Versatile DNA extraction from diverse plant taxa using ionic liquids and magnetic ionic liquids: a methodological breakthrough for enhanced sample utility.

BACKGROUND: There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. RESULTS: In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. CONCLUSIONS: The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.

Isolation of DNA from plant tissues using a miniaturized matrix solid-phase dispersion approach featuring ionic liquid and magnetic ionic liquid solvents.

The isolation of high-quality plant genomic DNA is a major prerequisite in many plant biomolecular analyses involving nucleic acid amplification. Conventional plant cell lysis and DNA extraction methods involve lengthy sample preparation procedures that often require large amounts of sample and chemicals, high temperatures and multiple liquid transfer steps which can introduce challenges for high throughput applications. In this study, a simple, rapid, miniaturized ionic liquid (IL)-based extraction method was developed for the isolation of genomic DNA from milligram fragments of Arabidopsis thaliana plant tissue. This method is based on a modification of vortex-assisted matrix solid-phase dispersion (VA-MSPD) in which the trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14+][NTf2-]) IL or trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(hfacac)3-]) magnetic IL (MIL) was directly applied to treated plant tissue (∼1.5 mg) and dispersed in an agate mortar to facilitate plant cell lysis and DNA extraction, followed by recovery of the mixture with a qPCR compatible co-solvent. This study represents the first approach to use ILs and MILs in a MSPD procedure to facilitate plant cell lysis and DNA extraction. The DNA-enriched IL- and MIL-cosolvent mixtures were directly integrated into the qPCR buffer without inhibiting the reaction while also circumventing the need for additional purification steps prior to DNA amplification. Under optimum conditions, the IL and MIL yielded 2.87 ± 0.28 and 1.97 ± 0.59 ng of DNA/mg of plant tissue, respectively. Furthermore, the mild extraction conditions used in the method enabled plant DNA in IL- and MIL-cosolvent mixtures to be preserved from degradation at room temperature.

Phylogenomic loci define the generic boundaries of Gochnatieae and improve resolution at the species level in Moquiniastrum (Compositae).

Understanding the evolution of the tribe Gochnatieae (Compositae) has been the subject of considerable effort in the past decade. This is due to the key position of this tribe in the phylogeny of the sunflower family and the corresponding implications for biogeographic and morphological evolution of Compositae. Previous studies have confirmed the monophyly of this tribe as well as most of the genera that belong to it. However, phylogenetic resolution of Gochnatieae at both the genus- and species-level has remained poor. A subset of new phylogenomic loci used in this study has proven effective and has improved phylogenetic resolution in this group. The results of this work demonstrate Gochnatieae is a well-supported clade comprised of nine genera (Anastraphia, Cnicothamnus, Cyclolepis, Gochnatia, Moquiniastrum, Nahuatlea, Pentaphorus, Richterago, Tehuasca). One recently described genus, Vickia, was not included in this study; but its placement in Gochnatieae as a tenth genus in the tribe is well-justified. The monospecific Cyclolepis, which had been circumscribed within the tribe since its inception but was subsequently removed and designated as incertae sedis since 2014, is also shown to belong to Gochnatieae. We confirmed the monophyletic Moquiniastrum with two well-supported subclades. Ancestral area reconstruction analyses show that Gochnatieae originated in Eastern South America about 53 my. Apparently, except for Cyclolepis and Richterago, the ancestors of the other genera of Gochnatieae originated about 44 my from an area that now corresponds to the central Andes. The presence of the genera in the Chaco phytogeographic province, central Chile, and Mexico-United States-Caribbean is a result of dispersal from the central Andes. The ancestral distribution of Moquiniastrum corresponds to a large area comprising Eastern South America and the current central Andes, about 32 my. Ancestral character state reconstruction that included four characters indicates several states associated with complex plant reproductive biology such as gynodioecy, gynomonoecy, and polygamodioecy are derived in Gochnatieae as are heterogamous capitula (in Moquiniastrum and Richterago), dimorphic and subdimorphic corollas (in Cnicothamnus, Moquiniastrum, and Richterago), and the presence of marginal female corollas (in Moquiniastrum and Richterago). Within Moquiniastrum, two subclades (Densicephalum and Polymorphum) exhibit divergent patterns of trait evolution associated with these reproductive characters which suggests this genus can serve as a model to understand the sexual system evolution in plants.

Simple and efficient isolation of plant genomic DNA using magnetic ionic liquids.

BACKGROUND: Plant DNA isolation and purification is a time-consuming and laborious process relative to epithelial and viral DNA sample preparation due to the cell wall. The lysis of plant cells to free intracellular DNA normally requires high temperatures, chemical surfactants, and mechanical separation of plant tissue prior to a DNA purification step. Traditional DNA purification methods also do not aid themselves towards fieldwork due to the numerous chemical and bulky equipment requirements. RESULTS: In this study, intact plant tissue was coated by hydrophobic magnetic ionic liquids (MILs) and ionic liquids (ILs) and allowed to incubate under static conditions or dispersed in a suspension buffer to facilitate cell disruption and DNA extraction. The DNA-enriched MIL or IL was successfully integrated into the qPCR buffer without inhibiting the reaction. The two aforementioned advantages of ILs and MILs allow plant DNA sample preparation to occur in one minute or less without the aid of elevated temperatures or chemical surfactants that typically inhibit enzymatic amplification methods. MIL or IL-coated plant tissue could be successfully integrated into a qPCR assay without the need for custom enzymes or manual DNA isolation/purification steps that are required for conventional methods. CONCLUSIONS: The limited amount of equipment, chemicals, and time required to disrupt plant cells while simultaneously extracting DNA using MILs makes the described procedure ideal for fieldwork and lab work in low resource environments.

Green plant genomes: What we know in an era of rapidly expanding opportunities.

Green plants play a fundamental role in ecosystems, human health, and agriculture. As de novo genomes are being generated for all known eukaryotic species as advocated by the Earth BioGenome Project, increasing genomic information on green land plants is essential. However, setting standards for the generation and storage of the complex set of genomes that characterize the green lineage of life is a major challenge for plant scientists. Such standards will need to accommodate the immense variation in green plant genome size, transposable element content, and structural complexity while enabling research into the molecular and evolutionary processes that have resulted in this enormous genomic variation. Here we provide an overview and assessment of the current state of knowledge of green plant genomes. To date fewer than 300 complete chromosome-scale genome assemblies representing fewer than 900 species have been generated across the estimated 450,000 to 500,000 species in the green plant clade. These genomes range in size from 12 Mb to 27.6 Gb and are biased toward agricultural crops with large branches of the green tree of life untouched by genomic-scale sequencing. Locating suitable tissue samples of most species of plants, especially those taxa from extreme environments, remains one of the biggest hurdles to increasing our genomic inventory. Furthermore, the annotation of plant genomes is at present undergoing intensive improvement. It is our hope that this fresh overview will help in the development of genomic quality standards for a cohesive and meaningful synthesis of green plant genomes as we scale up for the future.

Author Correction: Microfluidic Enrichment Barcoding (MEBarcoding): a new method for high throughput plant DNA barcoding.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microfluidic Enrichment Barcoding (MEBarcoding): a new method for high throughput plant DNA barcoding.

DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high-throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages - many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases.

Data Release: DNA barcodes of plant species collected for the Global Genome Initiative for Gardens Program, National Museum of Natural History, Smithsonian Institution.

The Global Genome Initiative has sequenced and released 1961 DNA barcodes for genetic samples obtained as part of the Global Genome Initiative for Gardens Program. The dataset includes barcodes for 29 plant families and 309 genera that did not have sequences flagged as barcodes in GenBank and sequences from officially recognized barcoding genetic markers meet the data standard of the Consortium for the Barcode of Life. The genetic samples were deposited in the Smithsonian Institution's National Museum of Natural History Biorepository and their records were made public through the Global Genome Biodiversity Network's portal. The DNA barcodes are now available on GenBank.

A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta.

PREMISE OF THE STUDY: Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. METHODS AND RESULTS: Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. CONCLUSIONS: Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships.

An expanded nuclear phylogenomic PCR toolkit for Sapindales.

PREMISE OF THE STUDY: We tested PCR amplification of 91 low-copy nuclear gene loci in taxa from Sapindales using primers developed for Bursera simaruba (Burseraceae). METHODS AND RESULTS: Cross-amplification of these markers among 10 taxa tested was related to their phylogenetic distance from B. simaruba. On average, each Sapindalean taxon yielded product for 53 gene regions (range: 16-90). Arabidopsis thaliana (Brassicales), by contrast, yielded product for two. Single representatives of Anacardiaceae and Rutacaeae yielded 34 and 26 products, respectively. Twenty-six primer pairs worked for all Burseraceae species tested if highly divergent Aucoumea klaineana is excluded, and eight of these amplified product in every Sapindalean taxon. CONCLUSIONS: Our study demonstrates that customized primers for Bursera can amplify product in a range of Sapindalean taxa. This collection of primer pairs, therefore, is a valuable addition to the toolkit for nuclear phylogenomic analyses of Sapindales and warrants further investigation.

Development of novel, exon-primed intron-crossing (EPIC) markers from EST databases and evaluation of their phylogenetic utility in Commiphora (Burseraceae).

PREMISE OF THE STUDY: Novel nuclear exon-primed intron-crossing (EPIC) markers were developed to increase phylogenetic resolution among recently diverged lineages in the frankincense and myrrh family, Burseraceae, using Citrus, Arabidopsis, and Oryza genome resources. • METHODS AND RESULTS: Primer pairs for 48 nuclear introns were developed using the genome resource IntrEST and were screened using species of Commiphora and other Burseraceae taxa. Four putative intron regions (RPT6A, BXL2, mtATP Synthase D, and Rab6) sequenced successfully for multiple taxa and recovered phylogenies consistent with those of existing studies. In some cases, these regions yielded informative sequence variation on par with that of the nuclear ribosomal DNA internal transcribed spacer. • CONCLUSIONS: The combination of freely available genome resources and our design criteria have uncovered four single-copy nuclear intron regions that are useful for phylogenetic reconstruction of Burseraceae taxa. Because our EPIC primers also amplify Arabidopsis, we recommend their trial in other rosid and eudicot lineages.