Comparative With The Ribosomal Repeat Unit Of Five Veneridae Species And Their Phylogeny

The family Veneridae has the most species in the Veneroida,including many economic species.The research on economic species was focused on the breeding technology and the influence of environmental factors.Long-term artificial breeding has resulted in germplasm,poor stress resistance and genetic diversity lossing.The molecular biology technology study can provide theoretical basis for the genetic breeding,germplasm resources protecting and genetic diversity analysing.And it can also provide molecular basis for solving the disputes existing in the Veneridae species classification.The ribosomal gene belongs to nuclear gene and contains parental inherited information.Different regions of ribosomal repeat unit have different evolution rate,which can be used for phylogenetic analysis at different levels.In this study,five Veneridae species were chosen,including four economic species,collected form weihai,as Ruditapes philippinarum,Meretrix meretrix,Staxidomus purpuratus,Mercenaria mercenaria.and Austrovenus stutchburyi which was introduced from New Zealand.By comparision with the ribosomal repeat unit of five clams,could illustrate the characteristics of nrDNA,and reveal the phylogenetic relationship.In this study,the complete ribosomal repeat unit,contains 18S rDNA-ITS(ITS1-5.8S rDNA-ITS2)-28S rDNA-IGS(just including the partial IGS sequence).The sizes of a ribosomal repeat unit of R.philippinarum,M.meretrix,S.purpuratus and M.mercenaria anda A.stutchburyi were 7510 bp,13799 bp,7169 bp,10132 bp and 11484 bp,respectively.The length of the 18S rDNA sequence of them were 1896 bp,1824 bp,1828 bp,1830 bp and 1917 bp,respectively,and the sequence identity varied from 67.84%to 98.53%,and the genetic distance varied from 0.007 to 0.300.Two mutation surge zones are found by sequences comparison.The lengths of the complete ITS sequences of them were 1172 bp,1451 bp,1211 bp.1197 bp and 1020 bp,respectively.The lengths of ITS1 were between 553 and 878 bp,the sequence identity was 50.35%,while that of ITS2 varied from 242 bp to 416 bp,and sequence identity was 51.31%.The sequence identity of ITS sequences varied from 41.59%to 60.26%,the genetic distance ranged from 0.3798 to 0.5895.There were 928 mutation sites and 234 simple information sites,accounting for 62.3%and 15.7%of all information sites,respectively.The length of the 28S rDNA sequences were 3800 bp,3606 bp,3700 bp,3780 bp and 3738 bp,the sequence identity varied from 84.80%to 96.53%.The genetic distance was between 0.024 and 0.094.There are nine mutation surge zones,the longest one is 80bp.The 18S rDNA,ITS and 28S rDNA were used as molecular markers to analyze the phylogenetic analysis of Veneridae.The classification of R.philippinarum,M.meretrix and S.purpuratus are consistent with the current classification system,while M.mercenaria,belonging to the Venerinae,clustered with the Dosiniinae firstly,and then with A.stutchburyi.The study results supported the views of the Meretricinae stems from the Callistinae.The partial IGS sequences of five clams were obtained by PCR amplification and high-throughput sequencing.The length of them were ranged from 442 bp to 6560 bp.According to the transcriptional starter and transcription terminator position,the putative 3'ETS region of M.meretrix,M.mercenaria and A.stutchburyi were 56 bp,145 bp and 195 bp,respectively;the putative 5'ETS region of R philippinarum,M.meretrix and M.mercenaria were 571 bp,669 bp and 478 bp,respectively.No repeats segments were detected in ETS region,but a number of repeats were found in the NTS sequences.However there were no obvious correlation between repeat segment and repeat number.In addition to M.meretrix,CpG islands were found in the 5'ETS region of the other four clams.The ML tree and the NJ tree were constructed based on the 5'ETS sequences,the topology of them were not totally same,in which M.mercenaria and A.stutchburyi both clustered first,which is the same as the ML and NJ trees constructed by ITS.So the 5'ETS could be a potential molecular marker used to study the phylogenetic relationship among closely related species.