THE CIC LIBRARY - A LARGE INSERT YAC LIBRARY FOR GENOME MAPPING IN ARABIDOPSIS-THALIANA

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEP H, INRA and CNRS). Optimization of plant culture conditions and protop last preparation allowed the recovery of large amounts of viable proto plasts. Mechanical shearing of DNA was minimized by isolation of DNA f rom protoplasts embedded in agarose. Cloning of large inserts was favo red by including two successive size fractionation steps (after partia l EcoRI digestion and after ligation with the vector arms), which sele cted DNA fragments larger than 350 kb. The library consists of 1152 cl ones with an average insert size of 420 kb. Clones carrying chloroplas t DNA and various nuclear repeated sequences have been identified. Twe nty-one per cent of the clones are found to contain chloroplast DNA. T herefore, the library represents around four nuclear genome equivalent s. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5. 8%, respectively. Only one clone was found to carry the 160 bp paracen tromeric repeated element. Given the smaller size of clones carrying A rabidopsis repeated DNA, the average size of remaining clones is aroun d 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy o ut of 76 pairs of primers identified from one to seven YAC clones. Thu s at least 92% of the genome is represented in the CIC library. The su rvey of the library for clones containing unlinked DNA sequences indic ates that the proportion of chimeric clones is lower than 10%.