Abstract

Genetic analysis using restriction fragment length polymorphisms (RFLPs) of D-loop region in the mitochondrial DNA including the part of 12s RNA and cytochrome b genes was made to clarify genetic variations and relations and relationships among eleven populations of Japanese loach Misgurnus anguillicaudatus. Ten haplotypes were detected using seven restriction enzymes (Alu I, Hinc II, Msp I, EcoR I, Hinf I, Hae III and Taq I). The differences between each pair of the populations were significant (p<0,0001~p<0.05), except for the test involving populations Saito and Nikko, and Ueda and Futtsu (p=1). The haplotypic and nucleotide diversities within populations ranged from 0 to 0.889 and 0 to 22.222%, respectively. The average nucleotide diversity (p_xy) among 11 populations ranged from 0 to 15.255%, with a mean of 6.272 ±0.004% and net nucleotide divergence (d) ranged from 0 to 15.255%, with a mean of 5.312±0.004%. The net nucleotide divergence between the two northern populations (Memanbetsu and Naruko) were high (12.521%, ranged from 5.083 to 15.256%), in contrast, the net nucleotide divergence among southern populations were relatively low (3.190%, ranged from 0 to 8.478%). The bootstrapped UPGMA dendrogram and NJ tree, which were constructed based on the net nucleotide divergences, showed that loach diverged into two groups. The northern group comprised two populations (Memanbetsu, Hokkaido Prefecture and Naruko, Miyagi P.) and the southern group contained nine other populations. In the southern group, the Hashima (Gifu P.), Izumo (Shimane P.) and Tomari (Tottori P.) populations clustered into one subgroup whereas the other six populations, Yuya (Yamaguchi P.), Saito (Miyazaki P.), Nikko (Tochigi P.), Ueda (Nagano P.), Futtsu (Chiba P.), and Nasu (Tochigi P.), categorized into another subgroup. The homogeneity test and AMOVA indicated in the consensus tree reconstructed by NJ method, the two genetically groups were considerably differentiated (0.0001<p<0.05) to each other. However, the NJ tree and UPGMA dendrogram based on the genetic distances, also showed that despite the pronounced genetic differences among some populations, four populations form the southern group, i.e. Saito, Nikko, Ueda and Futtsu, were closely related.

Key words: RFLP, mtDNA, D-loop, misgurnus anguillicaudatus.