Screening Of DNA Makers From Flies Based On Transcriptoe Data

Insects of Diptera and Cyclorrhapha are collectively called flies,which have important economic significance and scientific research value.Because flies are generally small in size?existing a large number of similar species and lacking of effective morphological characteristics,so that it is difficult for some scholars to analyze their relationship.Based on the morphological dilemma and the development of molecular phylogeny,this study was based on the Next-generation transcriptome Sequencing.By comparative transcriptome methods,we identified the homologous genes among species.And finally we screened out a number of single-copy orthologous nuclear DNA sequence markers which are suitable for the study of flies' higher-order phylogeny,and then designed primers to verify them.These single-copy orthologous nuclear DNA sequence markers will attempt to resolve the phylogenetic relationship between the Phroidae and its related groups,which is studing by our team.Based on the Next-generation Sequencing,40803092 raw reads were obtained from the Illumina Hiseq?2500/MiSeq? platform by bidirectional sequencing;35638938 clean reads were obtained from sequence processing;36453 transcripts were obtained from transcription splicing;26079 Unigene transcripts were selected from gene databases widely used for subsequent analysis sequences,and their average length is 949.1bp,the number of sequencs that between 500bp-2000 bp is 9060.Based on the transcriptome data of Megaselia scalaris and the transcriptome data of 9 related groups downloaded by NCBI,a total of 10 species,5 families and 4 genera were selected.A total of 100 homologous genes with 500 bp or more were randomly screened from these transcriptome data.Comparing to the exon ratio,26 single copy orthologous nuclear DNA with high homology and more than 300 bp length were seclected without base bias.After saturation analysis and construction of NJ,MP and ML trees,15 DNA single-copy homologous sequence markers were obtained for the phylogenetic study of higher-order flies at genera.The primers were designed for 44 orthologous nuclear genes genes from 100 homologous regions(including 15 segments suitable for phylogenetic studies of flies)and the efficiency of PCR amplification was validated.19 species of flies(14 Aschiza flies,1 Acalyptratae and 4 Calyptratae of Schizophora)were selected as templates to verify the efficiency of PCR amplification.The amplification efficiency of 5 pairs of primers was higher than71.4% in 44 pairs,and that of 2 pairs was 100%.The results obtained enrich the fly gene database,and it is of great scientific significance in the field of insect phylogeny and evolution to screen out single-copy direct-line homologous DNA sequences suitable for higher order flies at genera.