Highly Variable Chloroplast Markers for Evaluating Plant Phylogeny at Low Taxonomic Levels and for DNA Barcoding

BACKGROUND: At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species. METHODOLOGY/PRINCIPAL FINDINGS: We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnS(UGA)-trnG(UCC), petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS(UGA)-trnG(UCC), trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species. SIGNIFICANCE/CONCLUSIONS: Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding

Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis

Abstract Background Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo. Results The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery. Conclusions Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery.https://deepblue.lib.umich.edu/bitstream/2027.42/146540/1/12951_2018_Article_425.pd

Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution

Recent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth–death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies.Universidad de Costa Rica/[814-B8-257]/UCR/Costa RicaUniversidad de Costa Rica/[814-B6-140]/UCR/Costa RicaIDEA WILD/[]//Estados UnidosSociedad Colombiana de Orquideología/[]/SCO/ColombiaFundação de Amparo à Pesquisa do Estado de São Paulo/[11/08308-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[13/19124-1]/FAPESP/BrasilSwiss Orchid Foundation/[]//SuizaRoyal Botanic Gardens, Kew/[]//InglaterraSwedish Research Council/[2019-05191]//SueciaSwedish Foundation for Strategic Research/[FFL15-0196]/SSF/SueciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

<p>Abstract</p> <p>Background</p> <p>Milkweeds (<it>Asclepias </it>L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (<it>Asclepias syriaca </it>L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing <it>A. syriaca </it>as a model in ecology and evolution.</p> <p>Results</p> <p>A 0.5× genome of <it>A. syriaca </it>was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: <it>accD, clpP</it>, and <it>ycf1</it>. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/<it>copia</it>-like retroelements are the most common repeat type in the milkweed genome. At least one <it>A. syriaca </it>microread hit 88% of <it>Catharanthus roseus </it>(Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the <it>A. syriaca </it>genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed.</p> <p>Conclusions</p> <p>The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and <it>A. syriaca </it>in particular, as ecological and evolutionary models.</p

Biogeographical patterns and speciation of the genus Pinguicula (Lentibulariaceae) inferred by phylogenetic analyses

Earlier phylogenetic studies in the genus Pinguicua (Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matK and rpl32-trnL), from up to ca. 80% of the taxa in the genus Pinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genus Pinguicula has involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus

A new subfamilial and tribal classification of the pantropical flowering plant family Annonaceae informed by molecular phylogenetics

The pantropical flowering plant family Annonaceae is the most species-rich family of Magnoliales. Despite long-standing interest in the systematics of Annonaceae, no authoritative classification has yet been published in the light of recent molecular phylogenetic analyses. Here, using the largest, most representative, molecular dataset compiled on Annonaceae to date, we present, for the first time, a robust family-wide phylogenetic tree and subsequent classification. We used a supermatrix of up to eight plastid markers sequenced from 193 ingroup and seven outgroup species. Some of the relationships at lower taxonomic levels are poorly resolved, but deeper nodes generally receive high support. Annonaceae comprises four major clades, which are here given the taxonomic rank of subfamily. The description of Annonoideae is amended, and three new subfamilies are described: Anaxagoreoideae, Ambavioideae and Malmeoideae. In Annonoideae, seven tribes are recognized, one of which, Duguetieae, is described as new. In Malmeoideae, seven tribes are recognized, six of which are newly described: Dendrokingstonieae, Fenerivieae, Maasieae, Malmeeae, Monocarpieae and Piptostigmateae. This new subfamilial and tribal classification is discussed against the background of previous classifications and characters to recognize subfamilies are reviewed. © 2012 The Linnean Society of London.link_to_subscribed_fulltex