficiency is high, and the resistance of Brassica napus to sclerotinia rot can be predicted. The selection target is clear and is not affected by the environment. Brassica napus single plants with sclerotinia rot resistance can be identified in the early growth stage of Brassica napus, and other single plants are eliminated. In summary, the QTL locus for sclerotinia rot resistance in Brassica napus leaves of the present invention has a high contribution rate to the resistance to sclerotinia rot of Brassica napus, plays a key role in the regulation of the resistance to sclerotinia rot of Brassica napus, can be used for map-based cloning and molecular marker-assisted selection, and is suitable for large-scale promotion and application.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0067]

[0068]

Claims

1. A SNP molecular marker for a QTL locus for resistance to sclerotinia sclerotiorum in Brassica napus, characterized in that: The SNP molecular marker is located between the 31813481st base and the 33945641st base of the C04 chromosome of Brassica napus.

2. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 31813481st base of the C04 chromosome of Brassica napus, and the 31813481st base has polymorphisms of two alleles, C and T.

3. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 33945641st base of the C04 chromosome of Brassica napus, and the 33945641st base shows polymorphism of two alleles, G and A.

4. The SNP molecular marker according to claim 1, characterized in that The SNP molecular marker is located at the 31890428th base of the C04 chromosome of Brassica napus, and the 31890428th base shows polymorphism of two alleles, G and A.

5. Use of the SNP molecular marker according to any one of claims 1 to 4 in any one of the following: (1) Detect and / or predict resistance to sclerotinia sclerotiorum in Brassica napus; (2) Selecting Brassica napus for its resistance to Sclerotinia sclerotiorum; (3) Molecular marker-assisted breeding of Brassica napus; (4) Accelerate the breeding process of Brassica napus varieties for resistance to Sclerotinia sclerotiorum.

6. A primer or probe, characterized in that The method comprises a primer or a probe for amplifying or detecting the SNP molecular marker according to any one of claims 1 to 4.

7. The primer or probe according to claim 6, characterized in that The primers are designed based on any one of the following DNA fragments: (1) DNA fragments containing 400 bp of sequence before and after bases 31813481 on the C04 chromosome of Brassica napus; (2) DNA fragments containing 400 bp before and after base 33945641 of the C04 chromosome of Brassica napus; (3) DNA fragments with a sequence of 400 bp before and after base 31890428 of the C04 chromosome of Brassica napus.

8. The primer or probe according to claim 7, characterized in that (1) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 1; (2) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 2; (3) The nucleotide sequence of the DNA fragment is shown in SEQ ID NO.

3.

9. A reagent or kit, characterized in that Comprising the primer or probe according to any one of claims 6 to 8.

10. A rapeseed breeding method, characterized in that: The following steps are involved: The genomic DNA of the rapeseed to be tested is extracted, the SNP molecular marker according to any one of claims 1 to 4 is tested, and plants with high resistance to sclerotinia sclerotiorum are screened out for breeding according to the test results.

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