Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.

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Chloroplast genomes: diversity, evolution, and applications in genetic engineering

Premise of the study: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high-throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user-friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae). Methods and Results: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus. Conclusions: HybPiper was designed for Linux and Mac OS X and is freely ...

https://bioone.org/journals/applications-in-plant-sciences/volume-4/issue-7/apps.1600016/HybPiper--Extracting-Coding-Sequence-and-Introns-for-Phylogenetics-from/10.3732/apps.1600016.pdf

HybPiper: Extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment.

Summary
The past 100 years of ecological research has seen substantial progress in understanding the natural world and likely effects of change, whether natural or anthropogenic. Traditional ecological approaches underpin such advances, but would additionally benefit from recent developments in the sequence-based quantification of biodiversity from the fields of molecular ecology and genomics. By building on a long and rich history of molecular taxonomy and taking advantage of the new generation of DNA sequencing technologies, we are gaining previously impossible insights into alpha and beta diversity from all domains of life, irrespective of body size. While a number of complementary reviews are available in specialist journals, our aim here is to succinctly describe the different technologies available within the omics toolbox and showcase the opportunities available to contemporary ecologists to advance our understanding of biodiversity and its potential roles in ecosystems.
Starting in the field, we walk the reader through sampling and preservation of genomic material, including typical taxonomy marker genes used for species identification. Moving on to the laboratory, we cover nucleic acid extraction approaches and highlight the principal features of using marker gene assessment, metagenomics, metatranscriptomics, single-cell genomics and targeted genome sequencing as complementary approaches to assess the taxonomic and functional characteristics of biodiversity. We additionally provide clear guidance on the forms of DNA found in the environmental samples (e.g. environmental vs. ancient DNA) and highlight a selection of case studies, including the investigation of trophic relationships/food webs. Given the maturity of sequence-based identification of prokaryotes and microbial eukaryotes, more exposure is given to macrobial communities. We additionally illustrate current approaches to genomic data analysis and highlight the exciting prospects of the publicly available data underpinning published sequence-based studies.
Given that ecology ‘has to count’, we identify the impact that molecular genetic analyses have had on stakeholders and end-users and predict future developments for the fields of biomonitoring. Furthermore, we conclude by highlighting future opportunities in the field of systems ecology afforded by effective engagement between the fields of traditional and molecular ecology.

/pdf/the-ecologist-s-field-guide-to-sequence-based-identification-3m8eqnb369.pdf

The ecologist's field guide to sequence‐based identification of biodiversity

The rapid expansion of next-generation sequencing has yielded a powerful array of tools to address fundamental biological questions at a scale that was inconceivable just a few years ago. Various genome-partitioning strategies to sequence select subsets of the genome have emerged as powerful alternatives to whole-genome sequencing in ecological and evolutionary genomic studies. High-throughput targeted capture is one such strategy that involves the parallel enrichment of preselected genomic regions of interest. The growing use of targeted capture demonstrates its potential power to address a range of research questions, yet these approaches have yet to expand broadly across laboratories focused on evolutionary and ecological genomics. In part, the use of targeted capture has been hindered by the logistics of capture design and implementation in species without established reference genomes. Here we aim to (i) increase the accessibility of targeted capture to researchers working in nonmodel taxa by discussing capture methods that circumvent the need of a reference genome, (ii) highlight the evolutionary and ecological applications where this approach is emerging as a powerful sequencing strategy and (iii) discuss the future of targeted capture and other genome-partitioning approaches in the light of the increasing accessibility of whole-genome sequencing. Given the practical advantages and increasing feasibility of high-throughput targeted capture, we anticipate an ongoing expansion of capture-based approaches in evolutionary and ecological research, synergistic with an expansion of whole-genome sequencing.

Targeted capture in evolutionary and ecological genomics

The systematics of grasses has advanced through applications of plastome phylogenomics, although studies have been largely limited to subfamilies or other subgroups of Poaceae Here we present a plastome phylogenomic analysis of 250 complete plastomes (179 genera) sampled from 44 of the 52 tribes of Poaceae Plastome sequences were determined from high throughput sequencing libraries and the assemblies represent over 287 Mbases of sequence data Phylogenetic signal was characterized in 14 partitions, including (1) complete plastomes; (2) protein coding regions; (3) noncoding regions; and (4) three loci commonly used in single and multi-gene studies of grasses Each of the four main partitions was further refined, alternatively including or excluding positively selected codons and also the gaps introduced by the alignment All 76 protein coding plastome loci were found to be predominantly under purifying selection, but specific codons were found to be under positive selection in 65 loci The loci that have been widely used in multi-gene phylogenetic studies had among the highest proportions of positively selected codons, suggesting caution in the interpretation of these earlier results Plastome phylogenomic analyses confirmed the backbone topology for Poaceae with maximum bootstrap support (BP) Among the 14 analyses, 82 clades out of 309 resolved were maximally supported in all trees Analyses of newly sequenced plastomes were in agreement with current classifications Five of seven partitions in which alignment gaps were removed retrieved Panicoideae as sister to the remaining PACMAD subfamilies Alternative topologies were recovered in trees from partitions that included alignment gaps This suggests that ambiguities in aligning these uncertain regions might introduce a false signal Resolution of these and other critical branch points in the phylogeny of Poaceae will help to better understand the selective forces that drove the radiation of the BOP and PACMAD clades comprising more than 999% of grass diversity

A 250 plastome phylogeny of the grass family (Poaceae): topological support under different data partitions

Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next-generation sequencing methods. In this study, we develop an easy and cost-effective protocol for in-solution enrichment hybridization capture of complete chloroplast genomes applicable at deep-multiplexed levels. The protocol uses cheap in-house species-specific probes developed via long-range PCR of the entire chloroplast. Barcoded libraries are constructed, and in-solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes.

Vincent Maillol

Papers

Cost-effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies.