| The Asiatic Toad(Bufo gargarizans)has a wide distribution in China,ranging from 120 m to 4300 m above sea level.The taxonomic status of B.gargarizans populations in China has been controversial for over half a century.Now,more and more molecular evidences suggest that this toad did not have subspecies differentiation.And as a heterotherm species with such a large elevation span,there may be some differences between the population and the habitat.The Asiatic toad has a unique adaptability in such a harsh environment,but there is relatively little research on the high plateau adaptation of the Asiatic Toad and other amphibian species.Therefore,in this experiment: 1)we use the mitochondrial D-loop partial sequence(565bp)as the molecular marker to detect and analyze the genetic diversity,genetic distance,population structure situation and taxonomic status of B.gargarizans,which have 102 samples collected from Sichuan(Danba,Qingchuan,Zoige,Shangli,Shimian,Wenjiang and Wangyu)and Shaanxi(Hanzhong),China;2)we sequenced the mitochondrial ND1,ND2,COX1,COX2,ATP6 and ATP8 genes of 18 B.gargarizans samples in areas at a high elevation(2940~3440m)and 20 B.gargarizans samples in areas at a low elevation(550m)and detect the potential relationship between mitochondrial ND1,ND2,COX1,COX2,ATP6 and ATP8 variants and high-altitude adaptation in B.gargarizans.The main results and conclusions of this experiment are as follows:1.The overall base composition of the 102 B.gargarizans D-loop partial sequence alignment fragment is T 33.0%,C 18.9%,A 37.3% and G 10.8%,the A+T content is higher than G+C content.The amount of variation(polymorphism)site was 92,accounting for 16.3% of all sites.This sequence alignment fragment can be divided into 31 types(H1~H31),accounting for 30.39%(31/102)of the total sample size,with a relatively small number of shared haplotypes,accounting for only 12.90%(4/31),and no haplotype is shared by all species.The ratio of the unique haplotype of the Shimian small population in Sichuan was the highest,at 70.00%.2.The genetic diversity level of the nine small population of B.gargarizans was higher,and the overall haplotype diversity was 0.878±0.023,and the total nucleotide diversity was 0.04918±0.00176.The Wangyu small population in Sichuan,by contrast,had higher genetic diversity,and its genetic resource is relatively more abundant,with the biggest haploidtype diversity and nucleotide diversity values(0.889±0.091,0.04800±0.01137).3.The K2 P genetic distance among the nine small population of B.gargarizans was 0.000~0.096,and the overall average K2 P genetic distance was 0.052.Molecular phylogenetic analysis showed that all samples from nine small geographic populations were nested with each other,they are not gathered into their respective independent geography branch,which was consistent with the other scholar's research results: there is no relevance between the genetic structure of B.gargarizans and their geographic and altitude,which supports the hypothesis about B.andrewsi,B.minshanicus and B.gargarizans are all belong to B.gargarizans.4.The size of ND1,ND2,ATP6,ATP8,COX1 and COX2 was 946 bp,1035bp,684 bp,165bp,1542 bp and 688 bp,respectively,which are consistent with the reference sequence.Compared with those of reference sequence,ND1,ND2,ATP6,ATP8,COX1 and COX2 had 32,36,36,3,21 and 17 mutation sites,including 4,11,2,1,0 and 3 missense mutation sites,respectively.By analyzing the frequency of the missense mutation in the high/low population,and through the Pearson chi-square,eventually,we only found that there have been a very significant positive correlation(P < 0.01)between the A4767 C and G5142 A missense mutation sites in ND2 gene and the high-altitude adaptation of the B.gargarizans population in areas at a high elevation,suggesting ND2 gene may be a candidate gene of B.gargarizans high-altitude adaptation. |
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