Comparison And Diversity Analysis Of Mitochondrial Cytochrome B Gene Between Natural Diploid And Tetraploid Weather Loach Misgurnus Anguillicaudatus

The objectives of the study were to analyze genetic diversity and phylogenetic relationships between diploid and tetraploid loaches using cyt b gene sequence analyses, thus estimating the evolution of weather loach Misgurnus anguillicaudatus and investigating the molecular geographical distribution of weather loach. The main contents are as follows:1. Comparison and evolution analysis of mitochondriai cytochrome b gene between diploid and tetraploid loaches in Hubei ProvinceAfter extended and alignment, the natural cytochrome b gene sequences of diploid and tetraploid loaches were both 1140 bp. There were 45 sites for all sequences in the 1140 basic sites. 23 of which were variable among diploid loaches and 14 among tetraploids. There were 380 amino acids deduced from the nucleotide sequences of the cyt b genes in both diploid and tetraploid loaches. Besides, within 18 amino acid substitutions, 10 were in the diploids while 8 were in tetraploid loaches. Among all loaches sequences, the nucleotide pairwise distance ranged from 0.001 to 0.378. The nucleotide pairwise distance between the tetraploid and diploid ranged from 0.001 to 0.025. Based on the AMOVA analysis, there were no significant genetic variations within diploid loaches (Fst=0.2529, P＞0.05) and within tetraploid loaches (Fst=0.0564, P＞0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst=0.7634, P＜0.05).NJ phylogenetic tree and MP phylogenetic tree of the cyt b genes from diploid and tetraploid M. anguillicaudatus in cypriniformes was reconstructed. The branches of two kinds of phylogenetic trees had the same topology and similar bootstrap probabilities. Phylogenetic analyses using NJ identified two major lineages supported by moderate to high bootstrap values: diploid and tetraploid loaches (51% and 99% bootstrap, respectively). In the MP tree, tetraploid (XG, CB, SS, WH) loaches were at the derivative clade, while diploid (TM, SY, YX, ES, ZG) loaches were at the basal clade of MP tree, which was similar to the NJ tree. The diploid branch in the bottom of NJ and MP phylogenetic tree and tetraploid branches at the top of the trees indicate that diploids are earlier existing cytotype than tetraploids in nature. This is the first report to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.2. Molecular geographical distribution of weather loach In the present study, we added samples from Shandong Province, Chongqing Province besides Hubei Province, Hunan Province. After extended and alignment the cyt b gene of loach, we found that the length of gene were also 1140 bp. Among 1140 nucleotides of the cyt b gene, there were 961 conservative sites and 179 variable sites. 380 amino acids were deduced from the nucleotide sequences of the cyt b genes in both diploid and tetraploid loaches. After comparison, 38 amino acid substitutions were found. Among all loach populations, the nucleotide pairwise distance ranged from 0.001 to 0.104. There are significant differences among different geographical populations (P＜0.05). In the phylogenetic trees, different geographical populations constitute seven branches, respectively, including Hubei, Hunan, Shandong, Shanxi, Guangxi, Sichuan, Chongqing.