A Comparative miRNAome Analysis Reveals Seven Fiber Initiation-Related and 36 Novel miRNAs in Developing Cotton Ovules

Abstract

An increasing number of microRNAs (miRNAs) have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miRNAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly identified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.

The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers

“…According to our results as well as those of other studies (Gou et al, 2007;Al-Ghazi et al, 2009;Fan et al, 2009;Wang et al, 2012), it is possible that lignin/lignin-like phenolics is a determinant of cotton fiber quality.…”

High-throughput sequencing-based genome-wide identification of microRNAs expressed in developing cotton seeds

“…Small RNA library preparation and highthroughput sequencing Small RNA library construction was performed as described previously [3]. Briefly, 15-30 nt small RNAs were gel-purified from 15% PAGE (7 mol L 1 urea), 5′ and 3′ RNA adaptors were added, and RT-PCR using primers with partial complementarity to the adaptors was performed.…”

“…The relative abundances of known and novel miRNAs in these two libraries were reported as normalized reads (reads per ten million, RPTM). The Student's t-test was used to assess the statistical significance of the differences between the highest and lowest abundance levels of each miRNA for miRNA families with reads greater than 100 RPTM, as previously described [3]. Secondary structures were predicted with MFOLD (http://mfold.rna.albany.edu) using sequences identified by mireap.…”

“…The mature fiber is the world's most important natural textile fiber, whereas the high-quality oil and proteins can further increase the agronomic and economic importance of cotton as a crop plant. Although the growth process of cotton fiber has been intensively studied [2][3][4], as an integral part of the seed, increasing evidence shows that fiber growth is indeed regulated by seed development [5]. Therefore, it is necessary to have an in-depth investigation into the regulatory molecules of cotton seed development.…”

“…Especially, the high throughput RNA-sequence technology was employed to identify 562 candidate miRNAs from 7-DPA fibers of allotetraploid cotton [9], while cotton DD-genome shotgun sequences were examined to annotate 348 miRNA candidate sequences [2]. In our previous studies, seven fiber initiation-related miRNAs were characterized by comparative miRNAome analysis in developing cotton ovules [3]. Subsequently, from four small RNA libraries constructed from upland cotton fibers ranging from 5-to 20-DPA, eight miRNAs were found to be related to cotton fiber elongation [4].…”