Gao, Wenxiang (2003-12). Wide-cross whole-genome radiation hybrid (WWRH) mapping and identification of cold-responsive genes using oligo-gene microarray analysis in cotton. Doctoral Dissertation.
The first part of this research focused on wide-cross whole-genome radiation hybrid (WWRH) mapping of the cotton (Gossypium) genome. Radiation hybrid mapping has been used extensively to map the genomes of human and certain animal species, but not plant species. In lieu of in vitro hybrid cell line technologies for plants, we developed a novel approach for radiation hybrid mapping based on wide-cross in vivo hybridization. Flowers from one species of cotton, either G. hirsutum or G. barbadense, were -irradiated and then used to pollinate the other species. The resulting hybrid plants were assessed as a mapping tool. Two WWRH mapping panels were constructed from 5- and 8-krad -irradiation treatments. Both panels demonstrated that the WWRH mapping method can be used to map the cotton genome, and that this method complements traditional linkage mapping approaches. The second part of this research focused on the identification of cold-responsive genes using spotted oligo-gene microarray analysis. Increased cold-tolerance in cotton would promote early and uniform seedling establishment, expand the growing season, decrease susceptibility to fungal infections and certain diseases, and increase fiber yield and quality. BLAST searches of the cotton database using amino acid sequences of 93 drought/cold-related genes from Arabidopsis and several other plant species led to 806 cotton orthologous cDNAs and expressed sequence tags (ESTs). Eight hundred and six cotton 70-mer oligos were designed and included in an oligo-gene microarray containing 1,536 70-mer oligos, each representing a cDNA or EST from cotton, or one of 121 chloroplast genes or 66 mitochondrial genes from Arabidopsis. Thirty-eight cotton cDNAs and ESTs were identified as cold-responsive genes based on experimental treatment and oligo-gene microarray analysis. Expression was up-regulated for 36 genes and down-regulated for two genes by cold treatment. Results from microarray analysis were tested and confirmed by northern blot analysis for 16 genes. Our data suggest that Arabidopsis orthologous genes can be used to identify homologous cotton genes. The oligo-gene microarray is a valid approach to study transcriptional changes in cotton.