Rapeseed submergence tolerance related gene BnaGASA13 and application thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a rapeseed salt tolerance related gene BnaGASA13 and application thereof.The present application provides a 3-base mutation of the BnaGASA13 gene related to the salt tolerance of rapeseed and an excellent haplotype thereof, and provides precise gene resources and haplotype germplasm for screening and breeding of salt-tolerant rapeseed varieties.The rapeseed salt tolerance identification method provided by the present application is simple and feasible, has high selection efficiency, and has important application prospects in the field of rapeseed salt tolerance improvement breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a rapeseed submergence tolerance related gene BnaGASA13 and application thereof. BACKGROUND

[0002] Brassica napus L. as a global important oil crop, its high yield and stable yield is of great significance to ensure the safety of edible oil supply. However, in its main planting area, soil submergence has become one of the core ecological stress factors restricting yield improvement. Submergence leads to soil pores filled with water, causing severe oxygen deficiency in the rhizosphere, forcing the root system to perform anaerobic respiration, not only the energy output efficiency is low, but also harmful metabolites such as ethanol are accumulated, causing cell metabolic disorder. At the same time, the root activity decreases, the ability to absorb water and nutrients is hindered, the synthesis of chlorophyll in leaves is inhibited, and the photosynthetic rate is significantly reduced, ultimately leading to the inhibition of plant growth and development, reduction of biomass accumulation, and significant reduction of yield and quality. This physiological damage caused by submergence and waterlogging occurs throughout the growth stages of rapeseed from seedling to silique maturation, which poses a continuous threat to the stability of production.

[0003] At present, the improvement of rapeseed submergence tolerance still relies on traditional breeding methods, mainly through phenotypic observation for screening. However, rapeseed submergence tolerance is a complex quantitative trait controlled by multiple genes, showing continuous variation and being easily affected by environmental factors, which makes the accuracy of phenotypic selection limited. Therefore, traditional breeding methods have the significant limitations of low selection efficiency, long breeding cycle and slow genetic gain. In addition, the degree and duration of soil submergence vary greatly from year to year, further increasing the difficulty and environmental sensitivity of phenotypic selection. These factors jointly restrict the breeding efficiency of submergence tolerant rapeseed varieties, which is difficult to meet the urgent demand for high yield and stable yield varieties in production. SUMMARY

[0004] Therefore, the present application provides a rapeseed submergence tolerance related gene BnaGASA13 and application thereof.

[0005] The technical scheme of the present application is realized as follows:

[0006] In a first aspect, the application provides an application of BnaGASA13 gene in identification of Brassica napus salt tolerance, wherein the BnaGASA13 gene contains three mutation sites, i.e. G / A base mutation at 21606957 bp of Brassica napus A01 chromosome, T / G base mutation at 21607330 bp, and A / T base mutation at 21607536 bp, based on Brassica napus Darmor.v4.1 genome as a reference genome; and a haplotype composed of genotypes GA, TG and AT of the three mutation sites is identified as an excellent haplotype significantly related to Brassica napus salt tolerance.

[0007] Further, in some embodiments, the application further comprises a step of detecting the genotype of the three mutation sites of the to-be-tested Brassica napus.

[0008] When the genotype of the three mutation sites is GA, TG and AT, the Brassica napus has high salt tolerance.

[0009] When the genotype of the three mutation sites is any one of the following, the Brassica napus has low salt tolerance:

[0010] (1) GG, TG and AT;

[0011] (2) GG, TG and AA;

[0012] (3) GG, TT and AA;

[0013] Wherein, the high / low salt tolerance of the Brassica napus is a relative concept, which can be determined by comparison.

[0014] Further, the genotype of the three mutation sites of the to-be-tested Brassica napus is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

[0015] In a second aspect, the application provides an application of BnaGASA13 gene in breeding of salt-tolerant Brassica napus, wherein the BnaGASA13 gene contains three mutation sites at 21606957 bp, 21607330 bp and 21607536 bp of Brassica napus A01 chromosome based on Brassica napus Darmor.v4.1 genome as a reference genome, the genotype of the three mutation sites is detected, and samples with genotypes of GA, TG and AT are screened as excellent haplotype of salt tolerance for breeding.

[0016] In a third aspect, the present application provides a method for identifying the tolerance to waterlogging of Brassica napus, which uses the Brassica napus Darmor.v4.1 genome as a reference genome, and detects the genotypes of three mutation sites at 21606957 bp, 21607330 bp and 21607536 bp on the A01 chromosome of Brassica napus.

[0017] When the genotypes of the three mutation sites are GA, TG and AT, the Brassica napus has high tolerance to waterlogging.

[0018] When the genotypes of the three mutation sites are any of the following, the Brassica napus has low tolerance to waterlogging:

[0019] (1) GG, TG and AT;

[0020] (2) GG, TG and AA;

[0021] (3) GG, TT and AA.

[0022] In a fourth aspect, the present application further provides a method for breeding Brassica napus with tolerance to waterlogging, which uses the Brassica napus Darmor.v4.1 genome as a reference genome, detects the genotypes of three mutation sites at 21606957 bp, 21607330 bp and 21607536 bp on the A01 chromosome of Brassica napus, and screens samples with genotypes of GA, TG and AT as excellent haplotypes of tolerance to waterlogging for breeding.

[0023] The present application has at least the following beneficial effects:

[0024] (1) The present application provides three base mutations of the BnaGASA13 gene related to the tolerance to waterlogging of Brassica napus and an excellent haplotype thereof, which provides precise genetic resources and haplotype germplasm for screening and breeding of Brassica napus with tolerance to waterlogging, and the method for identifying the tolerance to waterlogging of Brassica napus provided by the present application is simple and feasible, has high selection efficiency, and has important application prospects in the field of breeding of Brassica napus with improved tolerance to waterlogging.

[0025] (2) The present application precisely locates the SNP site significantly related to the tolerance to waterlogging of Brassica napus through whole-genome association analysis, identifies the candidate gene BnaGASA13 in the segment near the peak SNP through linkage disequilibrium (LD) analysis, further finds three key base mutations in the CDS sequence through haplotype analysis, and successfully identifies an excellent haplotype of tolerance to waterlogging. The present application provides important theoretical and technical support for the mining and application of other waterlogging tolerance-related genes, and promotes the process of genetic improvement of the tolerance to waterlogging of Brassica napus.

[0026] The invention relates to the definition of the terms

[0027] A haplotype, or haploid genotype, refers to the combination of alleles at multiple loci on the same chromosome that are inherited together. It also refers to a single nucleotide polymorphism (SNP) that is statistically associated within a single chromatid.

[0028] Haplotype analysis refers to the methods of using genetic markers to perform linkage analysis on family data, sequencing a specific chromatid, and using mathematical models and computational software to estimate the frequencies of various haplotypes in a population.

[0029] Waterlogging-tolerant germplasm refers to plant genetic resources that can maintain normal growth and development, yield formation, and physiological metabolic stability under certain waterlogging or soil oversaturation stress conditions. Sensitive germplasm (also known as waterlogging-sensitive germplasm) refers to genetic material that exhibits significant growth inhibition, metabolic disorders, or even death under the same waterlogging stress.

[0030] In this invention, the high / low waterlogging resistance of rapeseed is a relative concept and can be determined by comparison. Attached Figure Description

[0031] 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.

[0032] Figure 1 This invention provides a frequency distribution diagram of the logarithmic values ​​of the aboveground fresh weight waterlogging tolerance index of rapeseed populations.

[0033] Figure 2 Manhattan diagram showing the chromosomal segments associated with waterlogging tolerance in rapeseed identified by genome-wide association analysis in this invention;

[0034] Figure 3 This is the result of linkage disequilibrium analysis within the segment of the rapeseed A01 chromosome with a peak SNP of approximately 0.01 Mb.

[0035] Figure 4 The results show the expression level of the BnaGASA13 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, 24 h recovery (R24), 72 h recovery (R72), and 168 h recovery (R168). The four groups of samples are: waterlogging-tolerant germplasm leaves (TL), sensitive germplasm leaves (SL), waterlogging-tolerant germplasm roots (TR), and sensitive germplasm roots (SR).

[0036] Figure 5 Figure 1 is a BnaGASA13 gene structure and non-synonymous mutation site polymorphism map of the present application;

[0037] Figure 6 Figure 4 is an analysis result of the aboveground fresh weight and waterlogging tolerance index (WTI-SFW) of five haplotypes of BnaGASA13 gene of the present application. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be purchased in the market.

[0039] Table 1 Sequence information table

[0040]

[0041] Example 1 Mining of genes related to waterlogging tolerance of rapeseed

[0042] 1. Material selection and phenotype identification

[0043] The 126 rapeseed diversity germplasm resources used in the present application were provided by the National Oil Crop Germplasm Resource Center (Wuhan). The specific sample numbers (as shown in Table 2) correspond to the information in the literature “Hu, J., Chen, B., Zhao, J. et al. 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” Appendix S1. According to the literature, the original collection sites of the above-mentioned germplasm resources include Shanghai, Canada, Poland and other places in China. The specific collection information is detailed in the literature Appendix S1.

[0044] Table 2 List of rapeseed germplasm

[0045]

[0046] 126 accessions of Brassica napus were planted in the tolerance pool of the Wuhan Oilseed Field of Hubei Province. The 126 accessions of germplasm materials were completely randomized, and were uniformly sown in 32-hole plug trays filled with nutrient soil. Each material was set up 3 replicates, and 16 seeds were sown in each replicate. After thinning, 2 healthy seedlings were retained in each hole. When the oilseed rape grew to the two-leaf-one-heart stage, the waterlogging treatment was started, and lasted for 2 weeks, and then recovered for 1 week.

[0047] The phenotypic identification was measured by the fresh weight of the aboveground part. The fresh weight of the aboveground part of the plants after the waterlogging treatment recovery and under normal growth conditions was measured, and the waterlogging tolerance index (WTI-SFW) was calculated as the evaluation index of waterlogging tolerance (reference NY / T 3067-2016 Oilseed Rape Waterlogging Tolerance Identification Technical Specification; Construction of Brassica napus Extreme Waterlogging Tolerance Germplasm Evaluation and Identification System, Journal of Plant Genetic Resources, 2025, 26:1982-1994). The calculation formula of the waterlogging tolerance index is as follows:

[0048] Waterlogging tolerance index (WTI-SFW) = fresh weight of aboveground part of oilseed rape after waterlogging recovery / fresh weight of aboveground part of oilseed rape under normal conditions

[0049] The differences in the aboveground fresh weight phenotype of different Brassica napus germplasm resources after waterlogging stress recovery were evaluated by the above method.

[0050] The data were transformed by taking the logarithm of the aboveground fresh weight waterlogging tolerance index, and the data were normally distributed (P<0.05) Figure 1 ), and the transformed values were classified according to waterlogging tolerance, as shown in Table 3, 1 strong waterlogging tolerance accession, 5 waterlogging tolerance accessions, and 9 extremely sensitive accessions were obtained.

[0051] Table 3 Evaluation criteria for oilseed rape seedling waterlogging tolerance

[0052]

[0053] 2. Genotype data collection and analysis

[0054] The genotype data of all accessions were obtained by whole genome sequencing platform (Illumina HiSeq 4000 platform, sequencing depth of 17X), and SNP genotype data.

[0055] 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.

[0056] 3. Genome-wide association analysis and gene mining

[0057] 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 for WTI-SFW on chromosome A01.

[0058] Chaining disequilibrium (LD) analysis was performed on the segment near the peak SNP. Figure 3 The gene BnaA01g31950D (BnaGASA13) was found in the LD region linked to the peak SNP. Based on the annotation information of homologous genes in the Arabidopsis thaliana genome, this gene was found to encode gibberellin regulatory protein 6 in Arabidopsis thaliana.

[0059] The nucleotide sequence (CDS sequence, 336 bp in length) of the BnaGASA13 gene is shown in SEQ ID NO:1.

[0060] Example 2: Validation of BnaGASA13 gene expression level under waterlogging stress

[0061] To further validate the expression of the BnaGASA13 gene under waterlogging stress, leaves and roots were collected from highly waterlogged-tolerant and extremely sensitive germplasm at 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 were designated as waterlogged-tolerant germplasm leaves (TL), sensitive germplasm leaves (SL), waterlogged-tolerant germplasm roots (TR), and sensitive germplasm roots (SR). All samples were stored at -80℃ for subsequent transcriptome sequencing. The specific procedures are as follows:

[0062] Plants were grown 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 from both waterlogging-tolerant and waterlogging-sensitive germplasm 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.

[0063] Transcriptome sequencing results ( Figure 4 The results showed that the expression of the BnaGASA13 gene was tissue-specific, detected only in leaves. During the waterlogging stress stage, the expression level of this gene in leaves of both germplasms showed a trend of first decreasing and then increasing. During the recovery stage, the expression level of this gene in leaves of the waterlogging-tolerant germplasm (TL) exhibited a fluctuating pattern of first decreasing, then increasing, and then decreasing again; while in leaves of the sensitive germplasm (SL), its expression level showed a change of first decreasing and then increasing. Therefore, it can be concluded that there is a significant difference in the expression of the BnaGASA13 gene between the two germplasms under waterlogging stress, indicating that the BnaGASA13 gene is a leaf-specific waterlogging stress response gene, and its expression pattern is closely related to the waterlogging tolerance of rapeseed.

[0064] Example 3 Haplotype analysis of BnaGASA13

[0065] To further analyze whether the candidate gene BnaGASA13 is related to stain resistance, this embodiment used the genotypes of the aforementioned associated peak SNPs to genotype 126 materials, and then analyzed the phenotypic changes among different genotypes. The results are as follows: Figure 5 and Figure 6The CDS sequence of BnaGASA13 gene has 3 base mutations, and 91 materials successfully completed genotyping, according to the mutation type, as shown in Table 4, are divided into 5 haplotypes Hap1, Hap2, Hap3, Hap4 and Hap5. The WTI-SFW of Hap2 is significantly higher than that of other haplotypes, that is, the degree of decrease in aboveground fresh weight of Hap2 individuals after waterlogging treatment is significantly lower than that of other haplotypes, indicating that Hap2 is an excellent haplotype significantly related to the waterlogging tolerance of rapeseed relative to other haplotypes.

[0066] Table 4 Haplotype of BnaGASA13

[0067]

[0068] Note: 21606957, 21607330 and 21607536 represent the base position of rapeseed A01 chromosome. (Reference genome: Brassica napus Darmor.v4.1)

[0069] The results show that the CDS of gene BnaGASA13 has 3 base mutations, which are G / A base mutation at 21606957 bp of rapeseed A01 chromosome, T / G base mutation at 21607330 bp, and A / T base mutation at 21607536 bp, and the genotype of the three base mutation sites is GA, TG and AT, and the haplotype Hap2 is an excellent haplotype significantly related to the waterlogging tolerance of rapeseed.

[0070] The results show that the genotype based on the three mutation sites 21606957 bp, 21607330 bp and 21607536 bp of rapeseed A01 chromosome can accurately distinguish the waterlogging tolerance phenotype of rapeseed, and when the genotype of the three mutation sites is GA, TG and AT, it is identified as a waterlogging-tolerant material.

[0071] In summary, by detecting the genotype of the three sites 21606957 bp, 21607330 bp and 21607536 bp of rapeseed A01 chromosome, the rapid and accurate identification of waterlogging tolerance of rapeseed germplasm resources can be realized, and waterlogging-tolerant samples can be selected, and then rapid breeding of waterlogging-tolerant rapeseed can be realized.

[0072] The present application combines waterlogging tolerance phenotype analysis, whole genome association analysis, LD analysis, haplotype analysis and other diversified comprehensive means, not only discovers BnaGASA13 as a waterlogging-related key gene, but also provides a referenceable technical path for the excavation of other crop stress-resistant genes and haplotype breeding.

[0073] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. BnaGASA13 The use of genes in the identification of tolerance to stress in Brassica napus, characterized in that, In Brassica napus Darmor.v4.1 genome as the reference genome, the BnaGASA13 Three mutation sites were contained in the gene, which were G / A base mutation at 21606957 of A01 chromosome of Brassica napus, T / G base mutation at 21607330, and A / T base mutation at 21607536. A haplotype composed of genotypes GA, TG and AT of the three mutation sites was identified as an excellent haplotype significantly related to the salt tolerance of Brassica napus.

2. Use according to claim 1, characterized in that, detecting the genotype of the test rape at the three mutation sites; When the genotype of the three mutation sites is GA, TG and AT, the rape has high salt tolerance; When the genotype of the three mutation sites is any of the following, the rape has low salt tolerance: (1) GG, TG and AT; (2) GG, TG and AA; (3) GG, TT and AA.

3. Use according to claim 2, characterized in that, The genotype of the test rape at the three mutation sites is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

4. BnaGASA13 The use of genes in breeding for stress tolerance in Brassica napus, characterized in that, In Brassica napus Darmor.v4.1 genome as the reference genome, the BnaGASA13 The gene has mutations at the bases of 21606957, 21607330 and 21607536 of the A01 chromosome of Brassica napus, the genotypes of the three mutation sites are detected, and the samples with genotypes of GA, TG and AT are screened as excellent haplotypes of salt tolerance for breeding.

5. Use according to claim 4, characterized in that, The genotype of the test rape at the three mutation sites is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

6. A method for identifying a flooding tolerance in Brassica napus, characterized in that, With Brassica napus Darmor.v4.1 genome as the reference genome, the genotypes of the three mutation sites at positions 21606957, 21607330 and 21607536 on the A01 chromosome of Brassica napus were detected; When the genotype of the three mutation sites is GA, TG and AT, the rape has high salt tolerance; When the genotype of the three mutation sites is any of the following, the rape has low salt tolerance: (1) GG, TG and AT; (2) GG, TG and AA; (3) GG, TT and AA.

7. The method of claim 6, wherein, The genotype of the test rape at the three mutation sites is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

8. A method for breeding a waterlogging tolerant Brassica napus, characterized in that, With Brassica napus Darmor.v4.1 genome as the reference genome, the genotypes of the three mutation sites at positions 21606957, 21607330 and 21607536 on the A01 chromosome of Brassica napus were detected, and samples with genotypes of GA, TG and AT were screened as excellent haplotypes for salt tolerance for breeding.

9. The method of claim 8, wherein, The genotype of the test rape at the three mutation sites is detected by sequencing, PCR amplification, specific probe hybridization, specific primer extension or gene chip method.

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