CONSTRUCTION OF A BARLEY (HORDEUM-VULGARE L) YAC LIBRARY AND ISOLATION OF A HOR1-SPECIFIC CLONE

We have constructed an EcoRI-based YAC (yeast artificial chromosome) l ibrary from barley (Hordeum vulgare L. cv. Franka) using the vector pY AC4. The library consists of approximately 18000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corr esponding to 50% of the barley genome. Size fractionation after ligati on resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the c olonies showed homology to a plastome-specific probe; approximately 50 % of the colonies displayed a signal with a dispersed, highly repetiti ve barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-ho rdein storage proteins, the library was screened by polymerase chain r eaction and subsequently by the colony hybridization technique. A sing le YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with th e Hor1-specific probe. Restriction with the isoschizomeric enzymes Hpa II/MspI suggests a high degree of methylation of the Hor1 region in me sophyll cells but not in YAC-derived (yeast) DNA.