Capturing drifting species and molecules—Lessons learned from integrated approaches to assess marine metazoan diversity in highly dynamic waters

Marine community diversity surveys require a reliable assessment to estimate ecosystem functions and their dynamics. For these, non-invasive environmental DNA (eDNA) metabarcoding is increasingly applied in zoological studies to complement or even replace traditional morphological identification methods. However, uncertainties remain about the accuracy of the diversity detected with eDNA to capture the actual diversity in the field. Here, we validate the reliability of eDNA metabarcoding in identifying metazoan biodiversity in highly dynamic marine waters of the North Sea. We analyzed biodiversity from water (eDNA) and zooplankton samples with cytochrome c oxidase subunit 1 (COI) and 18S rRNA (18S) metabarcoding at Helgoland Roads and validated the optimal molecular resolution by morphological and molecular zooplankton identification (metabarcoding) with the result of merely a few false-negative detections. eDNA and zooplankton metabarcoding resolved 354 species from all major and in total 16 metazoan phyla. This molecular genetic species inventory overlapped by 95.9% (COI) and 81.9% (18S) with published inventories of local, morphologically identified species, among them neozoa and rediscovered species. Even though half of all species were detected by both eDNA and zooplankton metabarcoding, the methods differed significantly in their detected diversity. eDNA metabarcoding performed very well in cnidarians and annelids, whereas zooplankton metabarcoding identified higher numbers of fish and malacostraca. Species assemblages significantly differed between the individual sampling events and the cumulative number of identified species increased steadily over the sampling period and did not reach saturation. About a third of the species were detected only once while a core community of 22 species was identified continuously. Our study confirms eDNA metabarcoding to be a powerful tool to identify and analyze North Sea fauna in highly dynamic waters and we recommend investing in high sampling efforts by repetitive sampling and replication using at least 0.45 μm filters to increase filtration volume

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

TG dinucleotides functioning as alternative 3' splice sites were identified and experimentally verified in 36 human genes

Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing

<p>Abstract</p> <p>Background</p> <p>The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.</p> <p>Results</p> <p>Six PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.</p> <p>Conclusion</p> <p>Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.</p

A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome

Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequenc

Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ~200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

BACKGROUND There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. RESULTS A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. CONCLUSIONS We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.This work was financially supported by the following grants: project GABI-BARLEX, German Federal Ministry of Education and Research (BMBF), #0314000 to MP, US, KFXM and NS; Triticeae Coordinated Agricultural Project, USDA-NIFA #2011-68002-30029 to GJM; and Agriculture and Food Research Initiative Plant Genome, Genetics and Breeding Program of USDA’s Cooperative State Research and Extension Service, #2009-65300- 05645 to GJM

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

<p>Abstract</p> <p>Background</p> <p>The improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (<it>Secale cereale </it>L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology.</p> <p>Results</p> <p>More than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool.</p> <p>Conclusions</p> <p>The established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye.</p

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence

BACKGROUND: Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics. RESULTS: We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes. CONCLUSIONS: Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association

De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

<p>Abstract</p> <p>Background</p> <p><it>De novo </it>sequencing the entire genome of a large complex plant genome like the one of barley (<it>Hordeum vulgare </it>L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable.</p> <p>Results</p> <p>To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes.</p> <p>By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies.</p> <p>Conclusion</p> <p>The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.</p

A homolog of <i>blade-on-petiole</i> <i>1</i> and <i>2</i> (<i>BOP1/2</i>) controls internode length and homeotic changes of the barley inflorescence

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected