Research On Identification Of Main Species And Phylogenetic Relationship Of Spider Mite Genus Tetranychus Based On Morphological And Molecular Evidence In Gansu Province

Tetranychus belongs to Arachnida,Acari,Acariformes,Tetranychidae.It is an important agricultural pest that seriously harms food crops,cash crops,ornamental plants and trees all over the world.Tetranychus are very small and have many species,and their morphological characteristics are very similar among different species.In general,the shape of the external genitalia is the most effective morphological features for identifying mites.However,the morphological identification of mites has certain requirements for insect state,which requires the development of adults for the identification.The number of male mites is small,the identification takes a long time,and some species have little difference in morphological characteristics.All these limitations have brought great difficulties for the morphological characteristics of mites.In order to solve the difficulty of morphological identification,molecular biology technology is increasingly used in species identification research.In this paper,the morphological identification of mites collected from different areas of gansu province was carried out by using traditional morphological characteristics,the ITS1 and COI sequences were sequenced and analyzed to explore a method for the identification of mite species combining morphological and molecular evidence.At the same time,it is clear whether ITS1 and COI are effective molecular markers for studying the occurrence of spider mites.The main results are as follows: 1.Morphological identification of spider mites in Gansu Province.Identify 27 population of spider mites in different regions of Gansu Province by morphological methods,which were obtained 5 populations,such as Tetranychus truncatus,Tetranychus urticae,Tetranychus viennensis,Tetranychus kanzawai and Tetranychus cinnabarinus.2.Nucleotide sequence analysis of rDNA ITS1 in spider mites populations in Gansu Province.27 nucleotide sequences with a length of 596-688 bp were obtained from the spider mites populations in Gansu Province.Then analysis the composition of sequence and showed that the average contents of the bases A,T,C and G were 32.5%,31.0%,18.6% and 17.9%,respectively.The content of A+T(63.5%)was significantly higher than that of C+G(36.5%),with an obvious A/T bias.The diversity of nucleotide was 0.128.A total of 9 Haplotypes were detected and the haplotype index was 0.775.By calculating the genetic distance,the difference of nucleotides between genera was greater than that between species,and the difference between species was greater than that between species.The genetic distance within the genus Tetranychus ranged from 0.000 to 0.384,and the average genetic distance within species was less than 0.002,meeting the standard that the difference of nucleotides within species was less than 2%.3.Nucleotide sequence analysis of mtDNA COI in spider mites populations in Gansu Province.27 nucleotide sequences with a length of 422-447 bp were obtained from the spider mites populations in Gansu Province.Then analysis the composition of sequence and showed that the average contents of the bases A,T,C and G were 31.6%,43.9%,10.5% and 14.0%,respectively.The content of A+T(75.5%)was significantly higher than that of C+G(24.5%),with an obvious A/T bias.The diversity of nucleotide was 0.071.A total of 10 Haplotypes were detected and the haplotype index was 0.889.The average genetic distance of the five populations of T.urticae was 0.019.The genetic distance between T.cinnabarinus and T.urticae was only 0.005,The genetic distance between T.truncatus and P.citri is the largest,reach up to 0.131.Therefore,the nucleotide difference of ITS1 was was more significant than that of COI.4.Identification and Phylogenetic of spider mites Populations in Gansu Province Used ITS1 and COI Molecular MarkersAfter Blast alignment of NCBI site,the sequencing results confirmed the five species of spider mites identified by morphology method.The phylogenetic analysis of spider mites used ITS1 and COI as molecular markers showed that the genetic relationship of four populations identified by morphology method.The T.kanzawai had the highest genetic relationship with the T.truncatus,and the T.viennensis had the lowest genetic relationship with other Tetranychus species.The analysis tends to support the view that the T.truncatus and the T.cinnabarinus was the same kind but different color type,but the occurrence status of the T.kanzawai is not well established.5.Phylogenetic of spider mites in Gansu Province based on the combined data of ITS1 and COI sequences.The combined data of ITS1 and COI sequences of the spider mites in Gansu were analyzed.The average contents of bases A,T,C and G were 32.1%,36.5%,15.2% and 16.2%,respectively.The content of A+T(68.6%)was significantly higher than that of C+G,showing AT bias.The positions of the bases in the coding amino acid codon were significantly different,among which the content of A+T at the third site of the codon was the highest(76.1%).The genetic distance between 27 spider mites populations ranged from 0.000 to 0.356.The average genetic distance was 0.132.Among the species,the largest difference in nucleotide was 0.356 between the P.citri populations of Lanzhou and the T.truncatus populations of Dingxi,while the smallest difference was 0.006 between the T.kanzawai populations of Lanzhou and the T.urticae populations of Lanzhou.In summary,ITS1 and COI can be used as effective molecular markers for the study of spider mites identification or Phylogenetic.Combinated with the morphological and the evidences of molecular to dentificate species and clear phylogenetic relationship of spider mites,aim to provide the corresponding basis and reference for the study on the species identification and phylogenetic of the spider mites.