title: PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.
abstract: Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.
authors.affiliation: Département de biologie and Québec-Océan, Université Laval, Québec, QC, Canada.
authors.forename: Marianne
authors.initials: M
authors.lastname: Potvin
authors.forename: Connie
authors.initials: C
authors.lastname: Lovejoy
mesh_terms: D059001:Aquatic Organisms; D001483:Base Sequence; D044822:Biodiversity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004141:Dinoflagellida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004777:Environment; D056890:Eukaryota / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D020459:Genes, rRNA; D008969:Molecular Sequence Data; D015873:Northwest Territories; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D054679:Ribosome Subunits, Small / Q000235:genetics; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA; D013045:Species Specificity; D015874:Yukon Territory
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S