New primers for ampli cation of cytochrome c oxidase subunit I barcode region designed for species of Decapoda (Crustacea)

We designed 14 new primers for amplifi cation of the COI barcode region of decapod crustacean species. We tested, with high level of success, the generation of ~ 640 ± 49 base-pair sequences in selected groups of decapods (hermit crabs, squat lobsters, marine and freshwater crabs and shrimps), encompassing representatives of 27 genera of 15 families, 11 of Pleocyemata (Anomura, Brachyura, and Caridea) and 4 of Dendrobranchiata. Based on the results we expect the applicability of these primers for several studies with diff erent taxa within Decapoda. keY woRds COI, DNA Barcoding, molecular markers, molecular techniques. This article is part of the tribute offered by the Brazilian Crustacean Society in memoriam of Michael Türkay for his outstanding contribution to Carcinology CORRESPONDING AUTHOR Fernando L. Mantelatto mantel@usp.br SUBMITTED 26 September 2016 ACCEPTED 12 November 2016 PUBLISHED 12 December 2016 Guest Editor Célio Magalhães DOI 10.1590/2358-2936e2016030 sHoRt commUnIcAtIon


ABstRAct
We designed 14 new primers for amplifi cation of the COI barcode region of decapod crustacean species.We tested, with high level of success, the generation of ~ 640 ± 49 base-pair sequences in selected groups of decapods (hermit crabs, squat lobsters, marine and freshwater crabs and shrimps), encompassing representatives of 27 genera of 15 families, 11 of Pleocyemata (Anomura, Brachyura, and Caridea) and 4 of Dendrobranchiata.Based on the results we expect the applicability of these primers for several studies with diff erent taxa within Decapoda.

Nauplius
BRAZIlIAn cRUstAceAn socIetY

Nauplius
BRAZIlIAn cRUstAceAn socIetY During the last three decades, molecular techniques have become a large, and in some cases, indispensable ally for advances in our knowledge of biodiversity.There is sufficient available literature providing evidence for the suitability and credibility of DNA-based investigations at different taxonomic levels.Among those taxa for which molecular analyses have proven their efficiency and allowed innumerous advances in different areas is a diverse and species-rich group: decapod crustaceans.The molecular methodological support has helped to advance knowledge about many aspects of this taxon, including systematics, biogeography, ecology, conservation, and taxonomy by the identification of larvae and eggs, cryptic species, and damaged specimens.Since 2011, the Laboratory of Bioecology and Systematics of Crustaceans (LBSC) has been involved in two long-term projects aiming the characterization of the marine and estuarine decapod crustaceans biodiversity of the Brazilian coast, supported by the Brazilian agencies "Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)" through Biota-FAPESP Program and "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)" through Ciências do Mar II.Both projects made use of combined analysis techniques to elucidate various aspects of the life cycle and evolution in decapod crustaceans, and molecular analyses were one of the main tools to support the assumptions of these studies.To this end, we attempted to generate DNA sequences of two mitochondrial markers, the barcode region of cytochrome c oxidase subunit I (COI) and a fragment of 16S rRNA, of all decapod species sampled along the coast of São Paulo in order to develop a genetic library to serve as base line to all researchers in this field (F. Mantelatto et al., unpubl. data).However, particularly for the COI region, we had considerable difficulties in obtaining successful amplifications using some previous standard universal pairs of primers: LCO1-1490/HCO1-2198 (Folmer et al., 1994) or COL6b/COH6 (Schubart and Huber, 2006), designed for crayfishes based on the primers of Folmer et al. (1994).
Thus, we designed 14 new primers for the COI barcode region (Tab.1, Fig. 1), five of them with degenerate bases.Four nucleotides of the previously designed primer COL6b (5´-ACAAATCATAAAGATATYGG-3´) (Schubart and Huber, 2006) were replaced by variable bases to constitute the primer COL6b2.The primers COIAL2o, COIAH2o, and COIAH2m were designed using the software NetPrimer, available at the PREMIER Biosoft International website <http://www.premierbiosoft.com/netprimer>.The others were designed using the Primer-Blast software tool developed at NCBI, which generates target-specific primer pairs (available at <http://www.ncbi.nlm.nih.gov/tools/primer-blast/>)(see Ye et al., 2012 for further details).
The applicability of these primers was highly satisfactory for the amplification of fragments from 584 to 712 base pairs, given the diversity of species, genera and families used as models (see Tab. 1).These new primers also showed good performance for samples from different populations and geographic regions.
According to this scenario and considering many other projects and publications that are in progress by our team, we are convinced that the new primers presented herein were successful in amplifying the target species and have proven their utility for several studies with different taxa within Decapoda.In addition, these new primers may help in different ways: 1) tthey have been used and may be useful in future studies to obtain comprehensive phylogenies and/or biogeographical variability of specific target genera and species; 2) to avoid pseudogenes during amplifications, since the occurrence of pseudogenes strongly decreases when using taxon specific (optimized) primers (Schubart, 2009); 3) based on our experience from the data obtained during this research, and pending future tests, we can speculate that some of the present primers can be used for other related genera and species.
New primers for cytochrome c oxidase subunit I Nauplius, 24: e2016030 Table 1.New primers of cytochrome c oxidase subunit I and the taxonomic groups with successful amplifications showed by pairs of primers.F: forward primer; R: reverse primer.PMT: Primer Melting Temperature; *used with COH6 (Schubart and Huber, 2006).Degenerate bases: Y = C or T, R = A or G, W = A or T. † "Turk" is in reference to the past researcher Dr. Michael Türkay who contributed significantly to studies on crustaceans, in especial on freshwater crabs.
New primers for cytochrome c oxidase subunit I Nauplius, 24: e2016030 Our results evidenced that a successful amplification of the COI region from decapod crustaceans is not always achieved using the universal primers.Therefore, we are happy to share our new findings with the carcinological community.The new primers may contribute to improve the quality and efficiency of molecular markers, aiming to advance the knowledge of evolution of decapod crustaceans and leading to the solution of several systematic issues.