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Premise: Traditional methods of ploidal-level estimation are tedious; using DNA sequence data for cytotype estimation is an ideal alternative. Multiple statistical approaches to leverage sequence data for ploidy inference based on site-based heterozygosity have been developed. However, these approaches may require high-coverage sequence data, use inappropriate probability distributions, or have additional statistical shortcomings that limit inference abilities. We introduce nQuack, an open-source R package that addresses the main shortcomings of current methods., Methods and Results: nQuack performs model selection for improved ploidy predictions. Here, we implement expectation maximization algorithms with normal, beta, and beta-binomial distributions. Using extensive computer simulations that account for variability in sequencing depth, as well as real data sets, we demonstrate the utility and limitations of nQuack., Conclusions: Inferring ploidy based on site-based heterozygosity alone is difficult. Even though nQuack is more accurate than similar methods, we suggest caution when relying on any site-based heterozygosity method to infer ploidy., (© 2024 The Author(s). Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise: Digitized biodiversity data offer extensive information; however, obtaining and processing biodiversity data can be daunting. Complexities arise during data cleaning, such as identifying and removing problematic records. To address these issues, we created the R package Geographic And Taxonomic Occurrence R-based Scrubbing (gatoRs)., Methods and Results: The gatoRs workflow includes functions that streamline downloading records from the Global Biodiversity Information Facility (GBIF) and Integrated Digitized Biocollections (iDigBio). We also created functions to clean downloaded specimen records. Unlike previous R packages, gatoRs accounts for differences in download structure between GBIF and iDigBio and allows for user control via interactive cleaning steps., Conclusions: Our pipeline enables the scientific community to process biodiversity data efficiently and is accessible to the R coding novice. We anticipate that gatoRs will be useful for both established and beginning users. Furthermore, we expect our package will facilitate the introduction of biodiversity-related concepts into the classroom via the use of herbarium specimens., (© 2024 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise: Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples., Methods and Results: We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively., Conclusions: This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research., (© 2023 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise: Commonly used molecular techniques such as next-generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high-quality genomic DNA. The opportunities provided by high-throughput, next-generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high-yield, high-quality, and highly scalable DNA extraction method is still needed., Methods and Results: We present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi-column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms)., Conclusions: Application of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi-preps) or small (96-well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short-read DNA sequencing., (© 2021 Mavrodiev et al. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of the Botanical Society of America.)

Premise: Large phylogenetic data sets have often been restricted to small numbers of loci from GenBank, and a vetted sampling-to-sequencing phylogenomic protocol scaling to thousands of species is not yet available. Here, we report a high-throughput collections-based approach that empowers researchers to explore more branches of the tree of life with numerous loci., Methods: We developed an integrated Specimen-to-Laboratory Information Management System (SLIMS), connecting sampling and wet lab efforts with progress tracking at each stage. Using unique identifiers encoded in QR codes and a taxonomic database, a research team can sample herbarium specimens, efficiently record the sampling event, and capture specimen images. After sampling in herbaria, images are uploaded to a citizen science platform for metadata generation, and tissue samples are moved through a simple, high-throughput, plate-based herbarium DNA extraction and sequencing protocol., Results: We applied this sampling-to-sequencing workflow to ~15,000 species, producing for the first time a data set with ~50% taxonomic representation of the "nitrogen-fixing clade" of angiosperms., Discussion: The approach we present is appropriate at any taxonomic scale and is extensible to other collection types. The widespread use of large-scale sampling strategies repositions herbaria as accessible but largely untapped resources for broad taxonomic sampling with thousands of species., (© 2021 Folk et al. Applications in Plant Sciences is published by Wiley Periodicals LLC on behalf of the Botanical Society of America.)

Premise: Putatively single-copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly related Memecylon and Tibouchina (Melastomataceae)., Methods: We present a two-tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome-skimming data from within the focal clades., Results: We sequenced 193 loci common to Memecylon and Tibouchina . Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome-skimming data within a focal clade were more successful than probes designed from other sources., Discussion: Our pipeline successfully identified and targeted SCN loci in Memecylon and Tibouchina , enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources., (© 2020 Jantzen et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

The past six years have seen the rapid growth of studies of CRISPR/Cas9 in plant genome editing, a method that enormously facilitates both basic research and practical applications. Most studies have focused on genetic model species, but plant species that are not genetic models may also be economically important or biologically significant, or both. However, developing the CRISPR/Cas9 system in a nongenetic model is challenging. Here, we summarize CRISPR/Cas9 applications in 45 plant genera across 24 families and provide a reference for practical application of CRISPR in nongenetic model plant systems. Suggestions for selecting plant species and target genes are given for proof-of-principle CRISPR studies, and the processes of vector construction are reviewed. We recommend using transient assays to identify a desired CRISPR/Cas9 system in a nongenetic model. We then review methods of plant transformation and describe approaches, using regenerated transgenic plants, for evaluating CRISPR editing results. Lastly, potential future applications of CRISPR in nongenetic model plant species are discussed. This review provides a road map for developing CRISPR in nongenetic models, an application that holds enormous potential in plant biology., (© 2020 Shan et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

Premise of the Study: Phenological annotation models computed on large-scale herbarium data sets were developed and tested in this study., Methods: Herbarium specimens represent a significant resource with which to study plant phenology. Nevertheless, phenological annotation of herbarium specimens is time-consuming, requires substantial human investment, and is difficult to mobilize at large taxonomic scales. We created and evaluated new methods based on deep learning techniques to automate annotation of phenological stages and tested these methods on four herbarium data sets representing temperate, tropical, and equatorial American floras., Results: Deep learning allowed correct detection of fertile material with an accuracy of 96.3%. Accuracy was slightly decreased for finer-scale information (84.3% for flower and 80.5% for fruit detection)., Discussion: The method described has the potential to allow fine-grained phenological annotation of herbarium specimens at large ecological scales. Deeper investigation regarding the taxonomic scalability of this approach is needed.

Premise of the Study: Herbarium specimens provide a robust record of historical plant phenology (the timing of seasonal events such as flowering or fruiting). However, the difficulty of aggregating phenological data from specimens arises from a lack of standardized scoring methods and definitions for phenological states across the collections community., Methods and Results: To address this problem, we report on a consensus reached by an iDigBio working group of curators, researchers, and data standards experts regarding an efficient scoring protocol and a data-sharing protocol for reproductive traits available from herbarium specimens of seed plants. The phenological data sets generated can be shared via Darwin Core Archives using the Extended MeasurementOrFact extension., Conclusions: Our hope is that curators and others interested in collecting phenological trait data from specimens will use the recommendations presented here in current and future scoring efforts. New tools for scoring specimens are reviewed.

Building on centuries of research based on herbarium specimens gathered through time and around the globe, a new era of discovery, synthesis, and prediction using digitized collections data has begun. This paper provides an overview of how aggregated, open access botanical and associated biological, environmental, and ecological data sets, from genes to the ecosystem, can be used to document the impacts of global change on communities, organisms, and society; predict future impacts; and help to drive the remediation of change. Advocacy for botanical collections and their expansion is needed, including ongoing digitization and online publishing. The addition of non-traditional digitized data fields, user annotation capability, and born-digital field data collection enables the rapid access of rich, digitally available data sets for research, education, informed decision-making, and other scholarly and creative activities. Researchers are receiving enormous benefits from data aggregators including the Global Biodiversity Information Facility (GBIF), Integrated Digitized Biocollections (iDigBio), the Atlas of Living Australia (ALA), and the Biodiversity Heritage Library (BHL), but effective collaboration around data infrastructures is needed when working with large and disparate data sets. Tools for data discovery, visualization, analysis, and skills training are increasingly important for inspiring novel research that improves the intrinsic value of physical and digital botanical collections.

Premise of the Study: The One Thousand Plant Transcriptomes Project (1KP, 1000+ assembled plant transcriptomes) provides an enormous resource for developing microsatellite loci across the plant tree of life. We developed loci from these transcriptomes and tested their utility., Methods and Results: Using software packages and custom scripts, we identified microsatellite loci in 1KP transcriptomes. We assessed the potential for cross-amplification and whether loci were biased toward exons, as compared to markers derived from genomic DNA. We characterized over 5.7 million simple sequence repeat (SSR) loci from 1334 plant transcriptomes. Eighteen percent of loci substantially overlapped with open reading frames (ORFs), and electronic PCR revealed that over half the loci would amplify successfully in conspecific taxa. Transcriptomic SSRs were approximately three times more likely to map to translated regions than genomic SSRs., Conclusions: We believe microsatellites still have a place in the genomic age-they remain effective and cost-efficient markers. The loci presented here are a valuable resource for researchers.

Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

Premise of the Study: Microsatellites, despite being commonly used population-level markers, contain biases because scoring relies solely on fragment length. Their complexity can lead to homoplasy, the effects of which are poorly understood. We investigated the impact of using fragment lengths, repeats, or flanking region sequences on common population-level analyses., Methods: Five polymorphic microsatellite markers amplified across the central Florida scrub endemic Polygala lewtonii (Polygalaceae) and its close, widespread congener P. polygama. We genotyped 147 individuals of P. lewtonii and 156 of P. polygama, and sequenced the amplicons of four markers across all individuals. We ran basic statistics, spatial clustering analysis, historical demographics, and migration tests., Results: One population of intermediate morphology was genetically clearly identified as P. polygama, making it the southernmost population of this species. Statistics were comparable between the fragment length and repeat numbers, with some notable differences. Flanking regions exhibited surprisingly high polymorphism between species, and between geographically distant conspecific populations., Discussion: The increasing use of markers developed in one species and amplified in another is only a good practice if precautions are taken, notably the sequencing of the fragments between species and populations. Flanking region sequences are a useful marker at the interspecific level.

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundation's (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7-36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

Premise of the Study: Targeted sequencing using next-generation sequencing (NGS) platforms offers enormous potential for plant systematics by enabling economical acquisition of multilocus data sets that can resolve difficult phylogenetic problems. However, because discovery of single-copy nuclear (SCN) loci from NGS data requires both bioinformatics skills and access to high-performance computing resources, the application of NGS data has been limited., Methods and Results: We developed MarkerMiner 1.0, a fully automated, open-access bioinformatic workflow and application for discovery of SCN loci in angiosperms. Our new tool identified as many as 1993 SCN loci from transcriptomic data sampled as part of four independent test cases representing marker development projects at different phylogenetic scales., Conclusions: MarkerMiner is an easy-to-use and effective tool for discovery of putative SCN loci. It can be run locally or via the Web, and its tabular and alignment outputs facilitate efficient downstream assessments of phylogenetic utility, locus selection, intron-exon boundary prediction, and primer or probe development.

Premise of the Study: Visualizing flower epidermal cells is often desirable for investigating the interaction between flowers and their pollinators, in addition to the broader range of ecological interactions in which flowers are involved. We developed a protocol for visualizing petal epidermal cells without the limitations of the commonly used method of scanning electron microscopy (SEM)., Methods: Flower material was collected and fixed in glutaraldehyde, followed by dehydration in an ethanol series. Flowers were dissected to collect petals, and subjected to a Histo-Clear series to remove the cuticle. Material was then stained with aniline blue, mounted on microscope slides, and imaged using a compound fluorescence microscope to obtain optical sections that were reconstructed into a 3D image., Results: This optical sectioning method yielded high-quality images of the petal epidermal cells with virtually no damage to cells. Flowers were processed in larger batches than are possible using common SEM methods. Also, flower size was not a limiting factor as often observed in SEM studies. Flowers up to 5 cm in length were processed and mounted for visualization., Conclusions: This method requires no special equipment for sample preparation prior to imaging and should be seen as an alternative method to SEM.

Premise of the Study: We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS) of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms. •, Methods and Results: A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots), which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×), even for the two monocots. •, Conclusions: Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving ∼50× mean coverage). However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96) available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms.