YAC cloning and identification of rice (Oriza sativa L.) genomic DNA

Construction of the genomic library by using yeast artificial chromosomes (YAC) has been an approach to a map-based gene cloning strategy. We have obtained more than 2000 YAC clones for the construction of a rice (Oriza sativa L.) genomic library through a procedure that could be summarized as follows: a fractionate by the pulsed-field gel electrophoresis (PFGE), the rice nucleic high molecular weight (HMW) DNA which is partially digested with EcoRI, and recovered fragments larger than 200 kb; ligate the fragments to the EcoRI digested YAC vector pairs pJS97 and pJS98; transform competent spheroplasts prepared from yeast strain YPH252; and select transformants directly on Ura-Trp-double selective media. Southern hybridization results indicated that the sizes of inserts were in the range of 200-820 kb.