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 f rom nuclei and partially digested with Sau3AI. DNA fragments of 75-100 kb were selected by size fractionation in low melting agarose, concen trated by a spot-evaporation/dialysis method, and cloned in the pAd10s acBII 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 pl ant. This library can be screened rapidly by dot hybridization of plat e and well position pools. Characterization of the library by restrict ion analysis, screening with RFLP probes, RFLP mapping of insert end s equences, and chromosome walking shows that the library is of high qua lity with respect to insert site, completeness, and absence of chimeri c artifacts. With this library a contig of about 600 kb has been const ructed in the cer9 locus region. This P1 library is expected to be use ful for genome mapping and gene cloning in Arabidopsis research progra ms.