The Sequence Differences Of D-loop, ND5, Cyt B And 16S RRNA And Their Phylogenetic Relationships In Panthera Tigris Altaica, P.t. Amoyensis, And P.t.Tigris

The mitochondrial DNA (mtDNA) sequences (Cyt b 572bp, ND5 309bp, 16S rRNA 326bp and D-loop 500bp or so) were analyzed in tigers (Panthera tigris). Based on mtDNA sequences analysis, the genetic distances of Cyt b sequences between Pts01, Pta01 and other tigers were more than that of among other tigers, the same as the 16S rRNA sequence of Ptt1. So the three sequences were refused when analyzing the phylogenetic relationships in tigers. Thus, among three tiger subspecies (P. t. altaica, P. t. amoyensis, P. t. tigris), the D-loop sequences had 57 variable sites and their percent was 11.88%, the ND5 sequences had 37 variable sites and their percent was 11.97%, the 16S rRNA sequences had 2 variable sites and their percent was only 0.6%, yet among the existing five tiger subspecies (P. t. altaica, P. t. amoyensis, P. t. tigris, P. t. Sumatra, P. t. corbetti) the Cyt b sequences had 9 variable sites and their percent was 1.57%, which showed the 16S rRNA and Cyt b sequences were very conserved in tigers. At the same time, the phylogenetic trees of 16S rRNA and Cyt b can not reflect correctly the phylogenetic relationships of tiger subspecies. However, the phylogenetic trees of D-loop and ND5 can reflect correctly the relationships of tiger subspecies and their variable-sites were large and their results were steady. So only the D-loop and ND5 sequences were used to analyze the phylogenetic relationship of tigers.Based on mtDNA D-loop sequence analysis, continuing 22bp deletion were found in hypervanable segment I (HVS I) to the South China tiger, the same as to snow leopard (P. uncia)and leopard (P.pardus). The genetic distance of D-loop between the Bengal tiger, the Siberian tiger and Snow leopard, leopard was more than that of between the South China tiger and snow leopard, leopard, the same as ND5. So our results agreed that the South China tiger was the most ancient among the three subspecies of Panthera tigris.At the same time, using oligonucleotide primers loop R and loop F designed to match hypervariable segments I (HVS-1) of tigers (P. tigris) mitochondrial DNA (mtDNA), we amplified two different PCR products (5OObp or so and 287bp) in tigers; but got only one PCR product (287bp) in leopard (P.pardus). Sequence analyses indicated that the sequences of 287bp was D-loop-like nuclear mitochondrial sequences (Numt) that occurred approximately 16.7 MY (million year) ago in the nuclear of tiger and leopard. According to the 1.76% difference of the 287bp D-loop-like nuclear mitochondrial sequences between tiger and leopard, it was obtained that the diverging time between tiger and leopard was 3.75 MY. Although the 287bp D-loop-like nuclear mitochondrial sequences (Numt) were highly conserved, such sequences represented molecular "fossils" that could shed light on the evolution of the mitochondrial genome and might be the most appropriate outgroup for phylogenetic analysis, this was also proved through comparing the phylogenetic trees that were reconstructed by the D-loop sequence of snow leopard and the 287bp Numt as outgroup, respectively.