Generation Of Expressed Sequence Tags For Flowering Genes Discovery And Simple Sequence Repeat Markers In Upland Cotton (Gossypium Hirsutum L.)

Upland cotton, Gossypium hirsutum L., is one of the world's most important economic crops. In the absence of the entire genomic sequence, a large number of expressed sequence tag (EST) resources of upland cotton have been generated and used in several studies. However, rare information is available on the flower development of these species. To further clarify the upland cotton flower development molecular mechanism, a total of 22915 high-quality ESTs were generated and assembled into 14373 unique sequences consisting of 4563 contigs and 9810 singletons from a normalized and full-length cDNA library that constructed from pooled RNA isolated from shoot apexes, squares, and flowers. Comparative analysis indicated that 5352 new unigenes were added in the cotton public database. Furthermore, the result of functional annotation of these unigenes revealed that the majority of the BLAST hits were recorded for Ricinus (26.4%), Vitis (24.5%), and Populus (23.4%), whereas only 886 (7.3%) of the entries were identified for the cotton used in this study.34 upland cotton homologs with flowering-related genes were identified in our library from the functional annotation. These genes were included flowering determination genes, floral meristem identity genes and floral organ development genes. To get the gene information of upland cotton, the 173 Arabidopsis flower related genes were used as a query to tBLASTn search against all upland cotton EST database, and 158 homologs were identified in upland cotton. These genes were divided into photoperiodic flowering pathway, vernalization pathway, autonomous pathway, gibberellin pathway, floral pathway integrators and floral organ identification genes. We cloned a series of flower development-related genes, analyzed multiple sequences alignment, constructed phylogenetic trees and analyzed tissue expression pattern. They showed that most of these genes with other genes had conserved domain with high identity, and clustered together with Poplar and Arabidopsis. The expression patterns revealed that these genes highly expressed in flower development organizations and some genes also had a high profile in the fiber or the ovule. Moreover, 670 new putative microsatellites with flanking sequences sufficient for primer design were identified from the 645 unigenes.25 EST-SSRs were randomly selected for validating and testing transferability on 17 Gossypium sepices. The results revealed that 23 pairs were identified as true-to-type SSR loci and could amplify polymorphic bands from G. hirsutum L. and a high transferability among the Gossypium species. These EST resources should be a valuable foundation of microarrays for gene expression profiling analysis, functional analysis of newly discovered genes, genetic linkage and quantitative trait loci analysis (QTL).