Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2017
Publication Date: 6/14/2017
Citation: Miao, Q., Deng, P., Saha, S., Jenkins, J.N., Hsu, C., Abdurakhmonov, I.Y., Buriev, Z.T., Pepper, A., Ma, D. 2017. Genome-wide identification and characterization of microRNAs differenytially expressed in fibers in a cotton phytochrome A1 RNAi line. PLoS One. 12(6):e0179381. Interpretive Summary: Recently we reported that silencing phytochrome A1 gene (PHYA1) by RNA interference technique in Upland cotton generated PHYA1RNAi lines with simultaneous improvements in fiber quality (longer, stronger and finer fiber) and other agronomic traits. Characterization of the altered molecular processes in the fiber of RNAi genotypes and comparing it with wild-type controls will provide valuable information on the molecular basis of the cotton PHYA1RNAiphenotypes and fiber development. MicroRNAs (miRNAs), a special class of regulatory single-stranded RNA molecules approximately 22 nucleotides size, were shown to play important regulatory roles in cotton fiber development including fiber initiation, elongation and secondary cell wall synthesis. The objectives of this report were identification and characterization of miRNAs that were differentially expressed in PHYA1 RNAi cotton lines. We identified at the molecular level the target genes of the differentially expressed miRNAs in PHYA1RNAi lines involved in the metabolism and signaling pathways of phytohormones including Gibberellin, Auxin and Abscisic Acid. The expression of several MYB transcription factors, normally known as regulatory genes in fiber development, was also affected by miRNAs in PHYA1RNAi cotton lines. In addition, we reported for the first time 35 novel miRNAs (novel miR1-novel miR35) in fibers. The target genes of the majority of these novel miRNAs were also predicted. We used qRT-PCR technique to further confirmed expression results of several differentially regulated miRNAs. Our results provided useful information for understanding of molecular mechanisms in fiber development and altered phenotypes associated with the cotton PHYA1RNAi lines.
Technical Abstract: Silencing phytochrome A1 gene (PHYA1) by RNA interference in Upland cotton (Gossypium hirsutum L. cv. Coker 312) had generated PHYA1 RNAi lines with simultaneously improved fiber quality (longer, stronger and finer fiber) and other key agronomic traits. Comparative analyses of altered molecular processes in these RNAi genotypes and its wild-type controls is a great interest to better understand PHYA1 RNAi in cotton. In this study, a total of 77 conserved miRNAs belonged to 61 families were identified in a PHYA1 RNAi line and its parental Coker 312 genotype by using multiplex sequencing. Of these miRNAs, seven (miR7503, miR7514, miR399c, miR399d, miR160, miR169b, and miR2950) were found to be differentially expressed in RNAi cotton. The target genes of these differentially expressed miRNAs were involved in the metabolism and signaling pathways of phytohormones, which included Gibberellin, Auxin and Abscisic Acid. The expression of MYB transcription factors was also affected by miRNAs in RNAi cotton. In addition, 35 novel miRNAs (novel miR1-novel miR35) were identified in fibers for the first time in this study. Target genes of vast majority of novel miRNAs were also predicted. Of these, nine novel miRNAs (novel-miR1, 2, 16, 19, 26, 27, 28, 31 and 32) were targeted to cytochrome P450 like TATA box binding protein (TBP). The qRT-PCR confirmed expression levels of some miRNAs, and inverse expression patterns of four miRNAs-targets pairs had also been detected via RNA deep sequencing. Together, the results imply thatPHYA1 RNAi caused a miRNA mediated fine-tune gene regulation that might confer to the phenotype of thePHYA1 RNAi line(s) with improved fiber quality.