Construction and application of DNA barcoding in sea cucumber Holothurians

Abstract

DNA barcoding is a diagnostic technique in which short DNA sequences can be used for species identi-fication.The mitochondrial cytochrome c oxidase subunit I(CO I) sequence is used in DNA barcoding for species identification in many organisms.In this paper,the validation of the COI gene for DNA barcoding and species identification in sea cucumbers was studied.The CO I genes from seven species of sea cucumbers were amplified and sequenced.Data were analyzed using DNAstar,DNAMAN,and MEGA 4.1.The DNA base composition was analyzed,and the genetic variations among species and within species were calculated.It was found that the ge-netic distance among the seven species was higher than that within a species.Phylogenetic analysis using neighbor-joining and maximum parsimony methods generated tree topologies that indicated that all individuals of each species formed a strong monophyletic group.These results suggested that the mitochondrial CO I gene was a valid DNA barcoding gene for species identification in sea cucumbers.On the basis of the DNA barcoding,spe-cies-specific probes of Apostichopus japonicus were designed.Dot-blot hybridization showed that the probes could identify A.japonicus with high specificity and sensitivity.The design of further probes for different sea cu-cumber species,combined with dot-blot hybridization and microchip assays,will facilitate the identification of sea cucumbers at relatively low costs.