Plant & Animal Genome VI Conference

P18

CONSTRUCTION OF A PINE BAC LIBRARY

1. Dept. of Soil & Crop Sciences, Texas A&M University, College Station, Texas 77843
2. USDA-FS, PSW Station, 800 Buchanan St., Albany, CA 94710
3. SIFG, USDA-FS, 23332 Highway 67, Saucier, MS 39574, USA
4. PSE Station, USDA-FS, PO Box 245, Berkeley, CA 94701, USA
5. USDA-FS, 5985 County Rd., K. Rhinelander, WI 54501

Pine has a genome of about 48 pg/2C (24,000 Mb/1C) that is extremely rich in repetitive sequences. The pine needles are highly waxed and rich in polyphenolic substances. It is of significance to demonstrate the feasibility of isolating megabase DNA from these species and constructing a large-insert DNA BAC library for molecular analysis of pine genomes. To isolate megabase DNA from pine needles, slight modifications were made to our standard DNA isolation method (Zhang et al. 1995). DNA digestion experiments and pulsed-field gel electrophoresis indicated that the majority of the DNA was larger than 1 Mb, the DNA was readily digestable, and therefore, was amenable to BAC cloning and genome analysis. From the megabase DNA, we have successfully constructed a partial BAC library for loblolly pine. The insert sizes of the BACs ranged from 45 to 210 kb, with an average insert size of about 120 kb. BAC fingerprint analysis showed that the BACs were stable in host cells for over 150 generations. A high degree of variability in restriction patterns was also observed and indicated that pine BACs is amenable to fingerprinting.