Abstract
Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169–23.880% for COI, 1.336–23.434% for 12S, and 7.897–71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.
Highlights
Partial Mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis
Since the number of heteroplasmic copies and nuclear mitochondrial pseudogenes (NUMTs) may be much smaller than that of the genuine mtDNA molecules, one might expect that heteroplasmy and NUMTs have little negative impact on the quality of nucleotide sequences obtained by direct nucleotide sequencing
We have frequently had difficulty for obtaining good electropherograms produced by direct nucleotide sequencing for COI, 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) and hypothesized that heteroplasmy was the major suspect for the issue
Summary
Consistent problems with obtaining good electropherograms in PCR-amplified mtDNA cytochrome oxidase subunit I (COI) fragments have been reported in some crustacean s pecies[5,6,7], in which multiple DNA sequences similar to a COI gene were detected from a single individual. In addition to these technical issues, unnoticed incorporation of heteroplasmic copies and NUMTs may lead to overestimation of population diversity and the number of species[2,5,8]. To the best of our knowledge, this is the first investigation for the extent of heteroplasmy and NUMTs in the Japanese spiny lobster as well as their impact for direct nucleotide sequencing
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