ITS Region Sequence Analysis And Genetic Diversity Study Of Several Bivalves

Robosomal RNA gene internal Inscribed spacers ITS1 and ITS2 fragments of bloody clam (Scapharca broughtonif) coming from Japanese, Russion and Wei Hai city of China were amplified via Polymerase Chain Reaction. The PCR products were ligated into T-vector, cloned and sequenced. 421bp and 495bp nucleotide sequences were got respectively, and the contents of A, C, G, T is 21.85%, 25.87%, 27.23%, 25.04% in ITS1; 26.66%, 22.18%, 24.04%, 27.13% in ITS2. The content of A+T in ITS2 is higher than that in ITS1 obviously. 2 polymorphic sites and 3 haplotypes were found in ITS2 sequences from 7 bloody clams. Genetic distance between 7 clams is 0-0.2%. 9 polymorphic sites and 7 haplotypes were found in ITS1 sequences from 16 bloody clams, and 4 transition sites and 2 transvertion sites were involved. NJ and MP molecular phylogenetic trees were constructed which showed that there is no obvious clustering phenomenon between all the 16 specimens coming from different geographic populations. The primers of ITS-1 and ITS-2 proved to be very universal in a variety of mollusk species. The potential uses of the two sequences for genetic variation studies and phylogenetic research in Bloody Clam species were discussed.It has been presumed that there are four common Crassostrea oyster species along the coast of China: Pacific oyster (Crassostrea gigas), Zhe oyster (C.plicacula), Suminoe oyster(C. rivularis) and Dalianwan oyster (C. talienwhanensis). Classification and species identification of these oysters have been difficult because of morphological plasticity. In this article, phylogenetic analysis was performed to clarify taxonomic status of these species using ribosomal Internal Transcribed Spacer regions ITS-1 and ITS-2. Nucleotide sequences of 403-524bp ITS1 and 585-65 Ibp ITS2 after excluding the primer combined sequences and partial sequences at the two end of the fragments.32 haplotypes and 193 polymorphic sites were found in ITS1 sequences of 33 specimens coming from 5 Crassostrea oyster species (including C. virginica as outgroup) , in which 98 polymorphic sites were found in 4 Chinese oyster species. 21 haplotypes and 331 polymorphic sites were found in ITS2 sequences of 21 specimens coming from 5 Crassostrea oyster species (including C. virginica as outgroup), in which 121 polymorphic sites were found in 4 Chinese oyster species. The divergence between C. gigas and C. talienwhanensis was very low, as 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. ariakensis in another one. Our molecular data suggests that C. gigas and C. talienwhanensis may be the same species. However, the lack of divergence between C. plicatula and C, rivularis samples may indicate that the C. plicatula specimens we sampledcould actually be a morph of C. rivularis living in higher salinity habitats. More work is needed for confirmation.It has been presumed that there are several common Crassostrea oyster species and they are difficult to classify and identify clearly because of their morphological plasticity. There exists a lot of dispute especially on classification of C.plicacula. In this paper, phylogenetic analysis was performed to identify these small oyster species from different sites along the coast of China (QingDao, DaLian, NingBo, XiaMen) using ITS1 and ITS2 fragments. 493-510 bp ITS1 sequence and 585bp-651bp ITS2 sequence were amplified and sequenced. 36 haplotypes and 81 polymorphic sites were found in ITS1 sequences; 24 haplotypes and 120 polymorphic sites were found in ITS2 sequences. Phylogenetic analysis showed that haplotypes of QD (QingDao), DL(DaLian), XM(XiaMen) clustered in one clade and those of NB (NingBo) in another one, which showed the small oysters called "Zhe oyster" coming from different geographic sites may be different species. 2 or 3 haplotypes were chosen to do phylogenetic analysis based on the two fragments ITS1 and ITS2 between the small oysters mentioned above, C. gigas,...