A first complete phylogenomic hypothesis for diploid blueberries (Vaccinium section Cyanococcus).

PREMISE: The true blueberries (Vaccinium sect. Cyanococcus; Ericaceae), endemic to North America, have been intensively studied for over a century. However, with species estimates ranging from nine to 24 and much confusion regarding species boundaries, this ecologically and economically valuable group remains inadequately understood at a basic evolutionary and taxonomic level. As a first step toward understanding the evolutionary history and taxonomy of this species complex, we present the first phylogenomic hypothesis of the known diploid blueberries. METHODS: We used flow cytometry to verify the ploidy of putative diploid taxa and a target-enrichment approach to obtain a genomic data set for phylogenetic analyses. RESULTS: Despite evidence of gene flow, we found that a primary phylogenetic signal is present. Monophyly for all morphospecies was recovered, with two notable exceptions: one sample of V. boreale was consistently nested in the V. myrtilloides clade and V. caesariense was nested in the V. fuscatum clade. One diploid taxon, Vaccinium pallidum, is implicated as having a homoploid hybrid origin. CONCLUSIONS: This foundational study represents the first attempt to elucidate evolutionary relationships of the true blueberries of North America with a phylogenomic approach and sets the stage for multiple avenues of future study such as a taxonomic revision of the group, the verification of a homoploid hybrid taxon, and the study of polyploid lineages within the context of a diploid phylogeny.

Reproductive developmental transcriptome analysis of Tripidium ravennae (Poaceae).

BACKGROUND: Tripidium ravennae is a cold-hardy, diploid species in the sugarcane complex (Poaceae subtribe Saccharinae) with considerable potential as a genetic resource for developing improved bioenergy and ornamental grasses. An improved understanding of the genetic regulation of reproductive processes (e.g., floral induction, inflorescence development, and seed development) will enable future applications of precision breeding and gene editing of floral and seed development. In particular, the ability to silence reproductive processes would allow for developing seedless forms of valuable but potentially invasive plants. The objective of this research was to characterize the gene expression environment of reproductive development in T. ravennae. RESULTS: During the early phases of inflorescence development, multiple key canonical floral integrators and pathways were identified. Annotations of type II subfamily of MADS-box transcription factors, in particular, were over-represented in the GO enrichment analyses and tests for differential expression (FDR p-value < 0.05). The differential expression of floral integrators observed in the early phases of inflorescence development diminished prior to inflorescence determinacy regulation. Differential expression analysis did not identify many unique genes at mid-inflorescence development stages, though typical biological processes involved in plant growth and development expressed abundantly. The increase in inflorescence determinacy regulatory elements and putative homeotic floral development unigenes at mid-inflorescence development coincided with the expression of multiple meiosis annotations and multicellular organism developmental processes. Analysis of seed development identified multiple unigenes involved in oxidative-reductive processes. CONCLUSION: Reproduction in grasses is a dynamic system involving the sequential coordination of complex gene regulatory networks and developmental processes. This research identified differentially expressed transcripts associated with floral induction, inflorescence development, and seed development in T. ravennae. These results provide insights into the molecular regulation of reproductive development and provide a foundation for future investigations and analyses, including genome annotation, functional genomics characterization, gene family evolutionary studies, comparative genomics, and precision breeding.

Stepwise Optimization of Real-Time RT-PCR Analysis.

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R2 ≥ 0.9999 and efficiency (E) = 100 ± 5% for the best primer pair of each gene, which serves as the prerequisite for using the 2-ΔΔCT method for data analysis.

A set of SSR markers to characterize genetic diversity in all Viburnum species.

About 160 species are classified within the Viburnum genus and many of these are cultivated for horticultural purposes. The vast dispersal of Viburnum makes the genus a useful model for studying evolutionary history and inferring how species expanded into their current distributions. Simple sequence repeat (SSR) markers were previously developed for five Viburnum species that were classified within the four major clades (Laminotinus, Crenotinus, Valvatotinus, and Porphyrotinus). The ability of some of these markers to cross-amplify in Viburnum species has been scantly evaluated, but there has not been any genus-wide assessment for the markers. We evaluated a collection of 49 SSR markers for the ability to cross-amplify in 224 samples, including 46 Viburnum species, representing all 16 subclades, and five additional species in the Viburnaceae and Caprifoliaceae. A subset of 14 potentially comprehensive markers for Viburnum species was identified and evaluated for the ability to detect polymorphisms in species outside of their respective clades. The 49 markers had overall amplification success in 52% of the samples, including a 60% success rate within the Viburnum genus and 14% in other genera. The comprehensive marker set amplified alleles in 74% of all samples tested, including 85% of Viburnum samples and 19% of outgroup samples. To the best of our knowledge, this is the first comprehensive set of markers able to characterize species across an entire genus. This set of markers can be used to assess the genetic diversity and population structure of most Viburnum species and closely allied species.

Plant Promoters and Terminators for High-Precision Bioengineering.

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels. Gene transcription is tightly regulated by promoters and terminators. Promoters determine the timing, tissues and cells, and levels of the expression of genes. Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally, e.g., the 3'-end processing, stability, translation efficiency, and nuclear to cytoplasmic export of mRNAs. The promoter and terminator combination affects gene expression. In the present article, we review the function and features of plant core promoters, proximal and distal promoters, and terminators, and their effects on and benchmarking strategies for regulating gene expression.

Genotype-independent plant transformation.

Plant transformation and regeneration remain highly species- and genotype-dependent. Conventional hormone-based plant regeneration via somatic embryogenesis or organogenesis is tedious, time-consuming, and requires specialized skills and experience. Over the last 40 years, significant advances have been made to elucidate the molecular mechanisms underlying embryogenesis and organogenesis. These pioneering studies have led to a better understanding of the key steps and factors involved in plant regeneration, resulting in the identification of crucial growth and developmental regulatory genes that can dramatically improve regeneration efficiency, shorten transformation time, and make transformation of recalcitrant genotypes possible. Co-opting these regulatory genes offers great potential to develop innovative genotype-independent genetic transformation methods for various plant species, including specialty crops. Further developing these approaches has the potential to result in plant transformation without the use of hormones, antibiotics, selectable marker genes, or tissue culture. As an enabling technology, the use of these regulatory genes has great potential to enable the application of advanced breeding technologies such as genetic engineering and gene editing for crop improvement in transformation-recalcitrant crops and cultivars. This review will discuss the recent advances in the use of regulatory genes in plant transformation and regeneration, and their potential to facilitate genotype-independent plant transformation and regeneration.

An optimized protocol for stepwise optimization of real-time RT-PCR analysis.

Computational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene's primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2-ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R2 ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2-ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

In vitro Ploidy Manipulation for Crop Improvement.

In vitro regeneration systems provide a powerful tool for manipulating ploidy to facilitate breeding and development of new crops. Polyploid induction can expand breeding opportunities, assist with the development of seedless triploid cultivars, enhance ornamental characteristics and environmental tolerances, increase biomass and restore fertility in wide hybrids. In vitro ploidy manipulation is commonly induced using antimitotic agents such as colchicine, oryzalin and trifluralin, while many other antimitotic agents have been relatively unexplored. Successful induction requires a synergistic pairing of efficient penetration of the antimitotic agent and may be dependent the length of exposure and concentrations of antimitotic agents, tissue types, and interactions with basal media and plant growth regulators. In vitro conditions vary among taxa and individual genera, species, and cultivars, often requiring unique treatments to maximize polyploid induction. In some taxa, the induction of polyploidy influences in vitro growth, development, and root formation. Here we provide an overview of mitotic inhibitors and their application for in vitro ploidy manipulation for plant breeding and crop improvement.

Revision of Fothergilla (Hamamelidaceae), including resurrection of F. parvifolia and a new species, F. milleri.

Fothergilla is a small genus of deciduous shrubs native to the southeastern United States that depending on circumscription comprises two to four species. Recent treatments recognized only two species in the genus: F. gardenii (tetraploid) and F. major (hexaploid). Until recently, no diploid taxon of Fothergilla was known. However, recent investigations identified a number of diploid populations in Alabama, Florida, Georgia, and South Carolina. A subsequent phylogenomic analysis showed that the diploids segregated into two, well-supported lineages, corresponding to largely allopatric populations. A re-examination of the morphology of diploid plants, in combination with the genetic evidence, has led us to the recognition of two species of diploids in the genus - a resurrected F. parvifolia and a new species (F. milleri W.D. Phillips & J.E. Haynes, sp. nov.) - bringing the total number of recognized species in Fothergilla to four. A revised taxonomic treatment of the genus is provided.

Corrigendum: Revision of Fothergilla (Hamamelidaceae), including resurrection of F. parvifolia and a new species, F. milleri.

[This corrects the article DOI: 10.3897/phytokeys.144.49589.].

Microbiome Variation Across Two Hemlock Species With Hemlock Woolly Adelgid Infestation.

The hemlock woolly adelgid (Adelges tsugae, HWA), an invasive insect, is devastating native hemlock populations in eastern North America, and management outcomes have so far had limited success. While many plant microbiomes influence and even support plant immune responses to insect herbivory, relatively little is known about the hemlock microbiome and its interactions with pathogens or herbivores such as HWA. Using 16S rRNA and ITS gene amplicon sequencing, we characterized the needle, branch, root, and rhizosphere microbiome of two hemlock species, Tsuga canadensis and T. sieboldii, that displayed low and high levels of HWA populations. We found that both archaeal/bacterial and fungal needle communities, as well as the archaeal/bacterial branch and root communities, varied in composition in both hemlock species relative to HWA population levels. While host species and plant-associated habitats explained a greater proportion of the variance in the microbiome than did HWA population level, high HWA populations were associated with enrichment of 100 likely fungal pathogen sequence variants across the four plant-associated habitats (e.g., needle, branch, root, rhizosphere) compared to trees with lower HWA populations. This work contributes to a growing body of literature linking plant pathogens and pests with the changes in the associated plant microbiome and host health. Furthermore, this work demonstrates the need to further investigate plant microbiome effects across multiple plant tissues to understand their influences on host health.

Identity and distribution of residues of energetic compounds at army live-fire training ranges.

Environmental investigations have been conducted at 23 military firing ranges in the United States and Canada. The specific training facilities most frequently evaluated were hand grenade, antitank rocket, and artillery ranges. Energetic compounds (explosives and propellants) were determined and linked to the type of munition used and the major mechanisms of deposition.

Effects of an afternoon nap on nighttime alertness and performance in long-haul drivers.

The effects of an afternoon nap on alertness and psychomotor performance were assessed during a simulated night shift. After a night of partial sleep restriction, eight professional long-haul drivers either slept (nap condition) or engaged in sedentary activities (no-nap condition) from 14:00 to 17:00 h. Alertness and performance testing sessions were conducted at 12:00 (pre-nap baseline), 24:00, 02:30, 05:00 and 07:30 h, and followed 2-h runs in a driving simulator. In the nap condition, the subjects showed lower subjective sleepiness and fatigue, as measured by visual analog scales, and faster reaction times and less variability on psychomotor performance tasks. Electrophysiological indices of arousal during the driving runs also reflected the beneficial effects of the afternoon nap, with lower spectral activity in the theta (4-7.75 Hz), alpha (8-11.75 Hz) and fast theta-slow alpha (6-9.75 Hz) frequency bands of the electroencephalogram, indicating higher arousal levels. Thus, a 3-h napping opportunity ending at 17:00 h improved significantly several indices of alertness and performance measured 7-14 h later.

Hydrolysis of ozone pretreated energy grasses for optimal fermentable sugar production.

Ozonated energy grass varieties were enzymatically hydrolyzed to establish process parameters for maximum fermentable sugar production. Conditions for ozonolysis were selected on the basis of maximum delignification and glucan retention after pretreatment. To study the effect of lignin degradation products generated during ozonolysis on cellulolytic enzymes, hydrolysis was carried out for washed and unwashed pretreated solids. Washing the solids significantly (p<0.05) enhanced glucan conversion from 34.3% to 100% while delivering glucose yields of 146.2-431.9 mg/g biomass. Highest fermentable sugars were produced when grasses were ozonated for maximum delignification and washed solids were hydrolyzed using 0.1g/g Cellic® CTec2. In a comparative study on alkaline pretreatment with 1% NaOH for 60 min, Saccharum arundinaceum exhibited the highest glucan conversion with maximum sugar production of 467.9 mg/g. Although ozonolysis is an effective and environmentally friendly technique for cellulosic sugar production, process optimization is needed to ascertain economic feasibility of the process.

Potential of ozonolysis as a pretreatment for energy grasses.

This study investigated the effect of ozonolysis on Miscanthus × giganteus, Miscanthus sinensis 'Gracillimus', Saccharum arundinaceum and Saccharum ravennae, collectively referred to as 'energy grasses'. Studies were conducted at three different ozone concentrations (40, 50 and 58 mg/l) using two ozone flow configurations - uni-directional and reversed flow. Pretreatment conditions for each variety were optimized based on lignin content and glucan recovery in ozonated solids. Results showed that ozonolysis was effective in removing up to 59.9% lignin without cellulose degradation. However, subsequent hydrolysis of pretreated solids with Cellic® CTec2 at 0.06 g/g raw biomass provided glucan conversion lower than untreated samples suggesting enzyme inhibition by lignin degradation products formed during ozonolysis. Future studies investigating hydrolysis efficiency of washed pretreated solids with higher enzyme loadings are therefore warranted to optimize the hydrolysis process and make it functionally feasible.

Effects of voice technology on test track driving performance: implications for driver distraction.

This work compares the degradation in driving performance associated with secondary tasks performed with voice-based and visual/manual interfaces, including radio tuning, phone dialing, and more complex tasks involving a sequence of interactions with an in-vehicle computer system. Twenty-one participants drove an instrumented vehicle while performing a combination of car-following, peripheral target detection, and secondary tasks on a closed test track. Drivers compensated for increased task demands associated with secondary tasks by increasing their following distance. Performing secondary tasks also resulted in significant decrements to vehicle control, target detection, and car-following performance. The voice-based interface helped reduce the distracting effects of secondary task performance. Modest improvements were observed for measures of vehicle control and target detection but not for car following. The results indicated that performing in-vehicle tasks required diversion of both peripheral (visual and manual) and attentional (cognitive) resources from driving. The voice-based interface reduced the peripheral impairment but did not appreciably reduce the attentional impairment. Actual or potential applications of this research include improvements to the design of invehicle information systems and the development of evaluation protocols to assess their distraction potential.