Plant & Animal Genome IX Conference

Poster: Large Insert Libraries, Gene Isolation, Etc.
P02_20.html

DEVELOPMENT OF GENOMIC RESOURCES FOR GRAPE: BAC LIBRARY CONSTRUCTION, PRELIMINARY STC ANALYSIS, AND IDENTIFICATION OF CLONES ASSOCIATED WITH FLAVANOID AND STILBENE BIOSYNTHESIS

¹ Clemson Univ. Genomics Institute, Room 100 Jordan Hall, Clemson, SC 29634, USA

² Dept. Crop and Soil Sciences, Univ. Georgia, Athens, GA 30602, USA

³ Centre for Plant Conservation Genetics, Southern Cross University, PO Box 157 Lismore NSW 2480, Australia

We have constructed a grape BAC library using the cultivar Syrah. The library contains 55,296 clones stored in 144 384-well microtiter plates. A random sampling of 381 BACs indicated an average insert size of 144 kb with a range of 30 to 355 kb, and less than 4% of the clones do not contain inserts. Eighty nine percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 483 Mb, library coverage is 16.5 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.5%). Library screening was performed using 2 high-density BAC colony filters per hybridization allowing 11 genome equivalents to be screened at a time. Screening was performed using fifteen cDNA probes associated with flavanoid and stilbene biosynthesis genes which resulted in an average of 15 hits per probe (range = 5 to 31). To evaluate the utility of the library for sequence-tagged connector development, 2,304 BAC clones were end sequenced in both forward and reverse. End sequences were searched against the SwissProt and GenBank plant databases and sorted according to function.