Plant, Animal & Microbe Genomes X Conference Plant, Animal & Microbe Genomes X ConferenceJanuary 12-16, 2002
Town & Country Convention Center
San Diego, CA
Poster: Wheat, Barley, Rye, Oat, and related
Large insert genome libraries and high-resolution genetic maps are essential for genome analysis. However, construction of a genome library from plants with large genome sizes is a considerable task. We are now constructing a BAC library from the Japanese malting barley cultivar Haruna Nijo. The procedure for the construction is after that of Nakamura et al. (1997). In preparation of high molecular weight (HMW) DNA from plants, protoplast of green leaves was chosen because the higher quality of the DNA than those prepared from nuclei. The DNA in agar-plug was digested with Hind III, size selected (200-500kb) and ligated into the dephosphorylated pBAC-LAC vector. The DNA was electroporated into E. coli DH10B cells. White colonies were picked and arrayed onto 384-well microtiter plates by the colony picking robot Q-Pix (Genetix Ltd.). 179 barley BAC clones were randomly selected and analyzed the inserts size by CHEF electrophoresis after Not I digestion. Results of the investigation for size distribution of BAC inserts indicated that although 24% of clones included shorter inserts than 80kb, the most frequent size was 130-140kb. It was confirmed that the protoplast method is suited for HMW DNA preparation also in barley. We are currently trying to eliminate the short insert DNA (<80kb) and to cover 6 X haploid genome equivalent.