Construction and characterization of a BAC library for the molecular dissection of a single wild beet centromere and sugar beet (Beta vulgaris) genome analysis

We have constructed a sugar beet bacterial artificial chromosome (BAC) libr ary of the chromosome mutant PRO1. This Beta vulgaris mutant carries a sing le chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta p rocumbens and is transmitted efficiently in meiosis and mitosis. The librar y consists of 50 304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitoc hondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PRO1 genome can be found in th e library. Approximately 0.2% of the clones hybridized with centromeric seq uences of the PRO1 minichromosome. Using the identified BAC clones in fluor escence in situ hybridization experiments with PRO1 and B. procumbens chrom osome spreads, their wild-beet origin and centromeric localization were dem onstrated. Comparative Southern hybridization of pulsed-field separated PRO 1 DNA and BAC inserts indicate that the centromeric region of the minichrom osome is represented by overlapping clones in the library. Therefore, the P RO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis .