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TitleEST derived PCR-based markers for functional gene homologues in cotton
AuthorsPeng W. Chee, Junkang Rong, Dawn Williams-Coplin, Stefan R. Schulze, and Andrew H. Paterson
PublicationGenome 47: 449-462
AbstractWe investigated the utility of the Gossypium arboreum EST sequences in the Genbank database for developing PCR-based markers targeting known-function genes in cultivated tetraploid cottons, G. hirsutum and G. barbadense. 465 randomly selected ESTs from this library were subjected to BLASTN search against all Genbank databases, of which putative function was assigned to 93 ESTs based on high nucleotide homology to previously studied genes. PCR primers were synthesized for 89 of the known-function ESTs. A total of 57 primer pairs amplifiedG. arboreum genomic DNA but only 39 amplified in G. hirsutum and G. barbadense, suggesting that sequence divergence may be a factor causing non-amplification for some sites. DNA sequence analysis showed that most primer pairs were targeting the expected homologous loci. While the amplified products that were of larger size than the corresponding EST sequences contain introns, the primer pairs with a smaller amplicon than predicted from the flanking EST sequences did not amplify to the expected orthologous gene sequences. Among the 39 primer pairs that amplified tetraploid cotton DNA, 2 detected amplicon size polymorphisms and 9 detected polymorphisms after digestion with one of six restriction enzymes. Nine of the polymorphic loci were subsequently mapped to an anchor RFLP map. Digestion of PCR-amplified sequences offers one means by which cotton genes can be mapped to their chromosomal locations more quickly and economically than by RFLP analysis.

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.: 2009 :: Barry Marler :: Plant Genome Mapping Laboratory :: University of Georgia