Genome-wide identification and functional analysis of cupin_1 domain-containing members involved in the responses to Sclerotinia sclerotiorum and abiotic stress in Brassica napus

Abstract

Cupin_1 domain-containing proteins (CDPs) are ubiquitously present in higher plants, which are known to play essential roles in various biological processes. In this study, we carried out genome-wide characterization and systematic investigation of the CDP genes in Brassica napus. A total of 96 BnCDPs, including 71 germin-like proteins (GLPs; proteins with a single cupin_1 domain) and 25 CDP bicupins (proteins with two cupin_1 domains), were identified and clustered into six distinct subfamilies (I-VI) based on the phylogenic analysis, gene structure and motif distribution. Further analysis indicated that whole-genome duplication (WGD) and segmental duplication are main contributors to the species-specific expansion of the BnCDP gene family, and all the duplicated genes subsequently underwent strong purification selection. The promoter region of BnCDPs showed enrichment of cis-regulatory elements associated with development, hormone and stress, as well as transcription factor binding sites, which validates the prediction that BnCDPs are widely involved in plant growth and biotic and abiotic stress responses. The BnCDPs in different subfamilies exhibited obvious differences in expression among 30 developmental tissues/stages of B. napus, implying that BnCDPs may be involved in tissue- and stage-specific developmental processes. Similar trends in expression of most BnCDPs were observed under Sclerotinia sclerotiorum inoculation and four abiotic stresses (dehydration, cold, ABA and salinity), particularly the BnGLPs in subfamily I and III with single cupin_1 domain, revealing that BnCDPs are of great importance in the environmental adaption of B. napus. We then performed a genome-wide association study (GWAS) of 274 B. napus core germplasms on S. sclerotiorum resistance and identified four significantly associated loci harboring five BnGLPs. The expression levels of two candidate genes, BnGLP1.A08 and BnGLP1.C08, were significantly correlated with S. sclerotiorum resistance. Their functional responses to multiple stages of S. sclerotiorum inoculation and four abiotic stresses were further examined through qPCR. Overall, this study provides rich resources for research on the function and evolutionary playground of CDP genes.

Keywords: Brassica napus; GWAS; Sclerotinia sclerotiorum resistance; abiotic stress; cupin_1 domain; germin-like protein.

FIGURE 1

A neighbor-joining (NJ) phylogenetic tree of CDP proteins in Arabidopsis thaliana (At) and Brassica napus (Bn). All CDP proteins were clustered into six subfamilies, and each subfamily was represented by a different color. Light purple dot on a branch in the figure indicates that the bootstrap support is greater than 60%. Circles refer to BnCDPs, while filled stars refer to AtGLPs identified previously and empty stars refer to AtCDPs identified in this research; Blue and Red refer to CDPs with single or duplicated cupin_1 domain, respectively.

FIGURE 2

Duplication analysis of BnCDP genes in Brassica napus. The gene names are marked outward of the corresponding chromosomes. The different colors indicate different subfamilies of the BnCDP genes. The duplicated gene pairs are highlighted with connecting lines colored according to the subgenomes, purple indicates that both genes in the gene pair are from the A_n subgenome, yellow represents that both genes in the gene pair are from the C_n subgenome, while the blue shows that two genes in the gene pair come from different subgenomes.

FIGURE 3

The phylogenetic relationship, exon-intron architecture, and conserved motifs of 96 BnCDP in Brassica napus. (A) The phylogenetic relationships of BnCDP proteins based on the NJ method. (B) Gene structures of BnCDP genes. Yellow boxes represent the untranslated region (UTR), green boxes represent exons and the gray lines represent introns. (C) The conserved motif composition of BnCDP proteins. Scale bars represent gene length (bp) and protein sequence length (aa).

FIGURE 4

Cis-acting regulatory elements identified in promoters of BnCDP genes in Brassica napus. Boxes indicate development-related elements, down-wedges indicate hormone-related elements, and up-wedges indicate stress-related elements. Different colors indicate different elements.

FIGURE 5

Expression patterns of 96 BnCDP genes in 30 tissues of Brassica napus ZS11 variety. The expression data were processed with the log₁₀ normalization of fragments per kilobase million (FPKM). The color scale represents relative expression levels from low (blue color) to high (red color).

FIGURE 6

Expression profiles of 96 BnCDP genes under biotic (left) and abiotic (right) stress conditions. The left panel shows the expression level of BnCDP genes in ZY821 at 0 and 24 h after Sclerotinia sclerotiorum inoculation; The left panel shows the expression level of BnCDP genes under different abiotic stress conditions (dehydration, cold, ABA and salinity). The expression data were processed with the log₁₀ normalization of fragments per kilobase million (FPKM). The color scale represents relative expression levels from low (blue color) to high (red color).

FIGURE 7

Both BnGLP1.A08 and BnGLP1.C08 are associated with Sclerotinia sclerotiorum resistance in Brassica napus. (A) Frequency distribution of disease index of 274 B. napus accessions. (B) Manhattan plot of the disease index from association analyses by FarmCPU model. Each point represents a SNP, and the SNP that exceeds the threshold (red dotted line) –log₁₀ (1/n) = 6.377 is significant. (C) QQ plot for the disease index from association analyses. (D,E) Boxplots for disease index based on the two haplotypes of BnaGLP1.A08 (D) and BnaGLP1.C08 (E). The phenotypic differences between groups were tested using a two-tailed t-test (P < 0.01).

FIGURE 8

The expression validation of candidate BnGLP1.A08 and BnGLP.C08 genes in response to Sclerotinia sclerotiorum infection and four abiotic stress treatments (dehydration, cold, ABA and salinity) by qRT-PCR. The time points under the x-axis represent hours (h) after corresponding biotic and abiotic treatments. The error bars show the standard error of three replicates. Student’s t-test was used for statistical analysis, ** indicates significant differences at P < 0.01, all compared to the treatment at 0 h.