Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton

As a main natural fiber source, cotton plays an important role in human life. To identify genes preferentially expressed during early and late cotton fiber development, we constructed two fiber subtracted libraries on the basis of PCR-selected subtraction using a pool of nonfiber tissues as the same driver and 10 days postanthesis (DPA) and 20 DPA fiber cells as testers, respectively. Through differential screening, 292 clones in both libraries were identified as being preferentially expressed during fiber development. Sequence analysis showed that 31 unique sequences were found in the library of 10 DPA fibers and 48 unique sequences were obtained in the library of 20 DPA fibers. In addition, there were 13 unique clones in common in both libraries. Many previously reported cotton-fiber-related genes were included in both libraries. Northern hybridization was performed to further confirm the differential expression and to determine the pattern of mRNA accumulation of selected clones. As a result, all selected cDNAs showed highly preferential expression in developing cotton fibers. Four genes-putative gibberellin-regulated protein, putative tonoplast intrinsic protein, putative plasma membrane intrinsic protein, and Gossypium hirsutum putative membrane protein-were identified in the 10 DPA fiber subtracted library, and they were found to be expressed a lot during early fiber development. On the other hand, those genes screened out of the 20 DPA fiber subtracted library, like arabinogalactan protein and fiber glycosyl hydrolase family 19 protein, were found to be highly expressed in fibers with the maximal transcription level during the developmental switch from elongation to cellulose deposition. By subtraction between fibers and five nonfiber tissues, two sets of genes were identified, and their fiber-specific or fiber-preferential expression indicated that they are involved in the network that controls cotton fiber development.