Transcriptomic profiles of non-embryogenic and embryogenic callus cells in a highly regenerative upland cotton line (Gossypium hirsutum L.)

Abstract

Background: Genotype independent transformation and whole plant regeneration through somatic embryogenesis relies heavily on the intrinsic ability of a genotype to regenerate. The critical genetic architecture of non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells in a highly regenerable cotton genotype is unknown.

Results: In this study, gene expression profiles of a highly regenerable Gossypium hirsutum L. cultivar, Jin668, were analyzed at two critical developmental stages during somatic embryogenesis, non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells. The rate of EC formation in Jin668 is 96%. Differential gene expression analysis revealed a total of 5333 differentially expressed genes (DEG) with 2534 genes upregulated and 2799 genes downregulated in EC. A total of 144 genes were unique to NEC cells and 174 genes were unique to EC. Clustering and enrichment analysis identified genes upregulated in EC that function as transcription factors/DNA binding, phytohormone response, oxidative reduction, and regulators of transcription; while genes categorized in methylation pathways were downregulated. Four key transcription factors were identified based on their sharp upregulation in EC tissue; LEAFY COTYLEDON 1 (LEC1), BABY BOOM (BBM), FUSCA (FUS3) and AGAMOUS-LIKE15 with distinguishable subgenome expression bias.

Conclusions: This comparative analysis of NEC and EC transcriptomes gives new insights into the genes involved in somatic embryogenesis in cotton.

Keywords: Callus, embryo; Gossypium hirsutum L; Somatic embryogenesis.

Fig. 1

a 4% of the explants stay on NEC stage; b 96% of the explants can induced to form EC, surrounded by NEC. The examples of NEC (green circle) and EC (red circle) and the section of the callus used for RNA extraction and sequencing were circled

Fig. 2

A VENN diagram of expressed genes that overlap and are unique to NEC and EC developmental stages, respectively. Genes were considered expressed when FPKM ≥ 1

Fig. 3

a Volcano plot of significant genes with FDR corrected p-values (<=.001). b Heatmap of gene expression profiles of NEC and EC callus cells

Fig. 4

Gene ontology enrichment of a Significant upregulated genes (6786) and b Significantly downregulated genes in EC (6538)

Fig. 5

Hiarachal clustering and Gene Ontology enrichment of gene expression profiles. On the left, clustered genes whose expression profiles are at least 1 logFC and error-corrected p-value is ≤ .001. Genes in each subcluster were analyzed for gene ontology enrichment (right)

Fig. 6

Genes involved in the synthesis of phytohormones

Fig. 7

Evaluation of gene expression in embryogenic cells (EC) and non-embryogenic cells (NEC) by RT-qPCR analysis. Five of the selected genes were up-regulated (left); and the other five genes were down-regulated in embryogenic cells (right). GhPP2A1 and GhUb7 were used as the internal controls. Three biological replicates and three technical replicates were used for statistical analysis. Error bars indicate ±SE (n = 3). ΔΔCt method was used for qPCR analysis. Asterisks (*, ** or ***) indicate a significant difference between embryogenic cells (EC) and non-embryogenic cells (NEC) at P < 0.05, 0.01, or 0.001, respectively, by student’s t-test