Characterization of a library from a single microdissected oat (Avena sativa L.) chromosome

Abstract

A plasmid library of oat chromosome No21, the smallest chromosome of the complement, was constructed by microdissection and microcloning. The chromosome was deproteinized with proteinase K and digested with Sau3A and linker adaptors were ligated to the DNA fragments. From the single chromosome (less than 0.4 pg), 10 μg of DNA was obtained after 2 rounds of PCR amplification. Cloning experiments with the amplified DNA produced as many as 500 000 recombinant clones from the single chromosome. The 500 clones evaluated ranged in size from 150 to 1700 base pairs (bp) with an average size of 650 bp. These were approximately 41% high-copy and 59% low/unique copy clones. Tandem repeats were absent in the library and may have been selected against by a combination of the Sau3A 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 blot analysis revealed that the unique-copy clones were suitable for restriction fragment length polymorphism analysis and that they mapped to the pertinent oat nullisomic lines.