Comparison And Phylogenetic Analysis Using Sequences Of Mitochondrial Genes In Some Rutelidae Species

In this study, mitochondrial COâ… , Cyt b and 16S rRNA genes in some species of Rutelidae were used as molecular markers. These gene fragments amplified by PCR were sequenced. The genetic distances, the ratios of si/sv and the frequencies of codons and amino acids were calculated. The phylogenetic analysis was performed basing on the constructed molecular phylogenetic trees.There were three main contents in this experiment:extraction of total genomic DNA; amplification of mtDNA COâ… , Cyt b and 16S rRNA gene fragments; sequenceing and analyzing of these genes.The results were shown as follows:(1) The sequences of the mitochondrial COâ… gene (651bp), Cyt b gene (489bp) and 16S rRNA gene (825bp) for Rutelidae were obtained. The results of nucleotide composition showed that the average A+T and G+C contents respectively were 65.0% and 35.0% for COâ… (A=29.2%,T=35.8%,G=16.7%,C=18.3%),69.2% and 30.8% for Cyt b (A=31.2%,T=38.0%,G=12.6%,C=18.2%),72.8% and 27.2% for 16S rRNA (A=33.2%,T=39.6%,G=17.6%,C=9.6%). Many sites in these genes exhibited strong A+T nucleotide bias.(2) The results in nucleotide divergence showed that the variable sites in COâ… , Cyt b and 16S rRNA gene sequences were mostly concentrated on the third site, the numbers of variable sites were 167,118 and 120, accounting for 81.5%,74.6% and 39.2%, respectively. And the bast substitution occurred mainly between the T and A.The average genetic distances were 0.14,0.11 and 0.07 and the average rates of (TS/TV) were 1.1, 1.67 and 1.14 in COâ… , Cyt b and 16S rRNA fragments.(3) The amino acid sequence analysis showed that the frequencies of Leu were the highest in the COâ… (16.4), Cyt b (14.1) and 16S rRNA (17.6) percent, while the lowest frequencies of amino acid were 0; the most usage frequency of codon was UUA, respectively were 1.6.4 (COâ… ),15.1 (Cyt b),25.8 (16S rRNA) percent, and the lowest frequency of codon usage were 0, indicating that the amino acid and codon usage also has a preference.(4) Phylogenetic tree analysis:Based on the COâ… , Cyt b and 16S rRNA gene were constructed NJ tree, MP tree and UPMGA tree, respectively.The molecular tree were consistent with morphological taxonomy of Rutelidae. The results showed that the species in genus of the Anomala were first aggregated, then Mimela holosericea, Blitopertha pallidipennis, Popillia quadriguttata genus respectively were aggregated in turn, after that, the species of Potosia and Lucanidae were aggregated. The results indicated that the relationship among species within the genus were most close, the relationship among species the different genus were more close, the genetic distance between families was farther relatively. The conclusion revealed that COâ… , Cyt b and 16S rRNA gene.sequences could be suitable as a molecular marker for identifying the genetic distance among Rutelidae population.