Cell-free transcription-translation (TXTL) has recently emerged as a versatile technology to engineer biological systems. In this chapter, we show how an all E. coli TXTL system can be used to build synthetic cell prototypes. We describe methods to encapsulate TXTL reactions in cell-sized liposomes, with an emphasis on the composition of the external solution and lipid bilayer. Cell-free expression is quantitatively described in bulk reactions and liposomes for three proteins: the soluble reporter protein eGFP, the membrane proteins alpha-hemolysin (AH) from Staphylococcus aureus, and the mechanosensitive channel of large conductance (MscL) from E. coli.

Arid environments provide ideal ground for investigating the mechanisms of adaptive evolution. High temperatures and low water availability are relentless stressors for many endotherms, including birds; yet birds persist in deserts. While physiological adaptation likely involves metabolic phenotypes, the underlying mechanisms (plasticity, genetics) are largely uncharacterized. To explore this, we took an intra-specific approach that focused on a species that is resident over a mesic to arid gradient, the Karoo scrub-robin (Cercotrichas coryphaeus). Specifically, we integrated environmental (climatic and primary productivity), physiological (metabolic rates: a measure of energy expenditure), genotypic (genetic variation underlying the machinery of energy production) and microbiome (involved in processing food from where energy is retrieved) data, to infer the mechanism of physiological adaptation. We that found the variation in energetic physiology phenotypes and gut microbiome composition are associated with environmental features as well as with variation in genes underlying energy metabolic pathways. Specifically, we identified a small list of candidate adaptive genes, some of them with known ties to relevant physiology phenotypes. Together our results suggest that selective pressures on energetic physiology mediated by genes related to energy homeostasis and possibly microbiota composition may facilitate adaptation to local conditions and provide an explanation to the high avian intra-specific divergence observed in harsh environments. This article is protected by copyright. All rights reserved.

Premise Hybrid capture with high-throughput sequencing (Hyb-Seq) is a powerful tool for evolutionary studies. The applicability of an Asteraceae family-specific Hyb-Seq probe set and the outcomes of different phylogenetic analyses are investigated here. Methods Hyb-Seq data from 112 Asteraceae samples were organized into groups at different taxonomic levels (tribe, genus, and species). For each group, data sets of non-paralogous loci were built and proportions of parsimony informative characters estimated. The impacts of analyzing alternative data sets, removing long branches, and type of analysis on tree resolution and inferred topologies were investigated in tribe Cichorieae. Results Alignments of the Asteraceae family-wide Hyb-Seq locus set were parsimony informative at all taxonomic levels. Levels of resolution and topologies inferred at shallower nodes differed depending on the locus data set and the type of analysis, and were affected by the presence of long branches. Discussion The approach used to build a Hyb-Seq locus data set influenced resolution and topologies inferred in phylogenetic analyses. Removal of long branches improved the reliability of topological inferences in maximum likelihood analyses. The Astereaceae Hyb-Seq probe set is applicable at multiple taxonomic depths, which demonstrates that probe sets do not necessarily need to be lineage-specific.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next-generation or massively parallel sequencing has made it possible to routinely obtain nucleotide-level data with relative ease. These short-read sequencing platforms offer deep coverage with unparalleled read accuracy in high-complexity genomic regions but encounter numerous difficulties in the low-complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy-number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long-read sequencing data on a pocket-sized device known as the MinION. Nanopore-based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor-intensive, time-consuming, and costly mainstay deep-sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment-free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR-free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean-up Support Protocol 3: Suggested data analysis pipeline

Exon capture across species has been one of the most broadly applied approaches to acquire multi-locus data in phylogenomic studies of non-model organisms. Methods for assembling loci from short-read sequences (eg, Illumina platforms) that rely on mapping reads to a reference genome may not be suitable for studies comprising species across a wide phylogenetic spectrum; thus, de novo assembling methods are more generally applied. Current approaches for assembling targeted exons from short reads are not particularly optimized as they cannot (1) assemble loci with low read depth, (2) handle large files efficiently, and (3) reliably address issues with paralogs. Thus, we present Assexon: a streamlined pipeline that de novo assembles targeted exons and their flanking sequences from raw reads. We tested our method using reads from Lepisosteus osseus (4.37 Gb) and Boleophthalmus pectinirostris (2.43 Gb), which are captured using baits that were designed based on genome sequence of Lepisosteus oculatus and Oreochromis niloticus, respectively. We compared performance of Assexon to PHYLUCE and HybPiper, which are commonly used pipelines to assemble ultra-conserved element (UCE) and Hyb-seq data. A custom exon capture analysis pipeline (CP) developed by Yuan et al was compared as well. Assexon accurately assembled more than 3400 to 3800 (20%-28%) loci than PHYLUCE and more than 1900 to 2300 (8%-14%) loci than HybPiper across different levels of phylogenetic divergence. Assexon ran at least twice as fast as PHYLUCE and HybPiper. Number of loci assembled using CP was comparable with Assexon in both tests, while Assexon ran at least 7 times faster than CP. In addition, some steps of CP require the user’s interaction and are not fully automated, and this user time was not counted in our calculation. Both Assexon and CP retrieved no paralogs in the testing runs, but PHYLUCE and Hybpiper did. In conclusion, Assexon is a tool for accurate and efficient assembling of large read sets from exon capture experiments. Furthermore, Assexon includes scripts to filter poorly aligned coding regions and flanking regions, calculate summary statistics of loci, and select loci with reliable phylogenetic signal. Assexon is available at https://github.com/yhadevol/Assexon.

Abstract. Army ants are a charismatic group of organisms characterized by a suite of morphological and behavioral adaptations that includes obligate collective

Premise The genus Antennaria has a complex evolutionary history due to dioecism, excessive polyploidy, and the evolution of polyploid agamic complexes. We developed microsatellite markers from A. corymbosa to investigate genetic diversity and population genetic structure in Antennaria species. Methods and Results Twenty-four novel microsatellite markers (16 nuclear and eight chloroplast) were developed from A. corymbosa using an enriched genomic library. Ten polymorphic nuclear markers were used to characterize genetic variation in five populations of A. corymbosa. One to four alleles were found per locus, and the expected heterozygosity and fixation index ranged from 0.00 to 0.675 and −0.033 to 0.610, respectively. We were also able to successfully amplify these markers in five additional Antennaria species. Conclusions These markers are promising tools to study the population genetics of sexual Antennaria species and to investigate interspecific gene flow, clonal diversity, and parentage of Antennaria polyploid agamic complexes.

Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 89 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian Cyrtandra lineage, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high-throughput sequencing, and modern phylogenomics tools to test 31 Hawaiian Cyrtandra samples (22 species, two putative hybrids, four species with two samples each, one species with four samples) and two outgroups for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species-tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. We expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and species-tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Multiple hybridizations were inferred, but many were on deeper branches of the island lineages suggesting a long history of hybridization. We demonstrated the utility of high-throughput sequencing and a phylogenomic approach using 569 loci to understanding species relationships and gene flow in the presence of ILS. This article is protected by copyright. All rights reserved.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 (OsHV-1) have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve disease. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C.gigas. This article is protected by copyright. All rights reserved.

Abstract. The typical owl family (Strigidae) comprises 194 species in 28 genera, 14 of which are monotypic. Relationships within and among genera in the typica