Genome-Wide Identification And Function Analysis Of Cotton Micrornas During Fiber Initial Development In Gossypium Hirsutum L.

Cotton is the source of the most important renewable, natural textile fiber in the world and is of significant importance in the textile industry. Meanwhile, Cotton provides a novel system in which to better understand the genetic and biochemical control of cell elongation as well as cellulose biosynthesis. Cotton’fibers’are trichomes derived from epidermal cells of the developing seed. These trichomes share many similarities with those found on Arabidopsis thaliana leaves and which could serve as a model for elucidating the genetic mechanisms that control cotton fiber and seed development. MicroRNAs (miRNAs) are endogenous20-24nucleotides in length, non-coding RNAs, serving as a class of post-transcriptional regulators in eukaryotic organism. MiRNAs negatively regulate their target genes by mRNA cleavage or translation expression via extensive complementarity between miRNAs and their targets. Increasing evidence has demonstrated that plant miRNAs have functions in a wide range of developmental processes and biotic and abiotic stress.In this study, we sequenced seven small RNA libraries constructed from young ovules during fiber initial period using three fibreless mutants (XZ142FLM, MD17FLM, SL1-7-1FLM), two fuzzless mutants (N1NSM, n2NSM) and wild type TM-1. New MIRNAs precursors were predicted in cotton using genome sequence of Gossypium raimondii and Gossypium hirsutum. Some of the novel miRNAs and candidate target genes were validated by the Northern blot and5’RACE. MiRNAs associated with fiber initiation were identified according to different expression between WT and the five mutants. Targets genes expression were validated using transcriptome profiling and the cleavage sites were validated using5’ RACE. The main results were as follows:1. Genome-wide identification of miRNAs during fiber and ovule initial developmentThe Upland cotton Texas Marker-1(TM-1) was used to construct small RNA libraries from young ovules during lint and fuzz initial period. TM-LA was pooled from-3,-1,0and1DPA, and TM-LB was pooled from-1,0,1,3and5DPA. A total of33million small RNA sequences were obtained, and33known miRNA families were expressed in TM-1ovules. Overall,93new miRNA precursors were identified, of which28belonged to10known families and the other65were considered to be novel miRNAs.65precursors containing43small RNAs which were numbered in consecutive order from miR7234to miR7276. Northern blotting validated that miR7235, miR7244and miR7251expressed equally between-1DPA and3DPA as the same as the deep sequencing results. It was predicted miR7235could target a series of Actin-like ATPase superfamily protein, two of which were validated using5’RACE with cleavage sites between the10th and11th nucleotide of miR7235.2. Genome sequence of upland cotton was used in miRNAs identification.Three fibreless mutants (XZ142FLM, MD17FLM, SL1-7-1FLM) were used to construct small RNA libraries during fiber initial period (-3,-1,0and1DPA), and two fuzzless mutants (N1NSM, n2NSM) were used to construct small RNA libraries during fuzz initial period (-1,0,1,3and5DPA). Taken TM-LA and TM-LB together, seven libraries were constructed, each with more than16M reads. Using the genome sequence of upland cotton, a total of322new MIRNAs were identified in upland cotton. The precursors varied in length, from79to544nt, and had an average length of149.8nt. The majority of the miRNAs (74.2%) have a uridine at5’terminal. Among the322miRNAs,197belong to38known families, the other125have no homolog in miRBase (Release20), and we labeled them as novel miRNAs. Those novel MIRNA precursors produce102distinct miRNA mature sequence belonging to86families, and number them as ghr-miRn01-86.58of the new MIRNAs precursors produce mature sequences with variances in size or position. Distinct members of the same family have variances in start site or nucleotides. The expression of the members differs a lot. The members of miR156/535could target the same target genes with score and the cleavage products differ by two nucleotides depending on which miRNA guides the processing event.3. Function analysis of miRNA associated with fiber initiationTaken cotton known miRNA and the new miRNAs identified in this study together,62known families and86novel miRNA families were identified in upland cotton. MiRNA downregulated in wild type TM-1and upregulated in the five mutants include seven conserved families (miR156, miR160, miR160*, miR166, miR167, miR171, miR535, miR827), six less conserved miRNA families (miR2948*, miR3476, miR7495, miR7504, miR7505, miR7508) and six novel families (miRn13, miRn21, miRn51, miRn60, miRn77, miRn80). MiRNA upregulated in wild type TM-1and downregulated in the five mutants included seven conserved families (miR162, miR164, miR166*, miR172, miR396*, miR403, miR482*), four less conserved miRNA families (miR2949, miR7122, miR7502, miR7511) and two novel miRNAs (miRn08and miRn81). Compared with TM-1, miRNAs upregulated in fiberless mutants and downregulated in fuzzless mutants included miR169, miR172*, miR390, miR390*, miR394, miR396, miR482, miR2948, miRn07, miRn76, miRn85, miRn85*. Targets genes expression of the39differentially expressed miRNAs were validated using transcriptome profiling. Cluster analysis of227target genes showed that expression pattern of73genes were complementary with miRNA. Eleven target genes of eight miRNA were validated using5’RACE. Seven target genes of miR164, miR171, miR394and miR396were cleaved in the exact site between nucleotides10and11from the5’end of the miRNA. Target genes of miR397, miR530and miRn20were cleaved at6-1Ont upstream of complementary region. Target gene of miRn80was cleaved at80nt downstream of complementary region. We found cleavage sites away from the10th and11th nucleotide of miRNA were actually cleavage by other small RNA.