Plant & Animal Genome VI Conference

P75

RNA FINGERPRINTING OF PAPAYA CULTIVARS AND MOLECULAR CLONING OF GTP-BINDING PROTEIN

Okinawa Pref. Agri. Exp. Stn.

Papaya is one of the most important tropical fruits in tropical and subtropical areas. Accumulation of DNA markers for molecular breeding of papaya should be essential. RAPD analysis of 12 papaya cultivars was reported recently, but the resolution of the conventional RAPD is limited. Here we describe RNA fingerprinting of 15 papaya cultivars and the isolation of cDNA clones encoding GTP-binding protein and one of the ATPase subunits. Total RNA was prepared from young leaves and cDNA was synthesized. A portion of cDNA was subjected to PCR with four different primers having an arbitrary sequences under the low annealing conditions. PCR products were electrophoresed on a 8% polyacrylamide gel and visualized with ethidium bromide. DNA band profile composed of 100-1000 base pairs was obtained. Some of the PCR products showed polymorphism among the 15 cultivars. Based on these polymorphisms, a pylogenetic tree of the 15 papaya cultivars was constructed. Interestingly, RNA fingerprinting is also effective for distinction of papaya cultivars as DNA fingerprinting. For further characterization of the PCR products derived from expressed RNA, DNA bands were reamplified and cloned into plasmid vectors. Partial nucleotide sequencing revealed that one of the clones showes significant homology with those of GTP-binding protein, while the second one has high homology to the subunit of proton translocating-ATPase complex. These results indicate that RNA fingerprinting is an alternative cDNA cloning method for biologically important genes.