Sequence Variation And Phylogenic Analysis Of Greater Horseshoe Bat(Rhinolophus Ferrumequinum) In Yunnan And Jilin Provinces Based On Partial Mitochondrial DNA

The greater horseshoe bat ( Rhinolophus ferrumequinum ) distributed through most ofthe old world, and the genus includes a high incidence of species of conservation concernin all over Europe. Now it has been listed as a LR/nt species by 2004 IUCN Red List ofThreatened Species by International Union for Conservation of Nature and NaturalResource ( IUCN ). Eight individuals of greater horseshoe bats were captured from Ji'ancity in Jilin province and Kunming city of Yunnan province in China. The whole genomeDNA was extracted from their muscle tissues as templates by which the mitochondrialDNA control region and cytochrome b gene were amplified with polymerase chain reaction( PCR ). After being sequenced, the sequences of control region and cytochrome b genewere analyzed by MEGA and DnaSP to explore genetic variation and phylogeneticrelationships of the samples from Jilin and Yunnan provinces. The results were as follows:1. The control region sequences of eight individuals were obtained. The results ofalignment showed six haplotypes and nucleotide substitutions mainly occurred in extendedtermination associated sequences ( ETAS ) and conserved sequence block ( CSB ) domains,but there were only four substitutions ( ts=1, tv=3 ) in the conserved central domain ( CD ).The nucleotide variation of the eight mitochondrial DNA control regions was 9.71%.2. The cytochrome b ( cytb ) gene sequences of six individuals were obtained. Theresults of alignment showed four individuals from Jilin shared the same haplotype( TYPE-JA ) and two from Yunnan shared another same haplotype ( TYPE-YN ).Nucleotide variation of the six sequences was 3.68%. The two haplotypes were mostlysimilar in codon usage and showed a bias of adenine(A) and uracil(U).3. The six sequences of control regions of 1019bp were aligned and their geneticdistances were calculated by MEGA3. The results showed the average distances in samplesfrom Jilin and Yunnan were 0.003 and 0.006 respectively, but the distance between Jilinand Yunnan was 0.096. The results indicated that variation between the two areas is moredistinct than in each area. Additionally, the haplotype diversity and nucleotide diversitywere 0.800±0.172 and 0.00262±0.00056 in Jilin samples, respectively.4. A neighbour-joining tree was constructed for the phylogenetic analysis based onnucleotide sequences of cytochrome b gene. TYPE-JA haplotype and the sequences fromJapan were clustered in one clade, which suggested that TYPE-JA haplotype was judged asone of subspecies of greater horseshoe bats, Rhinolophus ferrumequinum nippon,. Thedegree of variation between TYPE-JA and TYPE-YN was high, and their genetic distancewas 0.040.