Molecular markers of BnaUGD1 gene SNP related to water resistance and their applications

Abstract

This invention relates to the field of biotechnology, and more particularly to the SNP molecular markers of the BnaUGD1 gene, which is associated with waterlogging tolerance, and their applications. This invention uses genome-wide association analysis to precisely locate SNP sites significantly associated with waterlogging tolerance in rapeseed. Combined with linkage disequilibrium analysis, candidate genes for the BnaUGD1 gene are identified in the region near the peak SNP. Further screening identifies SNP molecular markers highly associated with waterlogging tolerance in rapeseed. Based on these markers, the identification of waterlogging tolerance traits can be achieved, demonstrating significant application prospects in the field of breeding for improved waterlogging tolerance in rapeseed.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the molecular marker of the BnaUGD1 gene SNP related to water resistance and its application. Background Technology

[0002] Rapeseed (Brassica napus L.), an annual oilseed crop belonging to the Brassicaceae family, is one of the world's and my country's main sources of edible oil. In rapeseed production, waterlogging is a key abiotic stress factor restricting yield stability. Due to the underdeveloped aerenchyma of rapeseed roots, waterlogging in rainy, low-lying areas or under water-dry rotation planting patterns leads to root hypoxia, causing abnormal anaerobic respiration and metabolism, producing toxic metabolites, damaging cell structure integrity, inhibiting water and nutrient absorption efficiency, and ultimately hindering photosynthesis in the aboveground parts, resulting in stunted growth and a significant decrease in yield.

[0003] Current screening for waterlogging tolerance in rapeseed still relies primarily on traditional breeding methods, which suffer from low screening efficiency. Specifically, this is manifested in the need for long-term field observations across multiple environments and growth stages, resulting in lengthy screening cycles and high costs. Furthermore, it is susceptible to environmental fluctuations, making it difficult to accurately assess the genetic stability of waterlogging tolerance traits. Given these limitations, there is an urgent need to develop a novel molecular marker technology system to achieve early and precise screening and genetic improvement of waterlogging tolerance traits, breaking through the technical limitations of traditional breeding and providing efficient technical support for the rapid development of waterlogging-tolerant rapeseed varieties and ensuring a secure supply of edible oil. Summary of the Invention

[0004] In view of this, the present invention proposes a molecular marker for the BnaUGD1 gene SNP related to water resistance and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides the application of waterlogging-related SNP molecular markers in the identification of waterlogging tolerance in Brassica napus. The SNP molecular markers are located at 13693515 bp on chromosome C05 of Brassica napus and are A / T polymorphic sites. The waterlogging tolerance of Brassica napus carrying the AA genotype is higher than that of Brassica napus carrying the AT genotype. The reference genome is Brassica napusDarmor.v4.1.

[0007] Furthermore, in some specific embodiments, the genotype of the rapeseed to be tested for the SNP molecular marker is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension, or gene chip method.

[0008] Secondly, this invention provides the application of a reagent for detecting waterlogging-related SNP molecular markers in the identification of waterlogging tolerance in Brassica napus. The SNP molecular marker is located at 13693515 bp on chromosome C05 of Brassica napus and is an A / T polymorphic site. Rapeseed carrying the AA genotype has higher waterlogging tolerance than rapeseed carrying the AT genotype. The reference genome is Brassicanapus Darmor.v4.1.

[0009] Thirdly, this invention provides the application of waterlogging-resistant SNP molecular markers in the breeding of waterlogging-resistant Brassica napus. The SNP molecular markers are located at 13693515 bp on chromosome C05 of Brassica napus and are A / T polymorphic sites. The genotype of the SNP molecular markers is detected, and samples with the genotype AA are selected for breeding. The reference genome is Brassicanapus Darmor.v4.1.

[0010] Fourthly, this invention provides a method for identifying the waterlogging tolerance of Brassica napus, which detects the genotype of an SNP molecular marker located at 13693515 bp on chromosome C05 of Brassica napus. The SNP molecular marker is an A / T polymorphic site, and the rapeseed carrying the AA genotype has higher waterlogging tolerance than the rapeseed carrying the AT genotype. The reference genome is Brassica napusDarmor.v4.1.

[0011] Fifthly, this invention provides a method for breeding waterlogging-tolerant Brassica napus, which involves detecting the genotype of an SNP molecular marker located at 13693515 bp on chromosome C05 of Brassica napus, representing an A / T polymorphism site; detecting the genotype of the SNP molecular marker; and selecting samples with the genotype AA for breeding; the reference genome is Brassicanapus Darmor.v4.1.

[0012] Furthermore, the genotype of the rapeseed to be tested for the SNP molecular marker was detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

[0013] Furthermore, in some specific embodiments, the sample with the genotype AA is used as the donor parent and hybridized and / or backcrossed with the recipient parent. The SNP molecular marker is used to detect and screen the offspring genotype until a homozygous line with the genotype AA at the SNP molecular marker locus is obtained.

[0014] Furthermore, in some specific embodiments, haploid breeding is performed on samples with the genotype AA to obtain a double haploid population, and homozygous lines with the genotype AA at the SNP molecular marker locus are screened from the double haploid population using the SNP molecular marker.

[0015] Specifically, anthers or microspores of samples with genotype AA are cultured in vitro to induce haploid plants; chromosome doubling is performed on the haploid plants to obtain a double haploid population; homozygous lines with genotype AA at the specified SNP molecular marker site are screened from the double haploid population using the SNP molecular marker.

[0016] The beneficial effects of the present invention include at least the following:

[0017] (1) The BnaUGD1 gene SNP molecular marker related to rapeseed waterlogging tolerance provided by the present invention provides a precise gene resource for screening and breeding waterlogging tolerant rapeseed. The method for identifying waterlogging tolerance of Brassica napus provided by the present invention is simple, feasible and efficient, and has important application prospects in the field of rapeseed waterlogging tolerance improvement breeding.

[0018] (2) This invention precisely locates SNP sites significantly associated with rapeseed waterlogging tolerance through genome-wide association analysis, identifies the candidate gene BnaUGD1 in the region near the peak SNP using linkage disequilibrium analysis, and further screens and identifies SNP molecular markers highly associated with rapeseed waterlogging tolerance. Based on these markers, the waterlogging tolerance trait can be identified. This invention provides important theoretical and technical support for the discovery and application of other waterlogging tolerance-related genes, and promotes the process of genetic improvement of rapeseed waterlogging tolerance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 To identify the frequency distribution of the logarithmic value of the aboveground fresh weight waterlogging tolerance index of rapeseed populations;

[0021] Figure 2 Manhattan diagram showing the identification of chromosomal segments associated with waterlogging tolerance in rapeseed through genome-wide association analysis;

[0022] Figure 3 The results of linkage disequilibrium analysis for a peak SNP of approximately 0.01 Mb on the C05 chromosome of rapeseed.

[0023] Figure 4The results show the expression level of the BnaUGD1 gene during waterlogging stress and recovery. The vertical axis represents the transcription level (TPM value), and the horizontal axis represents the eight time points of waterlogging treatment (0 h, 2 h, 24 h, 72 h, 168 h), recovery (24 h, 72 h, R72), and recovery (168 h, R168). The four groups of samples are: TL (waterlogging-tolerant germplasm leaves), SL (sensitive germplasm leaves), TR (waterlogging-tolerant germplasm roots), and SR (sensitive germplasm roots).

[0024] Figure 5 Diagram showing the structure of the BnaUGD1 gene and polymorphisms at nonsynonymous mutation sites;

[0025] Figure 6 The results of the analysis of the aboveground fresh weight waterlogging tolerance index (WTI-SFW) of rapeseed based on different genotypes of the BnaUGD1 gene SNP locus (C05:13693515). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Example 1: Discovery of genes related to waterlogging tolerance in rapeseed

[0028] 1. Material selection and phenotypic identification

[0029] The 126 diverse germplasm resources of Brassica napus used in this invention were all provided by the National Oilseed Crop Germplasm Resource Mid-term Bank (Wuhan). Specific sample numbers (as shown in Table 1) and their corresponding information can be found in Appendix S1 of the literature “Hu, J., Chen, B., Zhao, J. etal. Genomic selection and genetic architecture of agronomic traits during modern rapeseed breeding. Nat Genet 54, 694–704 (2022). https: / / doi.org / 10.1038 / s41588-022-01055-6”. According to the literature, the original collection sites of the above germplasm resources included Shanghai, China; Canada; and Poland. Specific collection information can be found in Appendix S1 of the same literature.

[0030] Table 1 Rapeseed Germplasm List

[0031]

[0032] 126 rapeseed accessions were planted in the waterlogging-tolerant ponds of the transgenic nursery at the Wuhan Oilseed Research Institute in Hubei Province. The 126 accessions were randomly arranged and sown uniformly in 32-cell trays filled with nutrient soil. Each material was replicated in triplicate, with 16 seeds sown in each replicate. After thinning, two robust seedlings were retained per cell. Waterlogging treatment began when the rapeseed reached the two-leaf stage and lasted for two weeks, followed by a one-week recovery period.

[0033] Phenotypic identification used the aboveground fresh weight as the measurement index. The aboveground fresh weight of plants was measured after recovery from waterlogging treatment and under normal growth conditions, and the waterlogging tolerance index (WTI-SFW) was calculated as an evaluation index for waterlogging tolerance (referencing NY / T 3067-2016 Technical Specification for Waterlogging Tolerance Identification of Rapeseed; Construction of an Evaluation and Identification System for Extreme Waterlogging Tolerance Germplasm of Brassica napus throughout its Growth Period, Journal of Plant Genetic Resources, 2025, 26: 1982-1994). The formula for calculating the waterlogging tolerance index is as follows:

[0034] Waterlogging Tolerance Index (WTI-SFW) = Fresh weight of rapeseed on the ground after waterlogging recovery / Fresh weight of rapeseed on the ground under normal conditions

[0035] The above-mentioned methods were used to evaluate the differences in aboveground fresh weight phenotypes of different Brassica napus germplasm resources after recovery from waterlogging stress.

[0036] Data was transformed by taking the logarithm of the above-ground fresh weight waterlogging resistance index, and the data showed a normal distribution. Figure 1 The transformed values ​​were graded for water-resistance, as shown in Table 2. One strongly water-resistant germplasm, five water-resistant germplasms, and nine extremely sensitive germplasms were obtained through screening.

[0037] Table 2 Evaluation Criteria for Rapeseed Seedling Waterlogging Tolerance

[0038]

[0039] 2. Genotype data collection and analysis

[0040] Genotypic data for all germplasms were obtained by sequencing SNPs using a whole-genome sequencing platform (Illumina HiSeq 4000 platform, sequencing depth 17X).

[0041] Reference genome: The Brassica napus Darmor.v4.1 genome was used as the reference genome. The Brassica napus Darmor.v4.1 genome data can be accessed openly from the Brassica napus multi-omics information resource database BnIR (database access link: https: / / yanglab.hzau.edu.cn / BnIR). The access link for the Brassica napus Darmor.v4.1 genome is: https: / / yanglab.hzau.edu.cn / static / bnir / assets / / genomic_sequence / BnIRData / AACC.Brassica_napus / Darmor / v4.1 / Brassica_napus.Darmor.v4.1.genome.fa.gz.

[0042] 3. Genome-wide association analysis and gene mining

[0043] Genome-wide association analysis (GWIA) was performed on 126 WTI-SFW accessions using the efficient mixed-model software GEMMA v.0.94.1, combining logarithmic values ​​of the data with genotypic data. A mixed linear model (MLM) was employed: a P+K model (controlling PCA and Kinship). Manhattan plots were generated using the qqman package in R software to visualize the association results. The threshold for SNPs significantly associated with phenotypic data and resequencing data was set to −log10(p) = 4.0, and candidate regions were located within 100 kb before and after the significantly associated SNPs. Figure 2 As shown, a significantly associated signal was found in WTI-SFW on chromosome C05.

[0044] Chaining disequilibrium (LD) analysis was performed on the segment near peakSNP. Figure 3The gene BnaC05g20270D (BnaUGD1) was found in the LD region linked to peakSNP. Based on the annotation information of homologous genes in the Arabidopsis thaliana genome, it was found that this gene encodes UDP-glucosyltransferase in Arabidopsis thaliana.

[0045] The nucleotide sequence (CDS sequence, 2808 bp in length) of the BnaUGD1 gene is shown in SEQ ID NO:1.

[0046] Table 3 Sequence Information Table

[0047]

[0048] Example 2: Validation of BnaUGD1 gene expression level under waterlogging stress

[0049] To further verify the expression of the BnaUGD1 gene under waterlogging stress, leaves and roots of highly waterlogged-tolerant and extremely sensitive germplasm were collected during the waterlogging treatment (0 h, 2 h, 24 h, 72 h, and 168 h) and recovery (R24 h, R72 h, and R168 h) stages, respectively. These samples were designated TL (waterlogged-tolerant germplasm leaves), SL (sensitive germplasm leaves), TR (waterlogged-tolerant germplasm roots), and SR (sensitive germplasm roots). All samples were stored at -80℃ for subsequent transcriptome sequencing. The specific procedures are as follows:

[0050] Plants were irrigated to the two-leaf-one-heart stage, then subjected to 7 days of waterlogging treatment followed by 7 days of recovery. Leaf and root samples were collected at eight time points: 0 h, 2 h, 24 h, 72 h, 168 h of waterlogging treatment, and 24 h, 72 h, and 168 h of recovery. The collected samples were flash-frozen in liquid nitrogen and then stored at -80°C. All samples were sent to Beijing Berry Genomics Co., Ltd. for transcriptome sequencing.

[0051] Transcriptome sequencing results showed ( Figure 4 During the waterlogging treatment stage, the expression level of BnaUGD1 in waterlogged-tolerant germplasm leaves (TL) exhibited a fluctuating pattern of first increasing, then decreasing, and then increasing again; while in sensitive germplasm leaves (SL), it showed a trend of first decreasing, then increasing, and then decreasing again. During the recovery stage, the expression level of this gene in both germplasm leaves first increased and then decreased. In terms of root tissue, during the waterlogging stage, the expression levels of TR (waterlogged-tolerant roots) and SR (sensitive germplasm roots) both first decreased and then increased; during the recovery stage, the expression level of TR first increased and then decreased, while the expression level of SR first decreased and then increased.

[0052] This indicates that the BnaUGD1 gene exhibits stage-specific expression regulation under waterlogging stress, and its expression pattern is closely related to the waterlogging tolerance of rapeseed.

[0053] Example 3: Development of SNP molecular markers for the BnaUGD1 gene and intergroup comparison of different genotypes

[0054] To assess the potential function of candidate gene BnaUGD1 in waterlogging tolerance of Brassica napus, this application genotyped 126 materials based on peak SNPs obtained from previous association analysis and using the Brassica napus Darmor.v4.1 reference genome as a reference. Furthermore, the association between different genotypes and waterlogging tolerance phenotypes (characterized by the aboveground fresh weight waterlogging tolerance index WTI-SFW) was analyzed.

[0055] result( Figure 5 This indicates that an A / T base mutation was identified at position 13693515 on chromosome C05 within the coding region of the BnaUGD1 gene. Of the 95 samples successfully genotyped at this site, two genotypes were detected: 22 samples with genotype AT and 73 samples with genotype AA.

[0056] Statistical analysis was performed on WTI-SFW of different genotypes and box plots were drawn. Figure 6 The study found that the median WTI-SFW value of the AA genotype population was significantly higher than that of the AT genotype population (P < 0.05), indicating that the allelic variation at this SNP locus is significantly associated with the waterlogging tolerance of rapeseed, and that the AA genotype material has a stronger tolerance to waterlogging stress.

[0057] This invention provides a SNP molecular marker associated with waterlogging tolerance in Brassica napus. This marker is based on the Brassica napus Darmor-bzh v4.1 reference genome, located at locus 13693515 on chromosome C05 (BnaC05g20270D gene region), corresponding to the A / T base polymorphism site of the BnaUGD1 gene. Specifically, Brassica napus with the AA genotype exhibits significantly higher waterlogging tolerance than the AT genotype under waterlogging stress, and can be used as a molecular marker-assisted breeding tool for selecting waterlogging tolerance traits.

[0058] 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 within the protection scope of the present invention.

Claims

1. The application of reagents for detecting waterlogging-resistant SNP molecular markers in the identification of waterlogging resistance in Brassica napus, characterized in that, The SNP molecular marker is located at 13693515 bp on chromosome C05 of Brassica napus, and is an A / T polymorphic site. Rapeseed carrying the AA genotype has higher waterlogging tolerance than rapeseed carrying the AT genotype. The reference genome is Brassica napusDarmor.v4.

1.

2. The application as described in claim 1, characterized in that, The genotype of the rapeseed to be tested for the SNP molecular marker was detected by sequencing, PCR amplification or gene chip method.

3. A method for identifying the waterlogging tolerance of Brassica napus, characterized in that, The genotype of the SNP molecular marker was detected. The SNP molecular marker is located at 13693515 bp on chromosome C05 of Brassica napus. It is an A / T polymorphic site. The rapeseed carrying the AA genotype has higher waterlogging tolerance than the rapeseed carrying the AT genotype. The reference genome is Brassica napus Darmor.v4.

1.

4. The method as described in claim 3, characterized in that, The genotype of the rapeseed to be tested for the SNP molecular marker was detected by sequencing, PCR amplification or gene chip method.

5. A method for breeding waterlogging-tolerant rapeseed, characterized in that, The genotype of the SNP molecular marker was detected. The SNP molecular marker is located at 13693515 bp on chromosome C05 of Brassica napus and is an A / T polymorphic site. The genotype of the SNP molecular marker was detected, and samples with the genotype AA were selected for breeding. The reference genome was Brassica napusDarmor.v4.

1.

6. The method as described in claim 5, characterized in that, The genotype of the rapeseed to be tested for the SNP molecular marker was detected by sequencing, PCR amplification or gene chip method.

7. The method as described in claim 5, characterized in that, Using the sample with the genotype AA as the donor parent, hybridization and / or backcrossing are performed with the recipient parent. The offspring are then genotyped and screened using the SNP molecular marker until a homozygous line with the genotype AA at the SNP molecular marker locus is obtained.

8. The method as described in claim 5, characterized in that, The samples with the genotype AA were used for haploid breeding to obtain a double haploid population. The homozygous lines with the genotype AA at the SNP molecular marker locus were screened from the double haploid population using the SNP molecular marker.

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