Molecular Identification Of Some Species Of Sclerotidae And Scarabaeidae Based On CO Ⅰ And 28S RRNA Gene

Both Sclerotidae and Scarabaeidae are important pests in agricultural and forestry in China.Traditional morphological classification and identification are difficult to identify larvae,broken insects or similar species.Therefore,it is necessary to explore new techniques and methods to compensate for traditional morphology.Five kinds of methods were used to preserve Ips subelongatus.The kit method and the improved CTAB method were used for DNA extraction to explore the best DNA extraction methods and preservation methods.In order to solve the problem of difficult identification of larvae and adult residues of Sclerotidae and Scarabaeidae in production practice,13 species of Sclerotidae from 9 genera collected from Hebei,Heilongjiang and Shanghai,and 24 species of Scarabaeidae collected from 6 subfamilies from Anhui,Hebei,Hubei and Zhejiang were used as the research objects in this study.At the same time,with gene sequence analysis,the differences between different individuals in the same subfamily or genus of Sclerotidae and Scarabaeidae were discussed to determine their genetic relationship and evolutionary status,which provided theoretical basis for the study of phylogenetic relationship.The main research results are as follows:1.The total genomic DNA of Ips subelongatus with five preservation methods was extracted through the kit method and the improved CTAB method.The results of spectrophotometric determination of the total DNA and PCR amplification of the CO Ⅰ gene showed that:the difference of DNA concentration between the body preserved in 75%alcohol and dried was small.While the body stored in absolute ethanol and ultra-low temperature refrigerator,higher concentration DNA could be extracted through the modified CTAB method and the subsequent primer test results were better also.So the improved CTAB method was used for subsequent study.High-quality DNA can be extracted from the two storage methods of ultra-low temperature refrigerator and absolute ethanol.However,considering the portability for field samples,absolute ethanol was more suitable.2.Used CO I and 28S rRNA as the target genes,the gene sequence analysis of 13 species of Sclerotidae from 9 genera in Hebei,Heilongjiang and Shanghai showed that the length of the CO I gene fragment(without primers)was 651 bp,270 conservative sites(C)and 377 variant sites(V).The content of A+T(62.2%)was significantly higher than that of G+C(37.8%).The A+T base preference was obvious,which was consistent with the characteristics of insect and animal mitochondrial base preference.The transition of all sites mainly occured between C and T,the transversion mainly occured between A and T,the TS/TV(R value)was 0.8,and the total number of substitutions was 194 times,which mainly occured at the third position.The content of Leu,Ile and Gly in CO Ⅰ gene encoding protein amino acid was higher,and the frequency of UUA of codon encoding Leu was the highest.The length of 28S rRNA gene fragment(without primers)was 633 bp,317 conservative sites(C)and 216 variant sites(V).The content of G+C(50.8%)was significantly higher than that of A+T(49.2%).The G+C base preference was obvious,which was consistent with the characteristics of insect and animal mitochondrial base preference.The transition of all sites mainly occured between A and G,and the transversion mainly occured between T and A,the TS/TV(R value)was 1.1.The total number of replacements was 106 times,which mainly occured at the second position.The content of Ser,Arg and Leu in 28S rRNA gene encoding protein amino acids was higher,and the the frequency of CUU codon encoding Leu was the highest.The standard deviations of genetic distances between the two gene samples were both less than 0.050,and the differences were significant;the average genetic distance between the two groups in the northern and southern regions(Shanghai/Hebei,Heilongjiang)was greater than the average genetic distance within the two regions,indicating that there were geographic population differences.The phylogenetic tree constructed by CO Ⅰ and 28S rRNA gene was divided into two major branches in North and South regions,but 28S rRNA gene can distinguish the outer groups more clearly.3.Used CO Ⅰ and 28S rRNA as the target genes,the gene sequence analysis of 24 species of Scarabaeidae in 6 subfamilies collected from Anhui,Hebei,Hubei and Zhejiang showed that the length of the CO Ⅰ gene fragment(without primers)was 658 bp,311 sites(C)and 3 13 variant sites(V).The content of A+T(58.5%)was significantly higher than that of G+C(41.5%).The A+T base preference was obvious,which was consistent with the characteristics of insect and animal mitochondrial base preference.The transition of all sites mainly occured between C and T,the transversion mainly occured between A and T,the TS/TV(R value)was 0.9,and the total number of substitutions was 138 times,mainly occurring at the first position.The content of Ser,Leu and Pro in the amino acid of CO Ⅰgene encoding protein was higher,and the codon AGC encoding Ser had the highest frequency.The length of 28S rRNA gene fragment(without primers)of was 600 bp in length,286 conservative sites(C)and 248 variant sites(V).The content of G+C(53.4%)was significantly higher than that of A+T(46.6%).The G+C base preference was obvious,which was consistent with the characteristics of insect and animal mitochondrial base preference.The transition of all sites mainly occured between A and G,and the transversion mainly occured between T and C.The TS/TV(R value)was 1.2.The total number of substitutions was 94 times,which mainly occured at the first position.The 28S rRNA gene encoded a protein with a higher content of Arg,Ser and Leu,and the codon CUU encoding Leu has the highest frequency.The average genetic distances showed that the CO I gene of the Rutelinae and the Dynastidae were greater than 28S rRNA gene and the average genetic distances within the Cetoniinae and the Melolonthidae were consistent.Among them,the genetic distance between the Dynastidae and the Morphology was the smallest,and the maximum was between the Melolonthidae and the Morphology.The phylogenetic tree constructed by CO I and 28S rRNA genes was divided into two branches:Rutelinae and Morphology.Dynastidae,Melolonthidae and Cetoniinae etc were among them according to distance.4.Through the data joint analysis of mitochondrial CO Ⅰ and nuclear 28S rRNA gene,the results showed that molecular biology methods could be used to classify Scolytidae and Scarabaeidae.The differences within and between the 13 species of Scolytidae and 23 species of Scarabaeidae reflected by the nuclear gene 28S rRNA were smaller than the differences reflected by mitochondrial CO Ⅰ gene.It showed that CO Ⅰ gene evolved faster than 28SrRNA gene,indicating that CO Ⅰ gene was more suitable for classification of similar species and related species,and 28S rRNA gene was more suitable for classification for family level and above.