Dataset Information

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers.

ABSTRACT: BACKGROUND: The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms.Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers. RESULTS: The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism. CONCLUSION: The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.

SUBMITTER: Robeson MS

PROVIDER: S-EPMC2796995 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers.

Robeson Michael S MS Costello Elizabeth K EK Freeman Kristen R KR Whiting Jeremy J Adams Byron B Martin Andrew P AP Schmidt Steve K SK

BMC ecology 20091211

<h4>Background</h4>The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the dive ...[more]

PMID: 20003362

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Project description:BackgroundBdelloid rotifers are the oldest, most diverse and successful animal taxon for which males, hermaphrodites, and traditional meiosis are unknown. Their degenerate tetraploid genome, with 2-4 copies of most loci, includes thousands of genes acquired from all domains of life by horizontal transfer. Many bdelloid species thrive in ephemerally aquatic habitats by surviving desiccation at any life stage with no loss of fecundity or lifespan. Their unique genomic diversity and the intense selective pressure of desiccation provide an exceptional opportunity to study the evolution of diversity and novelty in genes involved in DNA repair.ResultsWe used genomic data and RNA-Seq of the desiccation process in the bdelloid Adineta vaga to characterize DNA damage reversal, translesion synthesis, and the major DNA repair pathways: base, nucleotide, and alternate excision repair, mismatch repair (MMR), and double strand break repair by homologous recombination (HR) and classical non-homologous end joining (NHEJ). We identify multiple horizontally transferred DNA damage response genes otherwise unknown in animals (AlkD, Fpg, LigK UVDE), and the presence of genes often considered vertebrate specific, particularly in the NHEJ complex and X family polymerases. While 75-100% of genes involved in MMR and HR are present in 0-2 copies, genes involved in NHEJ, which are present in only a single copy in nearly all other animals, are retained in 3-8 copies. We present structural predictions and expression evidence of neo- or sub-functionalization of multiple copy genes involved in NHEJ and other repair processes.ConclusionThe horizontally-acquired genes and duplicated genes in BER and NHEJ suggest resilience to oxidative damage is conferred in part by increased DNA damage recognition and efficient end repair capabilities. The pattern of gene loss and retention in MMR and HR may facilitate recombination and gene conversion between divergent sequences, thus providing at least some of the benefits of sex. The unique retention and divergence of duplicates genes in NHEJ may be facilitated by the lack of efficient selection in the absence of meiotic recombination and independent assortment, and may contribute to the evolutionary success of bdelloids.

Project description:Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%-9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex.

Dataset Information

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers.

Publications

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers.

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