Rapid Identification Of Cicadellidae In Southern Foothills Of Pamirs Plateau In China Based On DNA Barcodes

There are many species of Cicadellidae,some species are the transmission vectors of plant virus diseases,such as Wheat blue dwarf disease,Jujube witches’ broom disease,Maize wallabyear disease and Rice stripe mosaic virus,which seriously injured the development of agriculture and forestry industry.But it is difficult to defense effectively.Because of its small size and similar morphology,it is hard to identify it’s species;the traditional classification methods are difficult to rapidly and accurately identify the eggs,nymphs of Cicadellidae.DNA barcode technologyemerged as the times require because of it’s conveniece and efficiency,which could improve the identification speed of Cicadellidae and lay a foundation for effective defence of Cicadellidae.Pamir Plateau situates in the southeast of Central Asia,southwest of Xinjiang in China,and parts of Kashgar and Kizilsu are located at the south foot of Pamir Plateau,borders or adjoins to 8 countries.With the development of the Belt and Road,the trade flows are becoming more frequent,and the risk of prevention and control of pests in agriculture and forestry is increasing.However,there are few studies on insects in the region,and there are few reports on the barcodes of Cicadellidae.Therefore,it is very important to carry out rapid identification of Cicadellidae to provide basis for effective detection and prevention.In this study,we ascertained the species of unknown Cicadellidae by morphological classification firstly,and then used DNA barcoding technology,selected mtDNA COI,Cytb and ITS2 genes,amplified Cicadellidae’s sequences by Universal PCR,compared and analyzed the similarity of target gene sequences with the help of bioinformatics software,constructed phylogenetic tree with Gen Bank data,computed genetic distance and gained DNA barcodes.The rapid identification of different kinds of Cicadellidae came true,whicih will enrich the gene database of Cicadellidae in border areas and provide basis for effective prevention and treatment.The results are as follows:1.28 samples were identified by morphological characteristics,and 28 samples belonged to 7 subfamilies,14 genera and 15 species of Cicadellidaes.2.66 mtDNA sequences of Cicadellidae were obtained,including 26 COI genes,24 Cytb genes and 16 ITS2 genesof this study.The meancontent ratio of a A + T in COI gene was 68.9%in 28 samples,the mean content of A + T about Cytb gene in 12 samples was 71.4%,the mean scale of A + T content about COI and Cytb genes was higher than that of G + C,and there was A/T bias,which was consistent with the composition of insect mitochondrial gene in insects.The results indicated that the COI and Cytb gene sequences can be detected and analyzed at the molecular level,which can be used as DNA barcodes of Cicadellidae.3.39 new sequences were obtained in this study,there were many new sequences of Cytb and ITS2 genes,and few new sequences of COI genes.Among them,8 new COI sequences,16 Cytb and 15 ITS2 sequences were reported for the first time in this region.4.In this study,28 samples were identified by sequence similarity comparison,phylogenetic tree and genetic distance analysis.The highest similarity of mtDNA COI,Cytb and ITS2 genes in Gen Bank were 100%,99.78%,99.81% separately,whose highest similarity all related to Cicadellidae.The structure of the COI and Cytb gene molecular phylogenetic tree constructed by the two methods of NJ and ME was consistent basically.The phylogenetic tree of 15 species,14 genuses and 7 subfamilies in this study was clearly located and classified.The subfamilies,genus,and species formed their own independent monophyletic lines among the taxonomic stages.The average genetic distance of COI gene between interspecific and intraspecific was 0.197 and 0.0021 respectively,that was 0.205 and 0.0051 of Cytb gene respectively,the meaninterspecific genetic distance was greater than 2%,the meanintraspecific genetic distance was smaller than 1%,polymorphic sites genetic distance showed that there was significant genetic differentiation in the southern piedmont of Pamir Plateau.The results of genetic distance based on COI and Cytb gene were consistent with those of morphological identification,that distinguished 15 species of Cicadellidae.The results indicated that there were 15 species of Cicadellidae,such as Psammotettix confinis,cicadella viridis,singapora shinshana,hishimonoides recurvatis,macropsis notate and hecalus sp.5.The nymphs of Psammotettix confinis,cicadella viridisand hecalus sp.have been experimented by PCR based on mtDNACOI,Cytb and ITS2 gene sequences.The COI,cytb and ITS2 gene sequences of the 3 nymphs were acquired,and the nymphs of the three species were identified successfully.Previous studies on the nymphs of Cicadellidae are rare,and DNA barcodes of three species of nymphs have not been recorded in the literature.6.Sample L was identified as tiaobeinia sp.by morphological characteristics which is not same with the existing known Cicadellidae of mukarinini and it’s highest similarity with COI sequence of Zygina ordinaria was 94.06% in database,which dose not reached the identification standard is judged as a new species.