Sequence and expression analysis of microRNAs during ovule and fiber development in cotton

Cotton fibers are seed trichomes, and their development undergoes a series of rapid and dynamic changes from fiber cell initiation, elongation to primary and secondary wall biosynthesis and fiber maturation. Previous studies showed that cotton homologues encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs). miRNAs are ~21 nucleotide (nt) RNA molecules derived from non-coding endogenous genes and mediate target regulation by mRNA degradation or translational repression. Here we show that among ~4-million reads of small RNAs derived from the fiber and non-fiber tissues, the 24-nt small RNAs were most abundant and were highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 28 putative miRNAs families, including 25 conserved and 3 novel miRNAs were identified in at least one of the cotton tissues examined. Thirty-two pre-miRNA hairpins representing 19 unique families were detected in Cotton Gene Indices version 9 (CGI9) using mirCheck. Sequencing, miRNA microarray, and small RNA blot analyses showed that many of these miRNAs differentially accumulated during ovule and fiber development. The cotton miRNAs examined triggered target cleavage in the same predicted sites of the cotton targets in ovules and fibers as that of the orthologous target genes in Arabidopsis. Targets of the potential new cotton miRNAs matched the previously characterized ESTs derived from cotton ovules and fibers. The miRNA targets including those encoding auxin response factors were differentially expressed during fiber development. We suggest that both conserved and new miRNAs play an important role in the rapid and dynamic process of fiber and ovule development in cotton. Clone and sequence small RNAs from immature ovules fiber-bearing ovules and leaves.