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Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

ABSTRACT: Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum- or group-specific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

SUBMITTER: Jung JH

PROVIDER: S-EPMC3887769 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

Jung Jae-Ho JH Kim Sanghee S Ryu Seongho S Kim Min-Seok MS Baek Ye-Seul YS Kim Se-Joo SJ Choi Joong-Ki JK Park Joong-Ki JK Min Gi-Sik GS

Molecules and cells 20120906 4

Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We d ...[more]

PMID: 22965748

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Project description:The cellular architecture of ciliates is one of the most complex known within eukaryotes. Detailed systematic schemes have thus been constructed through extensive comparative morphological and ultrastructural analysis of the ciliature and of its internal cytoskeletal derivatives (the infraciliature), as well as of the architecture of the oral apparatus. In recent years, a consensus was reached in which the phylum was divided in eight classes as defined by Lynn and Corliss [Lynn, D. H. & Corliss, J. O. (1991) in Microscopic Anatomy of Invertebrates: Protozoa (Wiley-Liss, New York), Vol. 1, pp. 333-467]. By comparing partial sequences of the large subunit rRNA molecule, and by using both distance-matrix and maximum-parsimony-tree construction methods (checked by boot-strapping), we examine the phylogenetic relationships of 22 species belonging to seven of these eight classes. At low taxonomic levels, the traditional grouping of the species is generally confirmed. At higher taxonomic levels, the branching pattern of these seven classes is resolved in several deeply separated major branches. Surprisingly, the first emerging one contains the heterotrichs and is strongly associated with a karyorelictid but deeply separated from hypotrichs. The litostomes, the oligohymenophorans, and the hypotrichs separate later in a bush-like topology hindering the resolution of their order of diversification. These results show a much more ancient origin of heterotrichs than was classically assumed, indicating that asymmetric, abundantly ciliated oral apparatuses do not correspond to "highly evolved" traits as previously thought. They also suggest the occurrence of a major radiative explosion in the evolutionary history of the ciliates, yielding five of the eight classes of the phylum. These classes appear to differ essentially according to the cytoskeletal architecture used to shape and sustain the cellular cortex (a process of essential adaptative and morphogenetic importance in ciliates).

Dataset Information

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

Publications

Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.

Similar Datasets

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