Plant & Animal Genomes XI Conference Plant & Animal Genomes XI ConferenceJanuary 11-15, 2003
Town & Country Convention Center
San Diego, CA
Workshop: Citrus
An in-house funded Citrus EST project has been operational at the United States Horticultural Research Station in Ft. Pierce FL for 2 years. The effort has focused primarily on the sequencing of random clones from sweet orange (Citrus sinensis) and trifoliate orange (Poncirus trifoliata) cDNA libraries. The two species were chosen since they represented the primary scion and rootstock germplasm donors, respectively. Libraries have been made from similar tissues of both species to allow for direct comparison of orthologous EST sequences and the identification of single nucleotide polymorphisms. To date, 38,000+ total cDNA clones have been sequenced and archived from 11 different citrus libraries by random EST sequencing. Continued isolation of unique clones has switched from random sequencing to a colony-array method developed to reduce cost of unique clone discovery through prescreening analysis. This method employs automated arraying of a minimum of 10,000 colonies (E. coli colonies containing library clones) per nylon membrane, and prescreening for clones that do not hybridize to a pooled probe composed of previously sequenced clones. Robotic manipulation of colonies allows analysis of 100,000 to 200,000 colonies per library. Functional genomics research involved: 1) isolate, sequence, and archive clones from multiple cDNA libraries, and 2) produce high-density membrane arrays of PCR products representing these clones. PCR-product array templates were developed and imported into GeneSpringtm array analysis software for profiling transcript expression. Examples of expression data from plant wounding and insect feeding experiments will be presented along with real-time RT-PCR verification methods.