* Polish Academy of Sciences, Institute of Oceanology, Department of Genetics and Marine Biotechnology, Gdynia, Poland
School of Biological Sciences, University of Wales Swansea, Swansea, United Kingdom
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Abstract |
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Key Words: mtDNA recombination Mytilus D-Loop DUI
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Introduction |
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Using PCR amplification of a segment of the COIII gene, Ladoukakis and Zouros (2001) exploited this situation and demonstrated the existence of recombinant molecules within the gonadal tissue of heteroplasmic male Mytilus galloprovincialis from the Black Sea. It seemed likely that the recombinant molecules originated recently, perhaps within the individuals from which they were recovered. They were absent from the populations studied, and their relative fitnesses were not ascertained. The heteroplasmic males studied by Ladoukakis and Zouros (2001) were atypical in that the nonrecombinant genomes present were not as highly diverged as typical F and M genomes, and thus the M genomes were assumed to be recently role-reversed. It was suggested that the consequent lower sequence divergence was high enough to facilitate the detection of recombinant molecules but low enough to suppress mismatch repair to a level that did not inhibit homologous recombination. Males of Mytilus trossulus from the Baltic Sea are also heteroplasmic for two genomes that are not as highly diverged as typical F and M genomes (Wenne and Skibinski 1995), and evidence for role reversal has been obtained (Quesada, Wenne, and Skibinski 1999).
In the present study, we demonstrate the existence of two recombinant mtDNA variants from within the noncoding region of M. trossulus. The variants, derived from homoplasmic sperm samples, are at polymorphic frequencies within the Baltic population studied and behave as M genomes.
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Materials and Methods |
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Primers suitable for amplification of the major noncoding region from Mytilus were designed using the M. edulis sequence obtained by Hoffman, Boore, and Brown (1992) (GenBank accession number M83756). The forward primer (CBM1) was AGAACGGCGTGAGCTAGTTC (16S rRNA gene, nucleotide positions 3313 to 3332), the reverse primer (CBM2) sequence was ACCTTCACCAGGCGTTTAAG (cytochrome b gene, nucleotide positions 4833 to 4814). For checking for presence of the M genome characteristic of M. edulis, the specific primers M1 (AAACCCTTCGTCCACAAGG) and M2 (AGCCTTTTTGTCATCATTCTGT) were used. Approximately 20 ng of total DNA was used for PCR amplifications, which were carried out in a volume of 20 µl with primers at 0.4 µM, nucleotides at 200 µM, magnesium chloride at 1.5 mM, high fidelity DyNAzyme EXT2 DNA polymerase (0.5 U), and appropriate reaction buffer from Finnzymes. Thirty-three cycles were used with denaturation at 94°C (for 1 min, but 3 min for first cycle), annealing at 54°C, 30 s, and extension at 72° C for 1 min 30 s (but 5 min for the last cycle).
Selected PCR products were purified by alkaline phosphatase (shrimp alkaline phosphatase from Amersham) and exonuclease I (USB) treatment (1/13 U/µl SAP along with 20/13 U/µl exonuclease I for 45 min at 37°C followed by enzyme inactivation at 80°C for 15 min) and sequenced directly with BigDye kit from PE Applied Biosystems using the above PCR primers as first run sequencing primers. Sequencing reactions were resolved on an ABI 310 genetic analyzer or ABI 377 sequencer (both from PE). New primers were designed based on the sequences obtained and used in additional sequencing runs in order to obtain the full sequence of each PCR product. The SeqManII program from DNASTAR Lasergene facilitated sequence assembly. Sequences alignments were made using ClustalW (Higgins, Thompson, and Gibson 1996). All sequences obtained have been deposited in GenBank (accession numbers AY115479 to AY115482). Nucleotide diversity between sequences was calculated using DnaSP version 3.53 (Rozas and Rozas 1999). The Kimura two-parameter distance between sequences and the UPGMA trees were calculated using MEGA2 version 2.1 (Kumar et al. 2001). Statistical analysis of potential recombined fragments was performed by runs tests implemented in GENECONV version 1.81 (http://www.math.wustl.edu/sawyer) (Sawyer 1989).
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Results and Discussion |
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The present study considers the 1 and 1a variant molecules in an attempt to detect recombination. Genomes that occur in heteroplasmic males are the natural candidates for the detection of recombinant events. However, the possibility that strand switching during PCR amplification of heteroplasmic DNA might generate artifactual recombinant molecules should be considered. That variants 1 and 1a are such artifacts seems most unlikely for several reasons. First, Ladoukakis and Zouros (2001) provided direct evidence against artifacts by cloning of PCR product molecules from artificially heteroplasmic targets. Second, the sperm DNA samples used in the present study were observed to be homoplasmic, giving only a single PCR product. Even if there was some minor somatic contamination, the use of such samples reduces the possibility of artifacts arising from recombinational events within the PCR reaction, compared with the use of heteroplasmic somatic DNA. Finally, identical strand switching events would need to be postulated to repeatedly generate only variants 1 and 1a, in circumstances in which a hypothetical parental molecule within the PCR mix was not itself amplified.
One copy of each of the 1 and 1a variants was sequenced. In addition, the same primers were used to amplify and sequence the major noncoding region of the F genome (from eggs of an M. trossulus female) and the M genome (from sperm of an M. edulis male). The M. edulis M specific M1 and M2 primers failed to detect the M genome in M. trossulus. Pairwise dotplot comparisons of the 1 and 1a variants with both F and M sequences are shown in figure 1. It is clear that different regions of the 1 and 1a variants have differential similarity to the F and M sequences. A global alignment of all four sequences was constructed. Nucleotide diversity was used as a measure of genetic divergence between pairs of sequences: F versus M, F versus 1, F versus 1a, 1 versus M, and 1a versus M. Estimates were made over the whole alignment in a sliding window of 50 bp (fig. 2A). Four regions were selected for further analysis: a 5' flank comprising the very end of rRNA gene and the beginning of noncoding region (1 to 223), a middle region showing the highest M versus F divergence (376 to 786), a core that was quite highly conserved among all sequences (786 to 1191), and a 3' coding region having a complete tRNATyr gene and the beginning of the cytochrome b gene (1289 to 1456). The dotplot analysis (fig. 1) suggests that the segment between the 5' flank and middle region in variant 1 has high homology with a region in the vicinity of the tRNATyr gene of the M sequence; there are only three nucleotide differences over an aligned region of 42 bp. For this reason, the fragment (224 to 376) was excluded. Between the core and the 3' coding regions the M sequence has a 70 bp deletion not shared by any other sequence. Hence this region was also excluded.
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The GENECONV analysis was performed on the global alignment to detect potential recombinant fragments (table 1). The analysis provides good statistical support for the hypothesis that variants 1 and 1a are mosaic, resembling F or M in different regions. The breakpoints suggested by this analysis receive good support statistically and fit well with the regions identified above based on the distance analysis. However, there are some discrepancies. For example, the GENECONV analysis supports a number of fragments that begin or end within the core region. In addition, a nonhomologous exchange would be needed to transfer the short region in the vicinity of the tRNATyr gene from M to variant 1. Taking into account these considerations, the results of the distance analysis, and the GENECONV analysis, a consensus schematic representation of the similarities and differences between the four genomes is given in figure 2C. This indicates that several independent recombination events may have occurred within male lineages of the Baltic M. trossulus mtDNA. To obtain variant 1, a homologous exchange would be needed to replace the middle region of the F genome with that of the M genome. To obtain variant 1a, a homologous exchange would be needed to replace the 5' region of the core of the F genome with that of the M genome. Different recombinational events would be implicated if the ancestral relationships between the genomes were not as assumed above.
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In the study of Ladoukakis and Zouros (2001), recombinants were detected in a high proportion of heteroplasmic individuals studied. The recombinant genomes were produced in individuals heteroplasmic from two variants that had a combined frequency of 0.96 in the Black Sea population. Despite the suggestion of potential for a high rate of production of recombinant molecules, none of those detected had become established in the population or contributed to a high level of population diversity. This could be consistent with the recombinant molecules having relatively lower fitness, but as Ladoukakis and Zouros (2001) point out, more work is required to establish this.
There is evidence of introgression of M. edulis mtDNA into Baltic M. trossulus (Quesada, Wenne, and Skibinski 1995). The M. edulis M genome is however at very low frequency in the Baltic. It thus appears possible from the present study that some genomes produced by recombination between the M. edulis M and F genomes or their descendants in the Baltic have taken over the role as paternal genome. This in turn provides a possible explanation for the polymorphic frequencies of these recombinant genomes.
The noncoding region analyzed in the present study might, by analogy with other animal mtDNAs, contain regulatory sequences, for example, those associated with the initiation of replication. Both the recombinant genomes reported here have the noncoding region derived from the M. edulis M genome rather than from the F genome. RFLP studies of whole mtDNA and PCR amplified segments have shown high similarity of Baltic mtDNA to the M. edulis F rather than M genome (Quesada, Wenne, and Skibinski 1995; Wenne and Skibinski 1995). It is thus possible that this acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence. This would implicate the noncoding region in the maintenance of DUI. Thus, even if recombination is quite frequent in Mytilus, it may not have any phylogenetic consequences unless it involves regulatory elements resulting in the emergence of new paternal lines. Studies of recombination focusing on other regions of the mtDNA as well as the noncoding region are needed to further test these ideas.
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Footnotes |
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Fumio Tajima, Associate Editor
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