Plant Genome III Conference Plant Genome III Conference
A plasmid library from the smallest chromosome of the oat (Avena sativa L.) complement was constructed by microdissection and microcloning. The chromosome was deproteinized with proteinase K. digested with Sau 3A and linker-adaptors ligated to the DNA fragments. From the single chromosome (less than 0.4pg) 10ug of DNA was obtained after 2 rounds of FICR amplification. Cloning experiments with the amplified DNA produced as many as 500,000 recombinant clones. The 500 clones evaluated ranged from 150 to 1700 bp with an average insert size of 550 bp. These were estimated to be 41% high copy and 59% low copy. Tandem repeats were not present in the library and mu have been selected against by a combination of the Sau 3A digestion which is sensitive to C-methylation and the PCR amplification. Many low copy dispersed repetitive sequences were present in the library. These were present primarily on A and D genome chromosomes. Southern analysis revealed that the low copy clones were suitable for RFLP analysis and mapped to the pertinent oat nullisomic line. Detailed mapping of the chromosome is in progress.