Plant & Animal Genome VI Conference

P22

CONSTRUCTION OF A APPLE BACTERIAL ARTIFICIAL CHROMOSOMAL LIBRARY

Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, P. R. China

Zaai-76 apple, which is a mutant from Malus baccata Borkh, contains a major dwarf gene (DW). This gene is important in developing dwarf culture of apple. In order to clone the DW gene of apple by map-based cloning, we have constructed a bacterial artificial chromosomal (BAC) library with highly purified HMW DNA from Zaai-76 apple leaves. The library consists of 22,300 BAC clones. The average insert size is 115 kb and the capacity of this BAC library represent about 3.4 times apple haploid genome equivalents. During this research work, we developed a new simple method for preparing highly pure HMW DNA (>2.2Mb) with PFGE to overcome the difficulty in digestion by HindIII caused by plant tissue debris and polyphenolic compounds in plugs and to eliminate contamination with mtDNA and cpDNA. Moreover, this high pure HMW DNA is more suitable for construction of BAC library because the small nuclear DNA (<2.2 Mb) is removed. There is no positive clone in this library when hybridized by mtDNA and cpDNA as probes. The molecular markers linked to DW gene are being identified by BSA analysis with AFLP technology and will be used as probes for this BAC library screening.