Applications in Plant Sciences

[This corrects the article DOI: 10.1002/aps3.11536.].

PREMISE: We recognized the need for a customized imaging protocol for plant specimens at the time of collection for the purpose of three-dimensional (3D) modeling, as well as the lack of a broadly applicable photogrammetry protocol that encompasses the heterogeneity of plant specimen geometries and the challenges introduced by processes such as wilting. METHODS AND RESULTS: We developed an equipment list and set of detailed protocols describing how to capture images of plant specimens in the field prior to their deformation (e...

PREMISE: Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of Theobroma cacao , structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits...

PREMISE: There are relatively few studies of flower color at landscape scales that can address the relative importance of competing mechanisms (e.g., biotic: pollinators; abiotic: ultraviolet radiation, drought stress) at landscape scales. METHODS: We developed an R shiny pipeline to sample color from images that were automatically downloaded using query results from a search using iNaturalist or the Global Biodiversity Information Facility (GBIF). The pipeline was used to sample ca...

PREMISE: Quantitative plant traits play a crucial role in biological research. However, traditional methods for measuring plant morphology are time consuming and have limited scalability. We present LeafMachine2, a suite of modular machine learning and computer vision tools that can automatically extract a base set of leaf traits from digital plant data sets. METHODS: LeafMachine2 was trained on 494,766 manually prepared annotations from 5648 herbarium images obtained from 288 institutions and representing 2663 species; it employs a set of plant component detection and segmentation algorithms to isolate individual leaves, petioles, fruits, flowers, wood samples, buds, and roots...

PREMISE: Variation in seed traits is common within and among populations of plant species and often has ecological and evolutionary implications. However, due to the time-consuming nature of manual seed measurements and the level of variability in imaging techniques, quantifying and interpreting the extent of seed variation can be challenging. METHODS: We developed a standardized high-throughput technique to measure seed number, as well as individual seed area and color, using a derived empirical scale to constrain area in Arabidopsis thaliana, Brassica rapa , and Mimulus guttatus ...

PREMISE: Current methods for maceration of plant tissue use hazardous chemicals. The new method described here improves the safety of dissection and maceration of soft plant tissues for microscopic imaging by using the harmless enzyme pectinase. METHODS AND RESULTS: Leaf material from a variety of land plants was obtained from living plants and dried herbarium specimens. Concentrations of aqueous pectinase and soaking schedules were optimized, and tissues were manually dissected while submerged in fresh solution following a soaking period...

PREMISE: Field images are important sources of information for research in the natural sciences. However, images that lack photogrammetric scale bars, including most iNaturalist observations, cannot yield accurate trait measurements. We introduce FieldPrism, a novel system of photogrammetric markers, QR codes, and software to automate the curation of snapshot vouchers. METHODS AND RESULTS: Our photogrammetric background templates (FieldSheets) increase the utility of field images by providing machine-readable scale bars and photogrammetric reference points to automatically correct image distortion and calculate a pixel-to-metric conversion ratio...

PREMISE: Transposable elements (TEs) make up more than half of the genomes of complex plant species and can modulate the expression of neighboring genes, producing significant variability of agronomically relevant traits. The availability of long-read sequencing technologies allows the building of genome assemblies for plant species with large and complex genomes. Unfortunately, TE annotation currently represents a bottleneck in the annotation of genome assemblies. METHODS AND RESULTS: We present a new functionality of the Next-Generation Sequencing Experience Platform (NGSEP) to perform efficient homology-based TE annotation...

Recent technological advances in long-read high-throughput sequencing and assembly methods have facilitated the generation of annotated chromosome-scale whole-genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high-molecular-weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast-freeze newly collected living samples to conserve high-quality DNA can be complicated when plants are only found in remote areas...

PREMISE: The functional annotation of genes is a crucial component of genomic analyses. A common way to summarize functional annotations is with hierarchical gene ontologies, such as the Gene Ontology (GO) Resource. GO includes information about the cellular location, molecular function(s), and products/processes that genes produce or are involved in. For a set of genes, summarizing GO annotations using pre-defined, higher-order terms (GO slims) is often desirable in order to characterize the overall function of the data set, and it is impractical to do this manually...

PREMISE: Robust standards to evaluate quality and completeness are lacking in eukaryotic structural genome annotation, as genome annotation software is developed using model organisms and typically lacks benchmarking to comprehensively evaluate the quality and accuracy of the final predictions. The annotation of plant genomes is particularly challenging due to their large sizes, abundant transposable elements, and variable ploidies. This study investigates the impact of genome quality, complexity, sequence read input, and method on protein-coding gene predictions...

PREMISE: The HybPiper pipeline has become one of the most widely used tools for the assembly of target capture data for phylogenomic analysis. After the production of locus sequences and before phylogenetic analysis, the identification of paralogs is a critical step for ensuring the accurate inference of evolutionary relationships. Algorithmic approaches using gene tree topologies for the inference of ortholog groups are computationally efficient and broadly applicable to non-model organisms, especially in the absence of a known species tree...

[This corrects the article DOI: 10.1002/aps3.11527.].

PREMISE: We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results. METHODS: Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA...

PREMISE: Plants with stiff, leathery leaves pose a challenge for standard DNA extraction protocols. These tissues are recalcitrant to mechanical disruption via TissueLyser (or analogous devices) and are often high in secondary metabolites. These compounding factors result in low yields, which may be sufficient for PCR amplification but are generally inadequate for genomic applications that require large quantities of high-quality DNA. Cycads in the genus Encephalartos exemplify these challenges, as this group of plants is fortified for life in harsh, dry habitats with notoriously thick and rigid leaves...

PREMISE: A novel protocol for rapid plant DNA extraction using microneedles is proposed, which supports botanic surveys, taxonomy, and systematics. This protocol can be conducted in the field with limited laboratory skills and equipment. The protocol is validated by sequencing and comparing the results with QIAGEN spin-column DNA extractions using BLAST analyses. METHODS AND RESULTS: Two sets of DNA extractions were conducted on 13 species spanning various leaf anatomies and phylogenetic lineages: (i) fresh leaves were punched with custom polymeric microneedle patches to recover genomic DNA, or (ii) QIAGEN DNA extractions...

PREMISE: Detailed studies of the fungi associated with lycophytes and ferns provide crucial insights into the early evolution of land plants. However, most investigations to date have assessed fern-fungus interactions based only on visual root inspection. In the present research, we establish and evaluate a metabarcoding protocol to analyze the fungal communities associated with fern and lycophyte roots. METHODS: We used two primer pairs focused on the ITS rRNA region to screen the general fungal communities, and the 18S rRNA to target Glomeromycota fungi (i...

PREMISE: The preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high-quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples. METHODS: DNA was isolated from leaves preserved with 96% ethanol or from silica-desiccated leaf samples and herbarium fragments that were pretreated with ethanol...

PREMISE: The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse Metrosideros taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites. METHODS AND RESULTS: We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps...