RNA-seq Transcriptome Analysis Of Genes Related To Cotton Fiber Elongation

Cotton, one of the most important economic crops in the world, is the most importantfiber crop for the textile industry and the third important oil crop. It is also a model plantspecies for the research of cell development and the origin and evolution of polyploids.RNA-Sequencing (RNA-Seq) is one of the next-generation sequencing (NGS) techniqueswith the characteristics of high-throughput and low cost，providing a powerful technologyplatform for identigying genes related to cotton fiber development.In this study, six sequencing libraries were constructed from the early cotton fiberdevelopment stages including the initiation stage (0days post anthesis,3DPA) and the fastelongation stage (10DPA) from two near-isogenic backcross inbred lines (BILs) with asignificant difference in fiber length (32.58vs.27.06mm). The main results were as follows:1. We obtained more than51000000high quality reads, approximately4.6Gbtranscriptome data in each library. And98464unigenes, longer than200bp, were got by denovo assembly. The total length of unigene was about88.2Mb, and the average length ofunigene was896bp, N50length of unigene was1397bp.2. Unigene sequences were aligned to public databases such as NR (Non-redundantProtein Sequence Database in GenBank), Swiss-Prot (Swiss-Prot Protein Sequence Database),KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Cluster of Orthologous Groupsof proteins), GO (Gene Ontology) and NT (Non-redundant Nucleotide Sequence Database inGenBank), then76724unigenes,77.9%of total, got functional annotations.70094unigeneswere matched to NR protein database,24709unigenes were matched to25functionalcategory of COG database,32363unigenes were matched to GO database,37964unigeneswere matched to126pathway in KEGG database.3. Comparative analysis of transcriptome of different material at the same fiberdevelopment stages,1931different expressed genes (DEGs) were found in10DPA group,including1536up-regulated and394down-regulated genes,5031DEGs were found in3DPAgroup, including500up-regulated and4531down-regulated genes,17658DEGs were foundin0DPA group, including7579up-regulated and10079down-regulated genes. Comparativeanalysis of transcriptome of the same material at different fiber development stages,3610 DEGs were got in3DPA vs0DPA group, including1771up-regulated and1839down-regulated genes.14655DEGs were got in10DPA vs3DPA group, including147up-regulated and12908down-regulated genes.4. Gene Ontology functional enrichment analysis and pathway enrichment analysis showthat1931DEGs in10DPA group which mainly participated in lipid transport activity termand lipid metabolism pathway. It is hypothesized that genes related to lipid played animportant role in the process of cotton fiber elongation development. Through quantitativeRT-PCR analysis on different stages of cotton fiber of12DEGs including GhABP (Auxinbinding protein), GhLTP3(Lipid transfer protein3), GhPRP3(Propine rich ptotein3),GhRDL2(Dehydration-induced protein RD22-like protein2), alpha-tubulin8, E6-4, GhEXP2(Expansin2), GhDET2(Steroid5Alpha-Reductase), GhAnx1(Annexin), CL10581.Contig1_All, Unigene11613_All, CL12718.Contig1_All, we confirmed that many genes played part infiber elongation, but the molecular mechanism is still unknown.5. We got1004DEGs by comparative analysis of three fiber development stages. Theexpression pattern of DEGs was clustered by software Cluster3.0, which was divided intofour types, a1：the expression level gradually increased from0DPA to10DPA; a2:3DPA hasthe highest expression level; a3:3DPA has the lowest expression level; a4: the expressionlevel gradually reduced from0DPA to10DPA. Besides, there are many genes related to fiberelongation in part a1, and many transcription factor related to fiber initiation in part a4.6genes, GhMYB5(MYB transcription factor), GhCIPK1(CBL-interacting protein kinase),GhGPAT6(glycerol-3-phosphate acyltransferase6), GhFLA10(Fasciclin-like arabinogalaetanprotein10), Unigene35146_All, Unigene30578_All, were selected to do qRT-PCR to verifythe expression pattern on8fiber development stages from0to25DPA.（6）In each fiber development stage, the expression level of GhLTP3in NMGA-062was higher than in NMGA-105，and rise to the point at10DPA，then gradually decreased，which was consistent with previous research. Unigene11613_All had high expression level infiber elongation period, and had significant difference in the two materials. GhGPAT6alsohad high expression level in fiber elongation period, but had higher expression level inNMGA-105than in NMGA-062. All these genes may play important roles in fiber elongation,which need to be further study to illustrate the molecular mechanism.