Microsatellite markers for the endangered root holoparasite Dactylanthus taylorii (Balanophoraceae) from 454 pyrosequencing

? Premise of the study: Microsatellite loci were isolated and developed as polymorphic markers for the New Zealand endemic root holoparasite Dactylanthus taylorii for use in population and conservation genetics studies. ? Methods and Results: Shotgun 454 pyrosequencing was performed on genomic DNA pooled from three individuals of D. taylorii. From 61709 individual sequence reads, primers for 753 microsatellite loci were developed in silico and 72 of these were tested for consistent amplification and variability. Ten microsatellite loci were found to be polymorphic and consistently scorable when screened in 44 individuals from five geographically distant populations. The number of alleles per locus ranged from four to 16 with an average of 9.7, and average observed heterozygosity per locus was between 0.182 and 0.634. ? Conclusions: These polymorphic microsatellite markers establish an important resource for ongoing conservation initiatives and planned population genetic studies of D. taylorii.     

A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing

• Premise of the study: Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation. Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia. • Methods and Results: Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flanking primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further characterized by genotyping Dyckia samples at the level of populations and species. Three, five, and six cpSSRs were polymorphic among four individuals of D. limae, 12 individuals of D. dissitiflora, and 12 of D. pernambucana, respectively, with two to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per locus. Ten primer pairs cross-amplified with bromeliad genera from five subfamilies. • Conclusions: The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.     

Development of microsatellite markers in Fosterella rusbyi (Bromeliaceae) using 454 pyrosequencing

? Premise of the study: Polymorphic microsatellite markers were developed for Fosterella rusbyi (Bromeliaceae) to evaluate the population genetic structure and genetic diversity of natural populations of F. rusbyi and other Fosterella species in Bolivia. ? Methods and Results: 454 pyrosequencing technology was used to generate 73027 sequence reads from F. rusbyi DNA, which together contained 2796 perfect simple sequence repeats (SSRs). Primer pairs were designed for 30 loci, of which 15 were used to genotype 30 F. rusbyi plants from two geographical areas in Bolivia. All markers were polymorphic, with two to nine alleles in the overall sample. Cross-species amplification was tested in 10 additional Fosterella species. Seven loci showed consistent amplification in six or more species. ? Conclusions: The 15 SSR markers developed for F. rusbyi are promising candidates for population genetic analyses within F. rusbyi and other species of Fosterella.     

Culture independent survey of the microbiota of the glassy-winged sharpshooter (Homalodisca vitripennis) using 454 pyrosequencing

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is an invasive pest that has spread across the southern and western United States. H. vitripennis is highly polyphagous and voracious, feeding on at least 100 plant species and consuming up to 100 times its weight in xylem fluid daily. The insect is a vector of the phytopathogen Xylella fastidiosa (Wells), which is the causative agent of Pierce's disease in grapevines. To evaluate the microbial flora associated with H. vitripennis, total DNA extracts from hemolymph, alimentary canal excretions, and whole insect bodies were subjected to 16S rDNA pyrosequencing using the bTEFAP methodology and the resulting sequences (370-520 bp in length) were compared with a curated high quality 16S database derived from GenBank http://www.ncbi.nlm.nih.gov. Species from the genera Wolbachia, Delftia (formerly Pseudomonas), Pectobacterium, Moraxella, Serratia, Bacillus, and many others were detected and a comprehensive picture of the microbiome associated with H. vitripennis was established. Some of the bacteria identified in this report are initial discoveries; providing a breadth of knowledge to the microbial flora of this insect pest can serve as a reservoir of information for developing biological control strategies.     

Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing

In this study, 341, 246, and 386 positive clones with endo-β-1,4-glucanase, β-glucosidase, and endo-β-1,4-xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and K _m at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a cost-effective approach to obtain functional genes from metagenomic libraries.     

Lessons learned from microsatellite development for nonmodel organisms using 454 pyrosequencing

Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab‐tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time‐consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

Assessment of bacterial endosymbiont diversity in Otiorhynchusspp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

Background

Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.

Results

High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.

Conclusions

Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.

     

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Analysis of the bacterial community in a full-scale printing and dyeing wastewater treatment system based on T-RFLP and 454 pyrosequencing

In this study, the bacterial dynamics and structure compositions in the two-stage biological process of a full-scale printing and dyeing wastewater (PDW) treatment system were traced and analyzed by terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing techniques. T-RFLP analysis showed that the microbial communities experienced significant variation in the process of seed sludge adaptation to the PDW environments and were in constant evolution during the whole running period of the system, despite the constant COD and color removal effects. Pyrosequencing results indicated that the two-stage biological system harbored rather diverse bacteria, with Proteobacteria being the predominant phylum during the steady running period, although its microbial compositions differed. The first-stage aerobic tank was dominated by α-Proteobacteria (89.05% of Proteobacteria), whereas in the second-stage aerobic tank, β- and γ-Proteobacteria, besides α-Proteobacteria, were the dominant bacterial populations.     

Galactomannan from ambiguous Crazyweed (Oxytropis ambigua (Pall) DC)

A galactomannan with a molecular weight of 735 kDa was first isolated and purified from seeds of ambiguous crazyweed Oxytropis ambigua (Pall) DC. (family Leguminosae) with a yield of 3.6%. Its aqueous solutions displayed an optical activity ([alpha]D = 73.32 degrees) and high viscosity ([eta] = 644 ml g-1). Chemical analysis and 13C-NMR spectroscopy revealed the presence in the heteropolysaccharide of D-mannopyranose and D-glucopyranose at a molar ratio of 1.39:1. The linear backbone of its macromolecule consists of 1.4-beta-D-mannopyranose residues. Single beta-D-galactose residues substitute 72% of mannoses to form branches.