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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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I3-M11 partition.PCR products of 41 marine nematode species using 0.125 µM of primers JB3-JB5 (A) or 0.5 µM of primers JB2-JB5GED (B). Numbers in lanes correspond to the numbers in Table S1, background colours of the numbers indicate quality of PCR product: white  =  band of expected size and no aspecific products, grey  =  aspecific bands, black  =  no product. -  =  negative control.
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pone-0013716-g001: I3-M11 partition.PCR products of 41 marine nematode species using 0.125 µM of primers JB3-JB5 (A) or 0.5 µM of primers JB2-JB5GED (B). Numbers in lanes correspond to the numbers in Table S1, background colours of the numbers indicate quality of PCR product: white  =  band of expected size and no aspecific products, grey  =  aspecific bands, black  =  no product. -  =  negative control.

Mentions: The JB3-JB5 primer set clearly outperformed the degenerated JB2-JB5GED primer set in terms of amplification success and lack of aspecific products (Fig 1). Of the 41 species tested, three (7.3%) vs. 13 (31.7%) did not produce any products, respectively. Aspecific products were formed in seven (17.0%) vs. eight species (19.5%), respectively (Fig 1). Hence, amplification success was 87.8% vs. 53.6%, respectively. Interestingly, both primer sets were more or less complementary: species with weak or no amplification for primer set JB3-JB5 (samples 1, 28, 29, 30, 31, 32) generally produced stronger bands when the degenerated primer set was used (Fig 1). In view of the high amplification success of JB3-JB5, we used this primer set in those species for which we had more than one specimen (see Table S1). Many species showed a consistent amplification with strong bands, except Ascolaimus, Eleutherolaimus and Bolbolaimus for which amplification was relatively weak.


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)

I3-M11 partition.PCR products of 41 marine nematode species using 0.125 µM of primers JB3-JB5 (A) or 0.5 µM of primers JB2-JB5GED (B). Numbers in lanes correspond to the numbers in Table S1, background colours of the numbers indicate quality of PCR product: white  =  band of expected size and no aspecific products, grey  =  aspecific bands, black  =  no product. -  =  negative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013716-g001: I3-M11 partition.PCR products of 41 marine nematode species using 0.125 µM of primers JB3-JB5 (A) or 0.5 µM of primers JB2-JB5GED (B). Numbers in lanes correspond to the numbers in Table S1, background colours of the numbers indicate quality of PCR product: white  =  band of expected size and no aspecific products, grey  =  aspecific bands, black  =  no product. -  =  negative control.
Mentions: The JB3-JB5 primer set clearly outperformed the degenerated JB2-JB5GED primer set in terms of amplification success and lack of aspecific products (Fig 1). Of the 41 species tested, three (7.3%) vs. 13 (31.7%) did not produce any products, respectively. Aspecific products were formed in seven (17.0%) vs. eight species (19.5%), respectively (Fig 1). Hence, amplification success was 87.8% vs. 53.6%, respectively. Interestingly, both primer sets were more or less complementary: species with weak or no amplification for primer set JB3-JB5 (samples 1, 28, 29, 30, 31, 32) generally produced stronger bands when the degenerated primer set was used (Fig 1). In view of the high amplification success of JB3-JB5, we used this primer set in those species for which we had more than one specimen (see Table S1). Many species showed a consistent amplification with strong bands, except Ascolaimus, Eleutherolaimus and Bolbolaimus for which amplification was relatively weak.

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