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Identification of genetic variation for salt tolerance in Brassica napus using genome-wide association mapping

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Abstract

Soil salinity negatively impacts rapeseed (Brassica napus) crop production. In particular, high soil salinity is known to hinder seedling growth and establishment. Identifying natural genetic variation for high salt tolerance in Brassica napus seedlings is an effective way to breed for improved productivity under salt stress. To identify genetic variants involved in differential response to salt stress, we evaluated a diverse association panel of 228 Brasica napus accessions for four seedling traits under salt stress to establish stress susceptibility index (SSI) and stress tolerance index (STI) values, and performed genome-wide association studies (GWAS) using 201,817 high-quality single nucleotide polymorphic (SNP) markers. Our GWAS identified 142 significant SNP markers strongly associated with salt tolerance distributed across all rapeseed chromosomes, with 78 SNPs in the C genome and 64 SNPs in the A genome, and our analyses subsequently pinpointed both favorable alleles and elite cultivars. We identified 117 possible candidate genes associated with these SNPs: 95/117 were orthologous with Arabidopsis thaliana genes encoding transcription factors, aquaporins, and binding proteins. The expression level of ten candidate genes was validated by quantitative real-time PCR (qRT-PCR), and these genes were found to be differentially expressed between salt-tolerant and salt-susceptible lines under salt stress conditions. Our results provide new genetic resources and information for improving salt tolerance in rapeseed genotypes at the seed germination and seedling stages via genomic or marker-assisted selection, and for future functional characterization of putative gene candidates.

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Abbreviations

GWAS:

Genome-wide association studies

SLAF- seq:

Specific length amplified fragment sequencing

SNPs:

Single nucleotide polymorphisms

GLM:

General linear model

MLM:

Mixed linear model

SSI:

Stress Susceptible Index

STI:

Stress Tolerance Index

GR:

Germination

RL:

Root length

SDW:

Shoot dry weight

SVI:

Seed Vigor Index

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Acknowledgments

This research was funded by the National Natural Science Foundation of China, grant number 31860417.

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Author notes

  1. Ghulam Mustafa Wassan and Hira Khanzada contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Crop Physiology, Ecology, and Genetic Breeding, Ministry of Education, Jiangxi Agricultural University, Nanchang, 330045, China

    Ghulam Mustafa Wassan, Hira Khanzada, Qinghong Zhou, Saba Khanzada, Abdul Malik Solangi, Donghui Fu & Haohua He

  2. College of Agronomy, Jiangxi Agricultural University, Nanchang, 330045, China

    Ghulam Mustafa Wassan, Hira Khanzada, Qinghong Zhou, Saba Khanzada, Abdul Malik Solangi, Donghui Fu & Haohua He

  3. Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany

    Annaliese S. Mason

  4. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Ayaz Ali Keerio

  5. Faculty of Crop Production, Sindh Agriculture University Tando Jam, Sindh, Pakistan

    Ayaz Ali Keerio & Muhammad Faheem

  6. College of Land Resources and Environment, Jiangxi Agricultural University, Nanchang, 330045, China

    Saba Khanzada & Muhammad Faheem

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  1. Ghulam Mustafa Wassan
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Contributions

GMW and HK performed the experiments, analyses the data, and wrote the paper. QZ provides seed material. AAK, SK, AMS, and MF provide technical support in data analysis, critical reading, and suggestions regarding the manuscript. DF and HH conceived the project and supervised this study. All of the authors discussed the results and commented on the manuscript. ASM, revised the whole manuscript for the language.

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Correspondence to Donghui Fu or Haohua He.

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Supplementary Information

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438_2020_1749_MOESM1_ESM.tif

Supplementary Fig. S1 Quantile-quantile plot for the GWAS using a generalized linear model (GLM) and mixed linear model (MLM) based on salt stress susceptibility and salt stress tolerance indices in four seedling traits in Brassica napus. (A) Germination % GLM and MLM by stress susceptibility index (SSI) (B) Germination % GLM and MLM by stress tolerance index (STI) (C) root length of GLM and MLM by SSI (D) root length of GLM and MLM by STI (E) shoot dry weight of GLM and MLM by SSI (F) shoot dry weight of GLM and MLM by STI (G) seed vigor index of GLM and MLM by SSI and (H) seed vigor index of GLM and MLM by STI (TIF 16627 KB)

438_2020_1749_MOESM2_ESM.tif

Supplementary Fig. S2 Manhattan plots of genome-wide association studies (GWAS) using a generalized linear model (GLM) for four seedling traits related to salt stress of Brassica napus based on stress susceptibility index (SSI) and stress tolerance index (STI). The log 10 (P-value) is a measure of the significance with which a SNP is associated with a trait. The blue horizontal dotted lines represent the genome wide significance threshold of p < 0.05 after FDR correction. Each chromosome is represented by a different color. (A) germination % using SSI (B) germination % using STI (C) root length in SSI (D) root length in STI (E) shoot dry weight in SSI (F) shoot dry weight in STI (G) seed vigor index in SSI and (H) seed vigor index in STI (TIF 16077 KB)

438_2020_1749_MOESM3_ESM.tif

Supplementary Fig. S3 Manhattan plots of genome-wide association studies (GWAS) using a mixed linear model (MLM) for four seedling traits related to salt stress of Brassica napus based on stress susceptibility index (SSI) and stress tolerance index (STI). The log 10 (P-value) is a measure of the degree to which a SNP is significantly associated with a trait. The blue horizontal dotted lines represent the genome-wide significance threshold. Each chromosome is represented by a different color. (A) germination % using stress susceptibility index (SSI) (B) germination % using stress tolerance index (STI) (C) root length based on SSI (D) root length based on STI (E) shoot dry weight based on SSI (F) shoot dry weight based on STI (G) seed vigor index based on SSI (H) seed vigor index based on STI (TIF 16080 KB)

438_2020_1749_MOESM4_ESM.xlsx

Supplementary Table S1 Detailed information about the 228 lines along with their subgroup of Brassica napus (XLSX 21 KB)

438_2020_1749_MOESM5_ESM.xlsx

Supplementary Table S2 Phenotypic variations among the lines in pilot experiment under various salt treatments (XLSX 11 KB)

438_2020_1749_MOESM6_ESM.docx

Supplementary Table S3 Correlation coefficients (r) between germination and seedling traits under control/non-stress conditions (CK) above the diagonal in blue, while under salt stress below diagonal with pink color in rapeseed natural population (DOCX 14 KB)

Supplementary Table S4 SNPs associated with germination by GLM and MLM in Brassica napus (DOC 81 KB)

Supplementary Table S5 SNPs associated with SDW by GLM and MLM in Brassica napus (DOC 63 KB)

Supplementary Table S6 SNPs associated with RL by GLM and MLM in Brassica napus (DOC 103 KB)

Supplementary Table S7 SNPs associated with SVI by GLM and MLM in Brassica napus (DOC 37 KB)

Supplementary Table S8 SNPs associated with RL and SVI by GLM and MLM in Brassica napus (DOCX 19 KB)

Supplementary Table S9 Estimation of favorable and unfavorable alleles detected by GLM-SSI analysis (XLSX 47 KB)

Supplementary Table S10 Identification of hot spot regions on the chromosomes (DOCX 18 KB)

Supplementary Table S11 Genes identified by GO analysis associated with salt in Brassica napus (XLSX 118 KB)

438_2020_1749_MOESM15_ESM.xlsx

Supplementary Table S12 Candidate genes highly associated with seedling traits of salt tolerance in Brassica napus (XLSX 51 KB)

438_2020_1749_MOESM16_ESM.xlsx

Supplementary Table S13 Salt related genes tagged by the associated SNPs in Brassica napus and homologous with Arabidopsis thaliana genes (XLSX 21 KB)

Supplementary Table S14 List of primers for quantitative real-time-PCR (qRT-PCR) (DOCX 14 KB)

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Wassan, G.M., Khanzada, H., Zhou, Q. et al. Identification of genetic variation for salt tolerance in Brassica napus using genome-wide association mapping. Mol Genet Genomics 296, 391–408 (2021). https://doi.org/10.1007/s00438-020-01749-8

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