MICRODISSECTION AND MICROCLONING OF THE BARLEY (HORDEUM-VULGARE L) CHROMOSOME 1HS

We have applied a refined microdissection procedure to create a plasmi d library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The t echnical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection o f the collection drop to avoid evaporation, and a motorized and progra mmable microscope stage. Thirteen readily-discernible telocentric chro mosomes have been excised from metaphases of synchronized root-tip mit oses. After lysis in a collection drop (2 nl), the DNA was purified, r estricted with RsaI, ligated into a vector containing universal sequen cing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant p lasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analy sis after colony hybridization with labelled total barley DNA. Analysi s of 552 recombinant plasmids established that: (1) the insert sizes r anged between 70 and 11 50 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecific H. vulgare x H. spontaneum F2 population. One of these probes was found to be closely linked to the Mla locus conferring mildew resistance.