Complete Mitochondrial Genome Of The Genus Kaburagia And Phylogenetic Relationship

Kaburagia belongs to Insecta,Hemiptera,Aphididae,Eriosomatinae and Fordini,which is a genus of the Rhus gall aphid group,including one species with 4 subspecies.The species finish their life cycle by switching between the primary host-plant(Rhus potaninii and Rhus punjabensis)and the secondary host-plant(moss species).They live on the primary host-plant to form galls,which are rich in tannin and widely used in various area,and have important economic value.The shapes of the galls are variable and were often considered as the classified characteristics to identify the species or subspecies.However,there are the limited reports on its genetic variation and phylogenetic relationship of this genus.Mitochondrial genome is usually a closed circular DNA molecule,and has been used as a good molecular marker to study the phylogeny of the insect species due to its genetic characteristics,e.g.maternal inheritance,fast evolutionary rate,and less gene rearrangement.In this study,the complete mitochondrial genome sequences of 10 individuals from 4 subspecies of the genus Kaburagia was determined using the second-generation high-throughput sequencing technology,and the genomic sequence information and genetic variation were analyzed in detail.Combining the related sequences from the public database Gen Bank),we analyzed the sequence composition and structural variation at the subspecies,species,and genus level of aphids.The phylogenetic trees were constructed to analyze the relationships between different subspecies and their relatives.The main results are as followings:1.The complete mitochondrial genomes of the four subspecies in the genus Kaburagia are a closed circular double-stranded DNA molecule,containing 37 genes with a sequence length of about 16 kb.Kaburagia rhusicola ovatirhuicola(A1137)has a longest length of 16,187 bp,and Kaburagia rhusicola ovogallis(A901)has a shortest length of 16,055 bp.2.From the overall sequence,the AT% content is relatively higher thanGC% up to about 83%,which is a common feature of insect mitochondrial genome.In case of the base composition of each coding region,the protein coding region ND6 of all the three sequences(A1137,A1336,A1529)of Kaburagia rhusicola ovatirhuicola has the highest AT% content(89%)and the highest AT% content of this sequence is the ATP8 coding region for other subspecies.The AT% content of the non-coding control region is also relatively higher.3.The total length of the 13 protein-coding genes in the mitochondrial genomes of the genus Kaburaugia is 10,925 bp,accounting for 67%-68% of the total length,of which AT content accounts for about 81.2%.ATN is used as the start codon and TAA as the stop codon except for COI and ND4,which use a single T as the stop codon.Isoleucine(Ile)is the most common amino acid,following by leucine(Leu),and cysteine(Cys)accounts for the least.Among the three terminators,UAA is most frequently used,and the frequency of occurrence is more than 50%.4.The mitochondrial genome of Kaburagia contains 22 t RNAs with a length between 60bp-70 bp,and the content accounts for about 9% of the whole genome.There are eight t RNA genes(t RNA-Pro,His,Phe,Tyr,Cys,Gln,Val and Leu)located on the light chain(L).Using t RNAscan-SE software,we found that all the t RNAs Can be folded to form a clover structure except for t RNA-Ser1 missing D loop and D stem,and t RNA-Thr missing T?C loop.5.The analysis of the variation of the mitochondrial genome among the subspecies of the genus Kaburagia indicated that the variation between different individuals of the same subspecies was lower than that between species,and the three subspecies(codes A1137,A1336 and A1529)of Kaburagia rhusicola ovatirhuicola had a sequence similarity rate of 99.5%with the variable rate only 0.5%.The variations mainly occur at the presence or absence and the numbers of base A or T,and also there were few base substitutions in some coding regions.The four individuals(codes A901,A910,A1209,and A1536)of Kaburagia rhusicola ovogallis had a similarityrate of 99%,and the variation happened at the individual A1536 which had 2more bases of A than others between t RNA-As and t RNA-Asn,and A1209 missed a certain base sequence in D-Loop region.Samples of unknown subspecies of K.rhusicola.ssp(codes A1070 and A1087)had a similarity rate as high as 99.9% and a variation rate of only 0.1%.The similarity was extremely high and only a few bases were replaced.The similarity rate between different subspecies of the genus Kaburagia was as high as 98.8%,and the variable rate was 1.2%.The variable sites were mainly base deletion in t RNA-Asn,ND4,t RNA-Thr and D-Loop regions,and base substitution in other regions.6.We downloaded the mitochondrial genomic sequence of the genus Kaburagia and its related species from the Gen Bank database,and compared the sequence composition and structural variation.Our analysis showed that the length and content of each region were very similar.In case of the base composition,each region had a similar higher AT% contents than GC%.ATN was used as the start codon for all the protein coding genes and TAA as the stop codon except for COI and ND4 using a single T as the stop codon.Kaburagia,Meitanaphis elongallis and Meitanaphis flavogallis used the same start and stop codon for the same coding gene.7.We constructed the ML and BI phylogenetic tree of the genus Kaburagia and the sister genus Meitanaphis using the genus Floraphis species as the outgroups,and the two methods showed the similar topology.The results supported the monophyly of the genus Kaburagia and the four subspecies(different individuals)grouped together with a high support(BS-ML = 100%,PP-BI = 1.0).However,the relationship between different subspecies needs to choose more samples and markers for further research.Meitanaphis flavogallis has a sister relationship with the genus Kaburagia,and were remote from the two species Meitanaphis elongallis and Meitanaphis microgallis,which indicated that Meitanaphis flavogallis might a new species or a new subspecies of Kaburagia.It is necessary to collect more samples to determine the classification of this taxa.8.Comparative analysis of mitochondrial genomes was performed between the genus Kaburagia and its related species,Rhus gall aphids(Gen Bank).By synteny analysis,we found that all their genes had the similar length and the gene orders were consistent and no rearrangement occurred among all the subspecies of the genus.In summary,this study obtained the complete mitochondrial genomes of the genus Kaburagia by the high-throughput sequencing technology.We analyzed the sequence composition and structures,and constructed the phylogeny of the genus subspecies and the sister genus to analyze their relative relationship.Our results will afford more molecular information for the evolutionary analysis of aphids and development of this natural resource.