GWAS and WGCNA analysis uncover candidate genes associated with drought in Brassica juncea L

Abstract

Drought poses a major challenge to crop growth and yield, and exploring the drought tolerance of crops is an effective and economical approach to mitigating the effects of drought. To screen drought-tolerant germplasm resources and key functional genes related to drought tolerance in Brassica juncea L.(193 accessions), three treatments were applied at the germination and seedling stages:control(CK), moderate drought stress (M), and severe drought stress (S). Drought tolerance identification, GWAS, and RNA-Seq analysis of these materials under different treatments showed that drought stress significantly reduced the germination rate, aboveground and underground fresh weight at the seedling stage, harvest index at maturity, and expanded the root/shoot ratio. From the 193 materials, 24 drought-tolerant, 139 drought-tolerant medium, and 30 drought-sensitive materials were identified. The 77 SNPs identified by GWAS were associated with the relative germination rate at the germination stage, and the fresh weight of the aboveground and underground parts at the seedling stage, which could be integrated into 27 QTLs. WGCNA identified 15, 0, and 5 modules significantly related to drought tolerance in the aboveground and underground parts at the germination and seedling stages, respectively. By correlating the significant GWAS SNPs with the significant WGCNA modules, a total of 11 genes related to drought tolerance under moderate and severe drought stress were identified. These genes were involved in the regulation of auxin-responsive protein (SAUR), LEA protein, glucosidase, AP2/ERF, WRKY and GATA transcription factors, FLZ zinc finger domain, PRP, and b561 proteins. Among them, the BjuB035910 gene was detected in the underground parts of the seedling and germination stages under moderate drought stress. GWAS and selective sweep analysis jointly identified the 23.955-24.089 Mb region of chromosome B06, where four genes (BjuB022264, BjuB022292, BjuB022282, and BjuB022235) were located, as confirmed by WGCNA analysis. A total of 125 SNPs with high linkage disequilibrium were found in this region, and 12 haplotypes were detected, with Hap1 being present exclusively in drought-tolerant materials and Hap3-Hap12 distributed in drought-sensitive materials. These findings provide new insights into the drought tolerance mechanisms of B. juncea and will contribute to the breeding of drought-tolerant rapeseed varieties.

Keywords: Brassica juncea L.; GWAS; WGCNA; drought; genes; selective sweep analysis.

Figure 1

Phenotypic changes under different drought stress treatments. (a-e) are the relative values of each trait under moderate stress M (Moderate) and severe stress S (Severe). (f) is the cluster analysis diagram of the tested varieties based on the membership function calculated according to the relative values of traits at germination stage and seedling stage.

Figure 2

Genome-wide association analysis of Manhattan plot and QQ plot. (a-c) are the Manhattan plots of the relative germination rate, the aboveground and underground fresh weight at the seedling stage between 3 years (2019, 2020 and 2021) under moderate drought stress. (d-f) are the QQ plots of relative germination rate, aboveground and underground fresh weight at seedling stage in 3 years under moderate drought stress.

Figure 3

Differentially expressed genes between drought-tolerant and sensitive groups at germination and seedling stages. (A) is the germination stage; (B) is the underground part of the seedling stage; (C) is the aboveground part of the seedling stage. DM, drought-tolerant group under moderate drought stress; SM, sensitive group under moderate drought stress; DS, drought-tolerant group under severe drought stress; SS, Sensitive group under severe drought stress. The names of the genes marked in the figure are co-upregulated genes.

Figure 4

Weighted gene co-expression network analysis. (A) Dynamic cutting clustering diagram at germination stage; (B) Dynamic cutting clustering diagram of underground at seedling stage; (C) Dynamic cutting clustering diagram of aboveground at seedling stage; (D) Correlation between modules and treatments at germination stage; (E) Correlation between underground modules and treatments at seedling stage; (F) Correlation between aboveground modules and treatments at seedling stage. *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001.

Figure 5

Functional analysis of drought tolerance-related genes and association of SNP alleles with phenotype. (A) KEGG enrichment analysis of 8 candidate genes; (B) GO annotation analysis of 8 candidate genes; (C) Gene co-expression network of Megrey60, MeDarkslateblue, Meorangered4 and MEmediumpurple3 modules at germination stage; (D) Gene co-expression network of Medarkturquoise and Meblue modules at seedling stage; (E) Expression heat map of drought tolerance-related genes, G(S) indicates severe drought stress at the germination stage,G(M) indicates moderate drought stress at the germination stage,G(CK) indicates control at the germination stage,R(S) indicates severe drought stress at the germination stage, R(S) indicates roots at seedling stage under severe drought stress, R(M) indicates roots at seedling stage under moderate drought stress, R(CK) indicates roots at seedling stage under control, L(S) indicates leafs at seedling stage under severe drought stress, L(M) indicates leafs at seedling stage under moderate drought stress, L(CK) indicates leafs at seedling stage under control. (F-K) Association analysis of SNP alleles with phenotypes, t6 indicates germination rate, t9 indicates aboveground fresh weight, t10 indicates underground fresh weight, REF, reference; Missing, missing alleles. NS: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001.

Figure 6

The SNPs and haplotypes responsible for relative germination rate under drought stress during germination period in the region from 23.955-24.089Mb on B06 chromosome. (a) Local Manhattan plot showing the surrounding the candidate region. The position marked by the blue arrow was the significantly correlated SNP screened out. (b) LD heat map of the region of the candidate region. (c) The haplotype of the chromosome B06 region between 23.955 Mb and 24.089Mb. BjuB022275, BjuB022288, BjuB022289, BjuB022291, BjuB022292, and BjuB022293 are 6 genes that contain 33 SNPs. 1/1, 0/1, and 0/0 are allele types. 0/0 indicates that it is identical to the reference genome, 0/1 indicates partial divergence from the reference genome, and 1/1 indicates complete divergence from the reference genome. Hap1-Hap12 are 12 haplotypes.