A Genome-Wide Characterization of Differentially Expressed Genes Encoding mRNAs and miRNAs and Methylation Analysis of Phytochrome Genes in a Cotton Phytochrome A1 RNAi line
CommitteeJenkins, Johnie N.
Silencing phytochrome A1 gene (PHYA1) by RNA interference in upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with increased fiber length, strength and low micronair (finer fiber). In order to identify and characterize mRNAs and miRNAs that are differentially expressed in the RNAi plants, transcriptome and miRNAome analyses via high-throughput RNA sequencing were performed. Total RNA isolated from 10-DPA (days post anthesis) fibers and small RNAs isolated from 5-, 10-, and 15-DPA fibers of RNAi and Coker 312 lines were used to construct 6 RNA libraries and 18 small RNA libraries, respectively, which were sequenced using the Illumina HiSeq system. A total of 142 differentially expressed genes (DEGs) were identified in PHYA1 RNAi compared to Coker 312. GO analysis showed that these DEGs were mainly involved in metabolic pathways, binding and regulating enzymes (hydrolase, transferase, and oxidoreductase activities), and cell structures which were reported to play important roles in fiber development. Twenty-eight KEGG pathways were mapped for 142 DEGs, and the pathways related to glycolysis/gluconeogenesis and pyruvate metabolism were the most abundant, followed by cytochrome P450-involved pathways. Sixty-one conserved miRNA families and thirty-five novel miRNAs were identified in upland cotton. The targets of 6 conserved miRNAs, which expressed differentially in the RNAi line, were reported to participate in primary cell wall synthesis and phytohormone signaling pathways. The 35 novel miRNAs were identified in cotton for the first time, and their target genes were predicted. Nine novel miRNAs were identified to target cytochrome P450 TBP. Together, the results imply that miRNAs involved in fine-tune gene regulation might confer to the phenotype of the RNAi line with improved fiber quality. Besides characterizing mRNAs and miRNAs, the CpG site methylation status within coding regions of phytochrome genes in RNAi line in leaves and 10-DPA fibers was determined using bisulfite genomic sequencing. The PHYA1, PHYC and PHYE in RNAi line had higher methylation levels in leaves than those in Coker 312, but PHYB had lower methylation levels. In fibers, the methylation levels of PHYB also decreased in RNAi plants. However, the methylation of other phytochrome genes showed no significant changes.