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Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes.

Derycke S, Vanaverbeke J, Rigaux A, Backeljau T, Moens T - PLoS ONE (2010)

Bottom Line: The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively).A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

View Article: PubMed Central - PubMed

Affiliation: Marine Biology Research Group, Department of Biology, Ghent University, Ghent, Belgium. s.derycke@ugent.be

ABSTRACT

Background: The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology: Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion: The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

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NJ-tree of the Folmer partition based on K2P genetic distance.Higher taxon levels are indicated after the vertical lines and brackets.
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pone-0013716-g004: NJ-tree of the Folmer partition based on K2P genetic distance.Higher taxon levels are indicated after the vertical lines and brackets.

Mentions: For the Folmer partition, Blastx searches indicated that only the Panagrolaimus and Plectus sequences showed similarity to nematodes (91% and 72%, respectively). All other sequences showed blast hits with low similarities (65% to 76%) to a variety of organisms such as polychaetes, flatworms, spiders and wasps. The alignment showed one amino acid deletion in Panagrolaimus (position 119) and in Theristus (position 159) and three deletions in Araeolaimus (positions 162, 163 and 206). The NJ tree generally was congruent with known taxonomy, except for Ascolaimus and Araeolaimus, which showed long branches positioned closer to the basal node (Fig 4). These sequences were removed from the dataset for calculation of genetic distances. Consequently, from the 41 species that were tested, we obtained 16 high quality sequences (39.0%).


Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes.

Derycke S, Vanaverbeke J, Rigaux A, Backeljau T, Moens T - PLoS ONE (2010)

NJ-tree of the Folmer partition based on K2P genetic distance.Higher taxon levels are indicated after the vertical lines and brackets.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965665&req=5

pone-0013716-g004: NJ-tree of the Folmer partition based on K2P genetic distance.Higher taxon levels are indicated after the vertical lines and brackets.
Mentions: For the Folmer partition, Blastx searches indicated that only the Panagrolaimus and Plectus sequences showed similarity to nematodes (91% and 72%, respectively). All other sequences showed blast hits with low similarities (65% to 76%) to a variety of organisms such as polychaetes, flatworms, spiders and wasps. The alignment showed one amino acid deletion in Panagrolaimus (position 119) and in Theristus (position 159) and three deletions in Araeolaimus (positions 162, 163 and 206). The NJ tree generally was congruent with known taxonomy, except for Ascolaimus and Araeolaimus, which showed long branches positioned closer to the basal node (Fig 4). These sequences were removed from the dataset for calculation of genetic distances. Consequently, from the 41 species that were tested, we obtained 16 high quality sequences (39.0%).

Bottom Line: The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively).A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

View Article: PubMed Central - PubMed

Affiliation: Marine Biology Research Group, Department of Biology, Ghent University, Ghent, Belgium. s.derycke@ugent.be

ABSTRACT

Background: The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology: Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion: The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

Show MeSH