Genetic Diversity Of Tupaia Belangeri Based On Cytb And MHC DRB Genes

Genetic diversity(Genetic diversity)refers to the sum of biological genetic information.The genetic variation is usually studied among individuals in different species or in different populations.C ytochrome b gene(C ytb)is the most extensive part of mitochondrial DNA.It is a kind of molecular marker which is suitable for genetic analysis in population,because of its genetic characteristics,such as small molecular,maternal inheritance and rapid evolution.Major histocompatibility complex(MHC)gene is a group of very high polymorphism and closely linked gene group,which mainly in the DP,DQ,DR and other genes gathered a number of high polymorphism.Tupaia belangeri is a small mammal similar to squirrels,mainly distributed in the southwest of C hina,including Yunnan,Guizhou,Hainan and other places.At present,as a new type of experimental animal,it has been studied in many aspects.Based on the molecular technology of the C ytb and MHC DRB genes as molecular markers,we studied the genetic diversity and related population dynamics of Tupaia belangeri,which provided a certain genetic background basis for the new model animals.This paper mainly includes the following two parts:(1)Genetic diversity of Tupaia belangeri based on the Cytb gene:In this study,the genetic diversity of 12 geographic populations of Tupaia belangeri was studied based on the C ytb gene.MEGA software was used to calculate the genetic diversity index of each population and the total population(including variable ectopic number,haplotype diversity,average nucleotide variation,nucleotide diversity).Phylogenetic tree and haploid phylogenetic tree were constructed based on the K imura two parameters using the adjacency method.The Arlequin software was used to analyze the molecular variance of Tupaia belangeri populations.DnaSP software calculates haplotype,neutral detection,and mismatch profiles.And the haplotype intermediate network map of the Tupaia belangeri was drawn with Network software.The results showed that the C ytb gene of Tupaia belangeri(1037bp)a total of 245 variation sites,including A+T(55.6%)of the base ratio is higher than that of C+G(44.4%)of the base ratio,total of 65 haplotypes were defined.The C ytb gene of Tupaia belangeri has high genetic diversity: nucleotide diversity(π)reached 0.271%~5.22%;average nucleotide variation(K)3.127~57.00.Based on the Nucleotide haplotype phylogenetic tree,phylogenetic tree and Network network all can put the Tupaia belangeri in 12 populations can be divided into 4 populations: Daxin population,Hainan population,Tengchong and Pianma population and other population(Mengla、Dali、K unming、Leye、Xingyi、Hekou、Xichang、Luquan).The results of AMOVA analysis showed that 87.33% of the molecular variation occurred among populations,and the variation of the 12.67% occurred in the population.This variation of C ytb gene in Tupaia belangeri interspecific and intraspecific have occurred,but mainly occurred in populations.The gene flow among populations was less than 1,which may lead to a certain population differentiation.Neutral test results were not significant,indicating that Tupaia belangeri population refused expansion.(2)The genetic diversity of MHC DRB gene in Tupaia belangeri:MHC of Tupaia belangeri is a group of highly polymorphic genes.The recombination and replication of these genes increased their polymorphism.The primer sequences of MHC DRB intron 4 genes were searched in GenBank and designed base on the Primer.5.Intron itself does not encode proteins,so this experiment some simple genetic analysis on MHC DRB intron 4 genes.Genetic diversity was calculated using MEGA software.DnaSp software was used to calculate the ratio of non-synonymous / synonymous mutations,and the neutral test was calculated.In this study,MHC DRB exon 2 genes was amplified by PCR and then cloned by TSINGK E vector.The results showed that there were only three alleles.Therefore,the MHC gene as functional gene in our studied.we used MEGA software to test the genetic diversity of MHC DRB exon 2 genes,and construct phylogenetic tree based on K imura two parameters.DnaSP software was used to calculate the ratio of non-synonymous / synonymous mutations,neutral detection and mismatch distribution.The results showed that the MHC DRB intron 4 genes amplified a sequence of 168 bp,with high genetic diversity.Among them,nucleotide diversity(π)and average nucleotide variation,the haplotype diversity(Hd)of Hainan population was the highest.The lowest nucleotide diversity was found in Xingyi population,while the lowest in haplotype diversity was Mengla population.Non-synonymous / synonymous mutations(dN/dS)results show that Hainan and Mengla population and the overall sample is greater than one,Indicated that the MHC DRB intron 4 genes of Tupaia belangeri have acceptance positive selection,the other populations experienced negative selection.The overall sample also accepted positive selection.The results of neutral test showed that the Tajima’sD values of the other populations were not significant except the Leye population.Indicating that the Tupaia belangeri in the history of the process of denial of population expansion model.The experimental results are consistent with the experimental results of cytochrome b gene.The results showed that MHC DRB exon 2 genes had high genetic diversity.The maximum and minimum values of polymorphic loci were Xingyi population(87),Leye population(44).The population diversity of Daxin,Hainan,Mengla,Xichang and Luquan were 1.000;The average number of nucleotide variants was between 14.648 ~ 37.156.The maximum and minimum values of nucleotide diversity w ere also in the population of Xingyi population(0.08668)、Leye population(0.21986).MHC DRB exon 2 genes have a very high polymorphism,which is closely related to disease immunity in living environment.The ratios of Dali,Daxin,Kunming,Yueye and Mengla populations were less than one in the results of non-synonymous / synonymous mutations,and the ratios of the other populations were greater than one.The all samples test showed that MHC DRB exon 2 genes had been positive selection.Neutral test were not significant.The results showed that no population expansion was consistent with the results of the two genes.In summary,based on the C ytb gene,the genetic communication among the 12 geographic populations was weak,and there were obvious population differentiation.They could be divided into four groups: Daxing population,Hainan population,Tengchong and Pianma population,other populations.Based on MHC DRB gene,Tupaia belangeri has high genetic diversity.Through neutral detection and dN / dS ratio,the MHC DRB gene of Tupaia belangeri has been selected in the historical process and the population did not expand.Combined with C ytb and MHC DRB genes,the results showed that Tupaia belangeri did not receive population expansion and experienced positive selection in the course of history.The MHC DRB exon 2 gene polymorphism was further confirmed.In conclusion,based on the genetic diversity analysis of the three genes of Tupaia belangeri,the study provided a part of the genetic background for Tupaia belangeri.