Identify And Function Verify Of Cotton Fibere Enlongation Related MiRNAs

The cotton fiber is the most important raw material for the textile industry and an ideal model system for studying cell elongation.Numerous studies have reported genes including miRNAs during fiber developments;however,most studies were focused on fiber initiation,with a few studies on fiber elongation.Therefore,the natural genetic variation of fiber elongation in relation to gene expression including miRNA is poorly understood.In this study,two lines(named as "Long" and "Short")differing in fiber length from a backcross inbred line(BIL)population of Gossypium hirsutum × G.barbadense were selected to study the relationship between miRNAs and fiber length using miRNA-seq,genetic mapping and virus-induced gene-silencing(VIGS)techniques.1."Long" and "Short" were chosen as parents to construct a high-density genetic map in F2 which was used to fine map fiber length QTL in F2:3 population.The genetic map had a total size of 3462.8 cM,containing 9182 single-nucleotide polymorphisms(SNPs)based on genotyping-by-sequencing(GBS).Seven fiber length QTLs were identified,including two stable QTL on two chromosomes(qFL-A08-l on A08 and qFL-D03-1 on D03).In addition,nine recombination hotspots in this population were identified.Utilizing previously obtained RNA-seq data for the two BILs and qRT-PCR analysis,two candidate genes annotated as cytochrome b5(CB5,Gh_A08G1729)and microtubule end-binding 1C(EB1C,Gh_D03G0232)were identified,which may regulate fiber length during the fiber elongation stage.2.A high-throughput comparative miRNA-seq of "Long,and "Short,on 10 day post anthesis(DP A)fibers revealed differentially expressed miRNAs and their targets in rapid]y elongating fibers.A real-time quantitative PCR analysis was farther performed to validate the results.A total of 463 miRNAs were identified,47 of which were differentially expressed miRNAs.Nine differentially expressed miRNAs were co-localized with nine fiber length QTL identified in the G.hirsutum × G.barbadensepopulation.The relationship between the allopolyploid and its diploid ancestral species with respect to miRNAs and their targets was also characterized.These results will facilitate the understanding of the molecular genetic mechanism of fiber elongation with regards to miRNAs in cotton.3.The family of miR477 in G.hirsutum and G.barbadense was identified.The sequences of the miRNA family were compared between "Long" and "Short".Cotton plants with virus-induced gene-silencing(VIGS)constructs to over-express had significantly longer fibers as compared to their wild-type,while the VIGS plants with suppressed miR477b expression had significantly shorter fibers.The expression level of the target gene(DELLA)and related genes(RDL1 and EXPA1 for DELLA through HOX3 protein)in the two BILs and/or the VIGS plants were generally congruent,as expected.This report has identified a candidate miRNA(miR477)affecting natural variation of fiber elongation in cotton for the first time.4.The family of miR160 in G.hirsutum and G.barbadense'was identified.The expression level of the miR160 family between "Long" and "Short" was compared.miR160a-5p was chosen to conduct a more in-depth analysis.Cotton plants with VIGS constructs that over-expressed miR160a-5p had significantly longer fibers compared with the wild-type,while the VIGS plants with the suppressed miRNA expression had significantly shorter fibers.The expression levels of the target genes ARF17 and related genes GH3 were generally congruent,as expected.This report has identified miR160a-5p that could affect fiber elongation in cotton.