NUMTs And Mitophylogenomics Of Passerines

There are over 10,000 species of birds of the world,which involved 40 orders,247 families and 2312 genus,and more than 60% of them belong to the passerines clade.Aves,as one large taxonomic group of vertebrates,many of these traditional groups are polyphyletic,and that morphologically similar ecotypes have evolved independently in different parts of the world.Birds are closely linked to the survival and development of humans,which also plays a big part in maintaining ecological balance.Passeriformes was the largest family in the Aves,but its origination and phylogenetic relationship were still not clear,which owing to the majority of these species experienced a rapid evolution in Paleogene.Since the origin and evolution of mitochondria throughout the eukaryotic origin and evolutionary process,mitochondrial genomes have proved to be powerful tools in resolving phylogenetic relationships as they show the characteristics of maternal inheritance and high mutation rate as well.In this study,we sequenced eight complete mitochondrial genomes of Passeriformes by Sanger sequencing technology and five individuals' complete mitochondrial genomes from Urocissa erythrorhyncha based on Illumina High-Throughput Sequencing.In addition,we sequenced the genome of six passerines birds and one Psittaciformes by Next-generation high-throughput DNA sequencing techniques.Based on the analysis of the sequenced transcriptome data in our laboratory and the relevant public data in NCBI,four topics were discussed in this paper:(1)the structural features of passerines mitochondrial genome;(2)the detection,validation and analysis of the passerines Nuclear Mitochondrial pseudogene(Numts);(3)the research on mitophylogenetics of Passriformes;(4)the research on the types of mitochondrial genome rearrangement and phylogenesis of control region(CR).The main research results are as follows:1.The structural features of the passerines mitochondrial genomePCR amplification and Sanger sequencing technology were used to gain the mitogenome.And in this study,eight mitogenome were sequenced to discuss the structural features.Based on sequences analysis,eight mitogenome were all identified as circular DNA,containing 13 protein-coding genes,2 rRNAs,22 tRNAs and ranging from 16,765~17,936 bp.Apart from Aegithalos glaucogularis,Alcippe morrisonia and Melanocorypha mongolica,who owned two mitochondria control regions(CRs),the rest of all had only one CR.According to naming convention of Gibb,types of the mitogenome rearrangement of A.caudatus and A.morrisonia belong to d_CR,and the rearrangement of M.mongolica belongs to r_CR2,and the rest of all belong to “Ancestral avian”.2.Construction of a cDNA library,transcriptome sequencing and analysisIn this study,the transcriptome of 5 individuals of U.rythrorhyncha were sequenced,which were chosen as a representative species to retrive the Numts and to research the heterogeneity of mitogenome.Take the case of transcriptome data of K0739,one sample we named,234,805 transcripts were produced with the total GC content being 47.89% and N50 being 3,343 bp by Trinity.In this research,we obtained 5 different individuals' mitogenome of U.rythrorhyncha from transcriptome data by utilizing bioinformatics methods,and further analysing their heterogeneity.The results showed that the heterogeneity was under 1%(Var=0.45%),thus we suggested these heterogeneity sites did not affect avian phylogenetic analysis.3.Construction of a genomic library and genomic sequencingTo research the mitophylogenetics of Passeriformes and verify the NUMT extracted from the transcriptome at the genome level,the species of U.rythrorhyncha,Garrulax canorus,Passer montanus,Leiothrix lutea,Spodiopsar cineraceus,S.sericeus,and Agapornis fischeri,which acted as the outgroup,were selected for genome sequencing.Take the genome of S.sericeus for example,1,129,900 Scaffolds with GC content being 41.96% and N50 being 2,035 bp were generated by SOAP denove2.4.The detection,validation and analysis of the Passerines NumtsBLASTn was utilized to hunt for Numts in transcriptome data of 5 passerines,and the candidate Numts were checked in the relevant genomic data.Ultimately,25 Numts with transcriptional activity was discovered in this study.It was suggested that the Numts detected with omics approaches could be verified in genome by PCR and Sanger sequencing randomly;the results of RT-PCR provided the strong evidence that the Numts had transcriptional activity;we suggested that the Numt of passerines were mainly located in the chromosomes with a lower gene density by localization analysis;the analysis of the flanking sequences also revealed a high content of AT with mass insertion sequence at both flanks of Numts.This is the first time to detect the Numts of passerines at the level of genome and transcriptome.5.The mitophylogenetics of PasseriformesEight mitogenomes of passerines were amplificated and then sequenced.The six mitogenomes of passerines and one Psittaciformes birds were retrived from high-throughput sequencing data.And then the mitogenome and some related public data from NCBI were used to infer the mitophylogenomics of passerines.Finally,the total of 260 Passerines species(the inner group)belonging to 57 families were used to reconstruct ML tree with IQ-tree and RAxML software,and bayesian tree with Mrbayes.The results were as follows:(1)Passeriformes is supported to be divided into Oscines,Suboscines and New Zealand wren(Acanthisittidae),and the Acanthisittidae should be an independent group at the basal position of Passeriformes;(2)Corvida is proposed to be a paraphyletic group,basal Corvida is the basal phylogenetic position of Oscines,and crown Corvida is monophyletic group;(3)Garrulax perspicillatus is placed within the genus Ianthocincla,which was belonged to Garrulax,suggesting that these two genera were monophyletic group;(4)Suboscines could be divided into two monophyletic groups according to geographical distribution: the New world Suboscines and Old world Suboscines;(5)Pseudopodoces humilis is sister to Phoenicurus auroreus,both of which are located in Turdidae,and we suggested that P.humilis belonged to genus Phoenicurus of Turdidae;(6)Muscicapoidea is proposed to be a paraphyletic group;(7)The taxonomical status of Callaeidae,Notiomystidae,Melanocharitidae,Petroicidae,Mohoidae,Regulidae,Hyliotidae,Paridae and Remizidae is still unclear.6.The types of mitochondrial genome rearrangement and phylogenetics of CRBased on the results of mitogenome rearrangement and the characteristics of CRs of 260 passerines,66 species have duplicated control regions.There are 7 Suboscines species and 5 species from Petroica(Petroicidae)owned to the type of r_CR2;Notiomystis cincta and Turdus philomelos in Oscines are the type of d_tPro-CR;and the 51 species within Sylvioidea made up for 77.27% of the non-Ancestral avain were all d_CR or r_CR2;the rest of all the species belong to Ancestral avain.The clustering results showed that the CR1 and CR2 in all types of d_CR clustered in one clade by using conserved sequence of CR to analyze the phylogenesis of Sylvioidea,which indicated that the duplication of the mtCR had occured before speciation.In this study,the complete mitochondrial genomes of some passerines,which were sequenced by PCR,Sanger sequencing,and high-throughput sequencing at this time,and some related public data from NCBI were utilized to construct the phylogenetic tree in multiple ways to study the mitophylogenetics of passerines.There were as many as 260 passerines in this analysis,the results will contribute to deal with the debate of the internal phylogenetics of Passeriformes.Moreover,the results of the types of mitochondrial genome rearrangement associated with the phylogenetic relationship of Passeriformes provided more proof of the theory that the types of mitochondrial genome rearrangement originated independently many times,which also indicated that the types of mitochondrial genome rearrangement and CR had a great significance in phylogenesis.In addition,it is the first time to detect the Numts of passerines at genomics level,which will also provide significant theoretical value for futher studying the molecular mechanism,sequence features and evolution characteristics of avian Numts formation.