Gene co-expression network analysis reveals key pathways and hub genes in Chinese cabbage (Brassica rapa L.) during vernalization

Abstract

Background: Vernalization is a type of low temperature stress used to promote rapid bolting and flowering in plants. Although rapid bolting and flowering promote the reproduction of Chinese cabbages (Brassica rapa L. ssp. pekinensis), this process causes their commercial value to decline. Clarifying the mechanisms of vernalization is essential for its further application. We performed RNA sequencing of gradient-vernalization in order to explore the reasons for the different bolting process of two Chinese cabbage accessions during vernalization.

Results: There was considerable variation in gene expression between different-bolting Chinese cabbage accessions during vernalization. Comparative transcriptome analysis and weighted gene co-expression network analysis (WGCNA) were performed for different-bolting Chinese cabbage during different vernalization periods. The biological function analysis and hub gene annotation of highly relevant modules revealed that shoot system morphogenesis and polysaccharide and sugar metabolism caused early-bolting 'XBJ' to bolt and flower faster; chitin, ABA and ethylene-activated signaling pathways were enriched in late-bolting 'JWW'; and leaf senescence and carbohydrate metabolism enrichment were found in the two Chinese cabbage-related modules, indicating that these pathways may be related to bolting and flowering. The high connectivity of hub genes regulated vernalization, including MTHFR2, CPRD49, AAP8, endoglucanase 10, BXLs, GATLs, and WRKYs. Additionally, five genes related to flower development, BBX32 (binds to the FT promoter), SUS1 (increases FT expression), TSF (the closest homologue of FT), PAO and NAC029 (plays a role in leaf senescence), were expressed in the two Chinese cabbage accessions.

Conclusion: The present work provides a comprehensive overview of vernalization-related gene networks in two different-bolting Chinese cabbages during vernalization. In addition, the candidate pathways and hub genes related to vernalization identified here will serve as a reference for breeders in the regulation of Chinese cabbage production.

Keywords: Chinese cabbage; Gradient-vernalization; Hub genes; RNA sequencing; Weighted gene co-expression network analysis.

Fig. 1

Transcriptional relationship between samples. a Heatmap of correlation value (R square) of 42 libraries. b Principal component analysis based on all of the expressed genes, showing 14 distinct groups of samples. c Number of transcripts in the 2 Chinese cabbage accessions, based on the FPKM of different samples

Fig. 2

WGCNA of gene expression in ‘JWW’ (a) and ‘XBJ’ (b) during vernalization. Hierarchical cluster trees show the co-expression modules identified by WGCNA

Fig. 3

Co-expression modules for ‘JWW’. a Relationships between modules (left) and traits (bottom). Red and blue represent positive and negative correlations, respectively, with coefficient values and p-values. b Pairwise correlation coefficients between modules. Rows and columns are the module names, numbers represent coefficient values and p-values

Fig. 4

Co-expression modules for ‘XBJ’. a Relationships between modules (left) and Traits (bottom). Red and blue represent positive and negative correlations, respectively, with coefficient values and p-values. b Pairwise correlation coefficients between modules. Rows and columns are the module names, numbers represent coefficient values and p-values

Fig. 5

Gene expression levels in ‘JWW’ (a) and ‘XBJ’ (b) with their corresponding log₂FPKM module values. The color gradient from blue to red indicates high to low gene expression

Fig. 6

Significant GO terms and ontological relationships (annotated from ClueGO) in ‘JWW’. The sizes of the circles represent the degree of the positive relationship between the significant GO terms. Redundant terms were grouped and presented in the same color. Each leading term, which has the highest significance, is indicated by colored font

Fig. 7

Significant GO terms and ontological relationships (annotated from ClueGO) in ‘XBJ’. The sizes of the circles represent the degree of the positive relationship between the significant GO terms. Redundant terms were grouped and presented in the same color. Each leading term, which has the highest significance, is indicated by colored font

Fig. 8

Hub genes and expression profiles of ‘JWW’. a Co-expression gene networks with the greatest “hubness” in every module. Nodes are represented by dots surrounded by module colors. b log₂FPKM expression profiles of the hub genes in J1, J2, J3, J4, J5, J6, and JCK. The locations of each gene correspond with A. The darker the green color, the higher the expression level

Fig. 9

Hub genes and expression profiles of ‘XBJ’. a Co-expression gene networks with the greatest “hubness” in every module. Nodes are represented by dots surrounded by module colors. b log₂FPKM expression profiles of the hub genes in X1, X2, X3, X4, X5, X6, and XCK. The locations of each gene correspond with A. The darker the green color, the higher the expression level

Fig. 10

qRT-PCR verification of the hub genes related to flower development. The bar and line graphs represent the qRT-PCR and RNA-Seq data, respectively. Data are presented as mean ± standard error (SE)

Fig. 11

a Treatment process of two Chinese cabbage accessions: (a), (b), and (c) are ‘JWW’; (d), (c), and (f) are ‘XBJ’; (a) and (d) are before vernalization (0 DAT); (b) and (c) are during vernalization (25 DAT); (c) and (f) are after flowering. b ‘JWW’ flowering time promoted by vernalization. c ‘XBJ’ flowering time promoted by vernalization