The mtDNA D-loop and its adjacent regions were amplified and sequenced in 28 individuals from different haplotypes detected using PCR-RFLP in Ukraine scale carp(Cyprinus carpio),crucian carp(Carassius auratus),silver carp(Hypophthalmichthys molitrix),big head carp(Aristichthys nobilis),grass carp(Ctenopharyngodon idellus)and Ussuri bullhead(Pseudobgrus ussuriensis)by PCR.The amplified fragment length of mtDNA D-loop and its adjacent regions in the fish were varied from 1 500 bp to 1 800 bp,and are compared with the mtDNA complete sequences of five fish species from GenBank by Clustal 1.83 software.It was found that the amplified fragment was comprised of complete tRNAPro,D-loop,tRNAPhesequences and about 400 bp part of the 12S rRNA sequence.Sequences of 28 individuals were submitted to GenBank for serial numbers(KC292921-KC292948), and the base composition and difference of the six species,and the genetic distance were calculated based on Kimura's 2-Parameter model and NJ phylogenetic tree with five sequences from GenBank was constructed by Mega 4.0 software.The sequence structure analysis showed that the intraspecific and interspecific differences in D-loop region were higher in the four region sequences of six species than those in other species.NJ phylogenetic trees of six species were consistent with the traditional classification methods,showing useful for fish species classification and identification.In addition,restriction sites of all kinds of haplotypes were found in sequences,which is consistent with PCR-RFLP experimental results.More variation out of the restriction sites were observed compared with RFLP analysis.
HAO Jun, YANG Qiang, BAO Di, LIANG Ai-jun, ZHANG Xing-hua, DONG Shi.
The sequence comparison of mtDNA D-loop and adjacent regions in six fish species[J]. Journal of Dalian Fisheries University, 2013, 28(2): 160-165
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