Transcriptome And Small RNA Sequencing Analysis Of Dwarf Mutant Upland Cotton (Gossypium Hirsutum L.)

Cotton is one of the most economically important crops in China. Dwarf variety is lodging-resistant and utilizing efficiency of solar-thermal resources, precocity and appropriate for close planting. Therefore, rational use of the dwarfism in cotton,controling of height based on genetics and selection of the dwarf cotton with optimal plant architecture, was the most important target in future cotton breeding programs. Using the next generation high-throughput sequencing technology, three cDNA and small RNA libraries were constructedand sequenced withIllumina HiSeqTM2000system. Through comprehensive analysis withtranscriptome and miRNA regulation level, we expected to reveal the molecular mechanism underlyingdwarfism of Aril327. These data will be beneficial for cloning the dwarfism genes and lays the foundation and resource for breeding cotton dwarf genetype. Aftersequencing and analyzing of data, we got the main results and conclusion as follows:(1) Through the analysis of high-throughput mRNA sequencing of stem apex of five leaf stage of three samples,52583550clean reads were obtained after removing low quality reads with156848transcripts longer than200bp and70877unigenes by de novo assembly.(2) After comparing the transcriptome data of Aril327with Ari971and Ari3697,13919differentially expressed unigenes (DEGs) were indentified, of which5406up-regulated while8513down-regulated in Ari1327.Gene Ontology (GO) functional enrichment and KEGG pathway analysis revealed that the DEGs enriched in plant hormone signal transduction pathway, especially in gibberellins and auxin signal transduction pathways.(3) The16genes in plant hormone signal transduction pathway was validated and analysed using quantitative real time PCR (qRT-PCR). The results of gene expression obtained from qRT-PCR were consisted with that through transcriptome sequencing, and also validated the reality of sequence results.(4) Through the analysis of small RNA sequencing,226known conserved miRNAs belong to32families were obtained, twenty of which were known in upland cotton. After prediction with strict criteria,19novel miRNAs were identified. Target genes of conserved miRNAs were predicted with software, and531target genes were obtained belong to436GO terms and116KEGG pathways.Through conjoint analysis of transcriptome and small RNA sequencing, we identified that miRNA858bdown-regulated MYB and then modulate related plant hormone transduction pathway.(5) After analysis of the differential expression of miRNA families in each sample, we got18miRNA families which differentially expressed in both Ari1327and Ari3697. Interestingly,16miRNA families were up-regulated in Ari1327and down-regulated in Ari3697. In three miRNAs libraries, we obtained25miRNAs only expressed in Ari1327and13miRNAs only expressed in Ari3697. These16miRNA families and38specific expressed miRNAs may play significant role in regulating plant height of Ari1327.(6) Differentially expression miRNAs of Ari1327and Ari3697compared with Ari971were obtained, respectively. Target genes of these differentially expression miRNAs were annotated in A and D genomes, then the target genes were located in chromosome of A and D genomes. Target genes of miRNAs differentially expressed in Ari1327and Ari971enriched in the12th chromosome, and target genes of miRNAs differentially expressed in Ari3697and Ari971enriched in the5th chromosome.(7) Seven miRNAs were selected foranalysisusing stem-loop RT-PCR. The results were consisted with sequencing data, which indicated that stem-loop RT-PCR could be used as a method to validate the reality of small RNA sequencing. At the same time, we analysed two target genes using qRT-PCR and the results were consisted with transcriptome sequencing.After analyzing the transcriptome and small RNA sequencing of three samples, large numbers of useful information were obtained. Although some genes did not have annotations, the results of sequences were useful for further studies. The two plant hormones GA and IAA may play important roles in dwarfism of Ari1327. Since the study of relationship between hormone and miRNA was few, the miRNAs that regulated GA and IAA need further identification and confirmation.