| Cotton is an important economic crop in the world and natural cotton fiber is breathable, absorbefacient, environmentally friendly, comfortable, etc, making it become one of the most important raw materials in textile industry. Fiber development contains four stages:initiation, elongation, secondary cell wall synthesis and dehydration mature. Fiber quality is determined during the second and third periods and study of those periods facilitates revealing the molecular mechanism of quality formation. We used Sea Island cotton chromosome segment introgression lines (CSILs) in background of genetic standard line of Upland cotton TM-1, aiming to uncover the mystery of the molecular mechanism of superior fiber quality formation. Years of testing showed that CSIL-A, CSIL-B, CSIL-C and CSIL-D produced longer and stronger fiber compared with recurrent parent TM-1, while CSIL-E produced shorter fiber than TM-1. Using these five CSILs and TM-1 as plant materials, we sequenced the transcriptome of immature fiber at 5,10,15 and 20 DPA.We constructed and sequenced 24 libraries using the Solexa Genome sequencing Analyzer system, which got 21 bp length tags. More than 7 million of clean tags were obtained from each library, which represented 160 to 200 thousand distinct tags. We found that the percentage of distinct tags with high counts dropped dramatically, implying less gene species occupied most of the gene expression.EST sequences of Upland cotton and Island cotton were downloaded from NCBI and assembled respectively using the software TIGR assembler. After redundancy removal, a total of 66,672 genes were conserved as reference genes. About 70% total tags were mapped to reference genes and about 30% were unambiguous tags. Unambiguous tags were counted and normalized to the number of transcripts per million tags (TPM) to evaluate the gene expression level for further analysis.To identify genes that were differentially expressed (differentially expressed genes, DEGs) during fiber elongation development, we compared pairs of DEG profiles of the 24 libraries. Four fiber development periods of the five CSILs were compared with the same period of TM-1, respectively, and 20 comparisons were obtained. DEG was defined as FDR^ 0.001 and the absolute value of|log2Ratio| ≥1 to judge the significance of differences in transcript abundance. Among the 20 comparisons, the number of DEGs was between 1,277-6,643. Moreover, there were unique genes expressed in each CSIL. In total,18,940 genes were differently expressed in at least one CSIL and TM-1.DEGs of CSIL-A, CSIL-B, CSIL-C and CSIL-D were analyzed using cluster analysis, GO and Pathway enrichment analysis.18,421 DEGs from the four superior fiber CSILs were clustered into six main clusters. GO enrichment analysis showed that the most dominant pathways were response to chitin salicylic acid stimulus, ethylene and jasmonic acid mediated signaling pathway. The same analysis was done to the tinpot quality of CSIL-E.11,442 DEGs were clustered into four main clusters. A similar GO distribution was revealed in the DEGs of CSIL-E. However, DEGs of CSIL-E also enriched in several bioprocesses including glucuronoxylan biosynthetic process, actin filament binding, etc. Pathway enrichment analysis showed that no matter superior or tinpot quality the CSIL was, the DEGs were enriched in starch and sucrose metabolism, galactose metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis, etc.To analyze the common DEGs along the development period of each CSIL, the genes all-upregulated or all-downregulated at all four periods (5/10/15/20DPA) or 5/10/15DPA were identified. CSIL-A and CSIL-B had about two hundred this kind of common DEGs, but much fewer common DEGs were identified in the other CSILs, exceptionally CSIL-C which only got four this kind of common DEGs. GO enrichment analysis was done to these common DEGs, only genes upregulated in CSIL-A and CSIL-B enriched in xyloglucan metabolic process, xyloglucan-specific endo-beta-1,4-glucanase activity, cell wall organization, response to stimulus, etc.There were DEGs up-or down-regulated in more than one CSIL, many of which were involved in fiber enlongation development. CSIL-A and CSIL-B have the most common DEGs, including transcription factors (erf, zinc finger, wrky, etc), xyloglucan endotransglucosylase hydrolase protein (xth), glycosyltransferase, beta-galactosidase (bgal), actin, expansin, tubulin, vacuolar membrane proton pump, etc. To identify the key genes involed in fiber enlongation, we analyzed the DEGs downregulated in CSIL-E and upregulated in the other four CSILs, which including xth, cytoskeleton related genes, bgal,... |
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