AFLP Technique And Mitochondrial DNA Sequence Analyse To Clarify Taxonomic Status Of Crassostrea Oyster Species In China

It has been presumed that there are four common Crassostrea oyster species along the coast of China: the Crassostrea gigas, C. plicatula, C. rivularis and C. talienwhanensis. Classification and species identification of these Crassostrea oysters have been difficult because of morphological plasticity. It is necessary to use the molecule mark to clarify taxonomic status of these species.The AFLP(Amplified Fragment Length Polymorphism)technique is based on the selective PCR amplification of restriction fragments from genomic DNA digested by restriction enzymes completely. In this paper , simplized AFLP technique was performed to primary clarify taxonomic status and genetic diversity on genome among Crassostrea oysters: C. gigas, C. plicatula, C. rivularis and C. talienwhanensis. The feasibility of AFLP in the oyster's taxonomic was also investigated. In all 16 primers, 8 random primers were chosen and used to amplify the individuals' DNA of four species. The 8 primers all generated amplification bands, whose numbers ranged from 1 to 10 and sizes were from 500 to 2000bp. There are four special genetic markers obtained: two are for C. gigas,one is C. talienwhanensis and one is C. rivularis. Data processing is followed by RAPD. Parameters of genetic variation and genetic distance among the four species indicated that there is little variance in C.gigas which the pencent of polymorphic sites(6.9%) and shannon' index of phenotypic diversity (0.0183) are lower than others. Intre-species genetic diatance of four species of oyster shows the divergence between C. gigas and C. talienwhanensis was very low, so was that between C. plicatula and C. rivularis. Phylogenetic analysis showed that haplotypes of C. gigas and C. talienwhanensis clustered in one clade and those of C. plicatula and C. rivularis in another one. Our data suggests that C. gigas and C. talienwhanensis may be the samespecies. But the lack of divergence between C. plicatula and C. rivularis samples may indicate that the C. plicatula specimen we sampled could actually be a morph of C. ariakensis living in high salinity habitats. More work is needed for confirmation.DNA sequencing is very useful for the genetic diversity analysis at molecular level. In order to clarify some disagreements existing on taxonomic status of C. plicacula, phylogenetic analysis was performed to clarify taxonomic status of these species using mitochondrial DNA sequence data. Nucleotide sequences of a 488bp fragment of ribosomal RNA gene and a 649bp segment of cytochrome coxidase I gene were obtained through sequencing and used for analysis. Ten variable nucleotide positions were found variable and four haplotypes were defined for all 31 individuals in 16S rRNA gene and eighty-four variable nucleotide positions were found variable and twenty-eight haplotypes were defined for all 60 individuals in COL From the parameters of genetic variation among oysters from four sites we can find that the genetic variation in Dalian and Qingdao population was very low, however in Ningbo and Xiamen population was high .In all With C. gigas, C. talienwhanensis and C. rivularis as outgroups, genetic distances among all the haplotypes(16SrRNA and COI) of these small oysters and those of Crassostrea species mentioned above were computed, and NJ and MP phylogenetic trees for them were generated. All phylogenetic trees get the same result. These results suggested that the so-called Zhe oysters (C. plicacula) was not the correct species name for oysters collected along coastline from the south to north, and it was not a single species. It at least contained two species: one was C. gigas in the north, and another one C. sikamea from NB.