title: Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea.
abstract: Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Glaser
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Kuppardt
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Krohn
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Anett
authors.initials: A
authors.lastname: Heidtmann
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Harms
authors.affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany. Electronic address: antonis.chatzinotas@ufz.de.
authors.forename: Antonis
authors.initials: A
authors.lastname: Chatzinotas
mesh_terms: D044523:Chrysophyta / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D016830:Kinetoplastida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan
keywords: 18S rRNA gene; Bacterivorous flagellates; Chrysophyceae; Kinetoplastea; Soil protists; T-RLFP