CONSTRUCTION OF BAC AND cDNA LIBRARIES OF THE PACIFIC OYSTER AND INITIAL DNA SEQUENCING EFFORTS

¹Department of Molecular Biology, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku Tokyo 160-8582, Japan

²International Center for Aquaspecies Genomics, 314 Inarinoshita, Shishizaki, Miyazu, Kyoto

³ Department of Physiology, Dokkyo University, School of Medicine, 880 Kitakobayashi, Mibucho, Shimotuga, Tochigi

It has been known that the oysters are monoecious. The larvaparous oysters (O. edulis) change sexual phase regularly in a definite rhythm, while the oviparous oysters (C. gigas and C. virginica) seem to change sex during the interval between two breeding seasons. As a step toward understanding the mechanisms of sex differentiation and changes, we recently examined the sex distribution of the C. gigas populations by detailed anatomical and histological observations. We found that there are many hemaphrodites during early period of of maturation and their number declines toward reproductive season by increasing the number of individuals with distinct sex. To further examine the molecular and genetic basis of gonad differentiation, we have constructed genomic BAC and cDNA libraries of the Pacific oyster C. gigas. BAC library was made from high molecular weight DNA of 3rd year C. gigas gonads using pBAC-Lac vector. Over 20,000 clones were isolated and deposited in 224 microtiter plates of 96 wells. Two dozens of representative clones were analyzed on pulsed field gels and found that the average size of DNA inserts is 130 kb. Since genome size of oyster was estimated to be about one tenth of the human genome, this BAC library covers 10 times the oyster genome. We are preparing a simple and rapid screening system using high density replica filters and PCR assay. cDNA libraries were constructed from oyster gonads representing different stages of sex differentiation. We are now in the process of sequencing these DNA clones to establish EST and STS markers for QTL mapping and gene identification.