Comparative Study On Mitochondrial Genome Between Tibetan And Han Populations, And Tibetans In Three Different Zones: Interrogating Mitochondrial Genes For Signature Of Natural Selection

During the long period of human evolutionary process,it currently leaves a puzzle for what signature of natural selection casts in human genome.Since the appearance of Darwin's Evolution Theory,the intense curiosity and exploring desire possess human beings for their ambiguous past.The limitations underlying knowledge and technique hamper the process of exploring human's mystery.The competition of Human Genome Project and HapMap as well as the maturity of biotechnology in sequencing put the investigation of human evolution from a brand-new standpoint on the agenda.The analysis and explanation on most results of these studies regarding natural selection,however,remain complexity,mainly due to high recombination rate of nuclear genome which can destroy the effects of natural selection.In addition, another puzzle encountered in the investigation of human evolutionary history is to distinguish natural selection from demography,such as the effects of population expansion,population bottlenecks,population structures and so on.Forasmuch, results of most of these studies regarding natural selection remain controversial,with no consensus on their implications.Human mitochondrial DNA(mtDNA),the extra-nuclear genetic material,is characterized by high copy numbers,lack of recombination,high mutation rate and inheritance through purely maternal lines.For example,maternal inheritance can enhance the rapid segregation,expression and adaptive selection of beneficial mtDNA mutation;lack of recombination means that the beneficial mutation can accumulated with the genetic hitchhiking of mtDNA haplotype.Therefore,all these characteristics of mtDNA offer the potentials of investigating human origin and evolution.Previous studies indicated that mtDNA sequences bore strong geographic variation, which was traditionally attributed to the hypothesis of genetic drift.However,the effect of selection on characteristic variation in mtDNA has not yet been proved.The aim in this study by compared between Tibetan and Han populations,and Tibetans in different zones is to detect the signature for natural selection in mitochondrial genome, meaning which and what are the factors for natural selection.In this study,we focused on 40 Tibetans who generationally resided in Tibet and 50 Han Chinese from Beijing to thoroughly decipher and compare the mtDNA whole sequences between the two populations and the mtDNA sequences(～2 kb) of ATP6, ATP8 and Cyt b genes between the three zone Tibetans.The mtDNA sequences were performed using Applied Biosystems 3730 DNA sequencer.Data in mtDNA were analyzed using softwares phredPhrap 16.0,Network,DnaSP 4.20.2 and SPSS 15.0. The SSPro and SSPro8 softwares were used to predict the protein secondary structure; the 3Dpro software was used to calculate the alpha carbon atom coordinate in PDB format,further the MaxSprout was used to fill the coordinates of other atoms,and finally Rasmol and Swiss-pdbviewer softwares were adopted to compare the structure. The protein stability variation caused by single-locus mutation was predicted by aid of SVM algorithm which was developed by Cheng et al.The structure changes of RNA conferred by mutation were simulated using the RNAfold software.Our results showed that all the pooled 90 studied subjects pertained to the Macrohaplogroup M and N,and were classified into 13 haplogroups.No differences were observed among all haplogroups between the two populations except for M9 haplogroup.Principal component analysis indicated that the first and second principal components(PC1 and PC2) respectively accounted for 41.3%and 10.7%of the total variance with the added contribution of 52.0%.Further analysis indicated that the Tibetan and Han populations pitched different quadrants,and PC1 and PC2 respectively accounted for 77.1%and 10.1%of the total variance with a total contribution of 87.2%,suggesting obvious geographic differences.A total of 18 variants were detected by comparing the mtDNA whole sequences between Tibetan and Han populations;of those 5 variants were reported firstly in current study(http://www.mitomap.org) and 8 variants were defined at the internal branch.After predicting the structure changes conferred by the 7 dominated variants in Tibetans,we found that the structures of ND2 G4491A,CO2 G7697A,tRNA alanine T5628C and 12S rRNA A1041G changed significantly after mutation and the stability was increased,meaning the existence of adaptive selection in bioenergetics. Additionally,we constructed the haplotypes of 5 variants harboring D-loop region, and founded that 16145 locus preferred to be combined with the 16255 and 16284 loci, as well as 16234 with 16316.After comparing the replacement of nonsynonumous(N) versus synonymous(S) of 13 peptide-encoded genes among 40 Tibetans,50 Han subjects,144 south Asians and 48 east Asians,we found that the N/S values of the ATP6,ATP8,and Cyt b genes were relatively large(greater than 1) among Tibetans. Significant difference was found for Cyt b gene between Tibetans and south Asians.In addition,we compared the sequences(～2 kb) of three ATP6,ATP8 and Cyt b genes among Tibetans in three different zones and found that the D or F value was negative using Tajima's D test,Fu and Li's D test and F test,which significant difference was found for ATP6 gene by Fu and Li's F test.We further performed N/S (nonsynonymous(N) versus synonymous(S)) substitutions analysis for these three zones and it provides evidence for the existence of adaptive selection in ATP6 gene and the adaptive selection trend was increased with the increase of altitudes,whereas for the existence of purifying selection in Cyt b gene and the purifying selection trend was increased with the decrease of altitudes.The analyses of the mtDNA whole sequences and sequences(～2 kb) of three ATP6, ATP8 and Cyt b genes provide clues for the existence of adaptive selection for ATP6, ATP8,ND2(G4491A),CO2(G7697A),tRNA alanine(T5628c) and 12S rRNA (A1041G) genes in Tibetans.The primary factor of natural selection should be the special geographical environment(e.g.,high-altitude,hypoxia,extreme cold) which this Tibetan locates,namely the different geographical environment has the direct selective action on variation of mtDNA.