Screening,verification And Application Of Shellfish Barcode

There are many kinds of shellfish,with a wide range of distribution and a variety of habitat environment,which brings difficulty to the traditional morphological classification.DNA barcoding has lots of advantages,including high accuracy,efficient classification and others.Thus,it has become one of the areas of rapid development of molecular biology.DNA barcoding has been proposed as a method that will make species identification faster and more accessible using a small fragment of DNA sequence,particularly in species with complex accessible morphology,and has been successfully used in animals,plants and microorganism.At present,the DNA bar code commonly used in shellfish classification are mainly mitochondrial genes,nuclear genes and ribosomal genes.The most widely concerned is mitochondrial DNA,which has the characteristics of maturation and evolution rate of maternal gene compared with nuclear gene,mainly COI,12 S,16S gene part of the sequence.Of course,ribosomal 18 S and 28 S and other nuclear genes can also provide accessibility.Firstly,the COI,16 S,18S and 28 S were screened as DNA barcode,and the corresponding sequences of bivalvia and gastropoda were downloaded from NCBI,and the genetic diversity was analyzed.Based on the sequence analysis of COI gene,it was found that the differences among the families were between 30% and 70%,and the differences among the genera were between 20% and 50%,and intraspecific genetic distance were between 0.0% and 7.5%.COI gene can be divided into different families,different genera,and even the same species.Indicating that COI gene can be used as DNA barcode.Based on the 16 S gene,we found that the families difference was between 35% and 70%,and the genera difference was between 10% and 45%.The intraspecific difference was between 0.2% and 1.5%.As with the COI gene,16 S could also be used as a DNA barcode to identify the classification.In the 18 S and 28 S gene partial sequence,the inter-family differences were concentrated in 10%-50%,inter-genera difference of 5%-15%,and the difference was basically zero.It can be seen that the 18 S and 28 S genes have no advantage in distinguishing the dominant species and are only suitable for the molecular markers of high classification.In this paper,first of all,DNA of various shellfish from Qingdao,Jiaonan,Yantai,Dalian,Hainan,Xiamen and Ningbo were extracted and PCR was carried out using universal primers COI and 16 S to obtain partial sequences of COI and 16 S genes.The genetic diversity of this sequence was analyzed and the results were as follows:A total of 74 COI sequences(578bp in length)were amplified,including the same species from different geographic populations.The average interspecific genetic distance was 0.958,and the genetic distance between species was greater than that of intraspecific species.From the COI phylogenetic tree,it can be seen that the two species are divided into two distinct branches.The same species are clustered together,and the same species of different geographical groups are clearly together to distinguish the species in this study.A total of 686 different shellfish 16 S sequences were obtained.After cutting,the length was 421 bp,and the average interspecific genetic distance was 0.633.With the results of the COI sequence,the 16 S phylogenetic tree can also be distinguished from most of the individuals in this study,although it is divided into two distinct ones,but Bursa rana and Unio douglasiae)together.And can not distinguish between Solen lamarckii and Meretrix meretrix,indicating that 16 S in the species identification still have some limitations.The results showed that both COI and 16 S could be used as DNA barcodes to distinguish and identify species.Finally,general PCR technology was used for amplification of the mitochondrial COI ? 12 S rRNA and ribosome 18 S rRNA and 28 S rRNA partial sequences to analyze five groups(Jiaonan,Rongcheng,Changdao,Penglai and Hanguo)of S.broughtonii.The 67 sequences of COI,73 sequences of 12 S,75 sequences of 18 S rRNA and 75 sequences of 28 S rRNA from different populations were determined and analyzed.The sequences with length of 776 bp,443bp,909 bp and 894 bp nucleotide of COI,12 S,18S and 28 S were respectively obtained.The results showed that though the genetic variation of COI ? 12 S ? 18 S and 28 S was relatively high,the base composition of them was stable.The genetic diversity of the five populations was rich,which indicated that the germplasm resource of the species was in good condition.Based on partial 18 S gene,a total of 74 haploid type were detected among them.And analysis of the Korea population of Scapharca broughtonii,the total variation loci,average nucleotide differences index,nucleotide diversity index Pi,were 488.69.724 and 0.080 respectively.The genetic diversity of this population was the richest.Also,the number of variation sites of 18 S were the largest in Jiaonan population.As for partial 28 S gene,a total of 18 haploid type were detected among 75 sequences,and Penglai populations has the richest gene diversity.