GENOME MANIPULATION IN RECALCITRANT SPECIES - CONSTRUCTION AND CHARACTERIZATION OF A YEAST ARTIFICIAL CHROMOSOME (YAC) LIBRARY FROM ERYSIPHE-GRAMINIS F SP HORDEI, AN OBLIGATE FUNGAL PATHOGEN OF BARLEY

Extraction of DNA from organisms where spores are the only source of pure material is a major problem. Methods are described which allow the isolation of high-M(r) DNA, from small quantities of Erysiphe graminis f. sp. hordei (Egh) conidia, suitable for cloning in yeast artificial chromosomes (YACs). A YAC library of 1500 clones was constructed in the vectors, pYAC4 and pYACRC. The average size of YAC inserts is 220 kb and range from 70 to 500 kb, providing ten haploid genome equivalents. Multicopy RFLP markers and an Egh-specific repetitive SINE element were used to characterize the library. The SINE element is effective in fingerprint analysis and contig assembly. Four out of five representative clones containing more than one YAC were mitotically unstable.