GENERATION OF A HIGH-QUALITY P1 LIBRARY OF ARABIDOPSIS SUITABLE FOR CHROMOSOME WALKING

Using improved techniques, a representative P1 library of Arabidopsis was constructed and characterized. Megabase genomic DNA was prepared from nuclei and partially digested with Sau3AI. DNA fragments of 75-100 kb were selected by size fractionation in low melting agarose, concentrated by a spot-evaporation/dialysis method, and cloned in the pAd10sacBII P1 vector. The library contains 10080 clones individually stored in microtiter plates. With an average insert size of about 80 kb, the library represents about eight haploid genomic equivalents of this plant. This library can be screened rapidly by dot hybridization of plate and well position pools. Characterization of the library by restriction analysis, screening with RFLP probes, RFLP mapping of insert end sequences, and chromosome walking shows that the library is of high quality with respect to insert site, completeness, and absence of chimeric artifacts. With this library a contig of about 600 kb has been constructed in the cer9 locus region. This P1 library is expected to be useful for genome mapping and gene cloning in Arabidopsis research programs.