Identification Technology And Evaluation Of Gene Barcodes Of Some Endangered Species Of Dicroglossidae And Luehdorfia Chinensis

The wildlife resources of frogs in China are rich,and many species have economic value for edible and medicinal purposes after artificial breeding.However,due to the presence of imbalance between the expansion of market demand and the existing scale of farming,the lack of natural breeding,people's excessive hunting,some species turn into the endangered status.On the other hand,the huge supply gap drives illegal traders to sell counterfeits in the market,making it difficult to distinguish the authenticity of such resources in the actual trade.More than 99%of the so-called Quasipaa spinosa sold on the market at present are Quasipaa boulengeri,which have very low medicinal value.Luehdorfia chinensis,a rare butterfly endemic to China,is now listed as endangered due to the reduction of its habitat and environmental disruption.Therefore,it is urgent to accurately identify and protect this species.The DNA Barcode technology is used to identify species at the gene level with high accuracy.The present study aimed to evaluate the applicability of using COI and 16s rRNA genes as the barcodes in the identification of Dicroglossidae species such as Q.spinosa,Q.boulengeri,Hoplobatrachus rugulosus,and the endangered species L.chinensis,and verify the effectiveness of DNA barcodes and their analysis methods in related species identification by genetic distance analysis and phylogenetic analysis.The main research results obtained are as follows:1.Application of COI and 16S rRNA gene sequences as DNA barcodes in the identification of some endangered species of DicroglossidaeIn this study,DNA were extracted from muscle tissues of a total of 17 individuals from 3 species of frogs,and each of them was used as template to obtain COI and 16S rRNA gene fragments,COI sequence is 658bp,16S rRNA sequence is 555bp.All these fragments were sequenced and used for further analysis.The utility of these two genes as DNA barcodes in identification those species was evaluated from genetic distance analysis,gene barcode gap analysis and systematic cluster analysis.The results showed that the genetic distance between species within the same genus was significantly greater than the genetic distance within species,and a clear gap is formed between the two.The interspecies genetic distance(0.1681-0.2929)of the three frogs based on the COI gene was significantly greater than the intraspecies genetic distance(0.0005-0.0157).The interspecies genetic distance(0.0450-0.2232)of the three frogs based on the 16S rRNA gene was obviously greater than the genetic distance within the species(0.0005-0.0198).Each of the different species of frogs in the NJ tree and the ML tree formed a monophyletic group.The identification functions of the COI and 16S rRNA genes at the species and genus levels are consistent with traditional taxonomic identification methods,indicating that these two genes has appropriate application value in the identification of frog species above mentioned.2.Application of COI and 16S rRNA gene as gene barcode in the identification of endangered species Luehdorfia chinensisIn this study,DNA was extracted from the muscle tissue of three L.chinensis,and primers for the COI gene were designed by using Primer Premier 5.0.The COI and 16S rRNA gene sequences of L.chinensis were amplified and sequenced.The effectiveness of the obtained sequence fragments as DNA barcodes was evaluated by genetic distance analysis,gene barcode gap analysis and systematic cluster analysis.The results show that the genetic distance between species within the same genus is significantly greater than the genetic distance within species,and a clear gap is formed between the two.Among them,the genetic distance between species based on the COI gene(0.0569-0.1533)is significantly larger than the genetic distance within species(0.0022-0.0061).The length of the COI sequence amplified by a pair of primers HFD3F/HFD3R is 326bp,and the genetic distance between species is(0.0571-0.1533),which is significantly greater than the intra-species genetic distance(0.0014-0.0126);based on the 16S rRNA gene interspecies genetic distance(0.0883-0.1606),which is significantly greater than the intra-species genetic distance(0.0021-0.0123).In systematic clustering analysis,by constructing NJ tree and ML tree,L.chinensis form a monophyletic group,and other butterflies of different genus form monophyletic groups too,indicating that the length of COI sequence of 658bp,326bp and 555bp of 16S rRNA sequence as gene barcodes have good application value in identifying L.chinensis and its incomplete specimens.In this study,genetic distance analysis,gene barcode gap analysis,and systematic cluster analysis were used to analyze the barcode applicability of the COI and 16S rRNA genes of 3 samples of 17 frog species and 3 samples of L.chinensis.The results show that the obtained COI and 16S rRNA gene sequences can be used as DNA barcodes.Save and upload 20 COI and 16S rRNA sequences to the endangered species gene barcode database(http://47.102.211.219/data/all/)and generate barcodes.