Plant & Animal Genomes XI Conference

January 11-15, 2003
Town & Country Convention Center
San Diego, CA

Poster: Gene Isolation
            

USING CDNA-AFLP AND BULKED SEGREGANT ANALYSIS TO IDENTIFY AN UP-REGULATED TRANSCRIPT IN OVULES OF APOMICTIC CITRUS 10 DAYS AFTER ANTHESIS

University of California Riverside, Botany and Plant Sciences Dept., Riverside, CA 92521, USA

Developmental differences in the nucellus tissue as early as anthesis have been recorded in some citrus varieties with apomixis (nucellar embryony, Nu+) when compared to non-apomitic (Nu-) varieties. Citrus flowers were collected from individuals in a population known to be segregating for the trait of nucellar embryony at anthesis and 10 DAA (days after anthesis). Genomic AFLP markers tightly linked to the trait and phenotypic data were used to classify individuals Nu+, or Nu-. Five individuals at the extreme of each phenotype were chosen for analysis at the two time points. The cDNA-AFLP analysis was conducted using mRNA purified from dissected ovules and the Expression Analysis kit by Li-Cor, Inc. Templates were standardized and equal fractions of the 5 samples, at a given time point and classification, were bulked. Banding patterns from 64 different primer combinations were analyzed to identify and select 10 transcript-derived fragments (TDFs) for further investigation. Individuals comprising the bulks were then analyzed for their contribution to the differential expression in the bulk. A transcript strongly up-regulated near 10 DAA in each individual of the bulk with nucellar embryony was identified and sequenced. BACs previously confirmed as containing the most tightly linked genomic AFLP marker were converted into template using the Expression Analysis kit components. A fragment amplified from this BAC template has the same sequence as the TDF up-regulated in the Nu+ individuals 10 DAA.