Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S. polyrhiza, leading to triacylglycerol and carbohydrate accumulation. In FGD wastewater-treated plantlets, the degraded products of monogalactosyldiacylglycerol, primarily polyunsaturated fatty acids (18:3), were incorporated into triacylglycerols. Genes involved in early fatty acid biosynthesis, ß-oxidation, and lipid degradation were upregulated while genes involved in cuticular wax biosynthesis were downregulated by treatment. The transcription factor gene WRINKLED3 (SpWRI3) was upregulated in FGD wastewater-treated plantlets, and its ectopic expression increased tolerance to FGD wastewater in transgenic Arabidopsis (Arabidopsis thaliana). Transgenic Arabidopsis plants showed enhanced glutathione and lower malondialdehyde contents under stress, suggesting that SpWRI3 functions in S. polyrhiza tolerance of FGD wastewater-induced heavy-metal stress. These results provide a basis for improving heavy metal-stress tolerance in plants for industrial applications.

Effector Repertoire of the Sweetpotato Black Rot Fungal Pathogen Ceratocystis fimbriata.

In 2015, sweetpotato producers in the United States experienced one of the worst outbreaks of black rot recorded in history, with up to 60% losses reported in the field and packing houses and at shipping ports. Host resistance remains the ideal management tool to decrease crop losses. Lack of knowledge of Ceratocystis fimbriata biology represents a critical barrier for the deployment of resistance to black rot in sweetpotato. In this study, we scanned the recent near chromosomal-level assembly for putative secreted effectors in the sweetpotato C. fimbriata isolate AS236 using a custom fungal effector annotation pipeline. We identified a set of 188 putative effectors on the basis of secretion signal and in silico prediction in EffectorP. We conducted a deep RNA time-course sequencing experiment to determine whether C. fimbriata modulates effectors in planta and to define a candidate list of effectors expressed during infection. We examined the expression profile of two C. fimbriata isolates, a pre-epidemic (1990s) isolate and a post-epidemic (2015) isolate. Our in planta expression profiling revealed clusters of co-expressed secreted effector candidates. Based on fold-change differences of putative effectors in both isolates and over the course of infection, we suggested prioritization of 31 effectors for functional characterization. Among this set, we identified several effectors that provide evidence for a marked biotrophic phase in C. fimbriata during infection of sweetpotato storage roots. Our study revealed a catalog of effector proteins that provide insight into C. fimbriata infection mechanisms and represent a core catalog to implement effector-assisted breeding in sweetpotato. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Long-read sequencing genome assembly of Ceratocystis fimbriata enables development of molecular diagnostics for sweetpotato black rot.

Ceratocystis fimbriata is a destructive fungal pathogen of sweetpotato (Ipomoea batatas (L.) Lam.) that leads to losses at all stages of sweetpotato production. Accurate detection of C. fimbriata would allow for more efficient deployment of management tactics in sweetpotato production. To develop a diagnostic assay, a hybrid genome assembly of C. fimbriata isolate AS236 was generated. The resulting 31.7 MB assembly was near-chromosome level, with 18 contigs, 6,481 predicted genes, and a BUSCO completion score of 98.4% when compared to the fungi-specific lineage database. Additional Illumina DNA reads from C. manginecans, C. platani, and a second C. fimbriata isolate (C1421) were then mapped to the assembled genome using BOWTIE2 and counted using HTSeq, which identified 148 genes present only within C. fimbriata as molecular diagnostic candidates; 6 single-copy and 35 highly multi-copy (>40 BLAST hits), as determined through a self-BLAST-P alignment. Primers for PCR were designed in the 200 bp flanking region of the first exon for each candidate, and the candidates were validated against a diverse DNA panel containing Ceratocystis species, sweetpotato pathogens, and plants. After validation, two diagnostic candidates amplified only C. fimbriata DNA and were considered to be highly specific to the species. These genetic markers will serve as valuable diagnostic tools with multiple applications including the detection of C. fimbriata in seed, soil, and wash water in sweetpotato production.

Network Analysis of Publicly Available RNA-seq Provides Insights into the Molecular Mechanisms of Plant Defense against Multiple Fungal Pathogens in Arabidopsis thaliana.

Fungal pathogens can have devastating effects on global crop production, leading to annual economic losses ranging from 10% to 23%. In light of climate change-related challenges, researchers anticipate an increase in fungal infections as a result of shifting environmental conditions. However, plants have developed intricate molecular mechanisms for effective defense against fungal attacks. Understanding these mechanisms is essential to the development of new strategies for protecting crops from multiple fungi threats. Public omics databases provide valuable resources for research on plant-pathogen interactions; however, integrating data from different studies can be challenging due to experimental variation. In this study, we aimed to identify the core genes that defend against the pathogenic fungi Colletotrichum higginsianum and Botrytis cinerea in Arabidopsis thaliana. Using a custom framework to control batch effects and construct Gene Co-expression Networks in publicly available RNA-seq dataset from infected A. thaliana plants, we successfully identified a gene module that was responsive to both pathogens. We also performed gene annotation to reveal the roles of previously unknown protein-coding genes in plant defenses against fungal infections. This research demonstrates the potential of publicly available RNA-seq data for identifying the core genes involved in defending against multiple fungal pathogens.

Genome of tetraploid sour cherry (Prunus cerasus L.) 'Montmorency' identifies three distinct ancestral Prunus genomes.

Sour cherry (Prunus cerasus L.) is a valuable fruit crop in the Rosaceae family and a hybrid between progenitors closely related to extant Prunus fruticosa (ground cherry) and Prunus avium (sweet cherry). Here we report a chromosome-scale genome assembly for sour cherry cultivar Montmorency, the predominant cultivar grown in the USA. We also generated a draft assembly of P. fruticosa to use alongside a published P. avium sequence for syntelog-based subgenome assignments for 'Montmorency' and provide compelling evidence P. fruticosa is also an allotetraploid. Using hierarchal k-mer clustering and phylogenomics, we show 'Montmorency' is trigenomic, containing two distinct subgenomes inherited from a P. fruticosa-like ancestor (A and A') and two copies of the same subgenome inherited from a P. avium-like ancestor (BB). The genome composition of 'Montmorency' is AA'BB and little-to-no recombination has occurred between progenitor subgenomes (A/A' and B). In Prunus, two known classes of genes are important to breeding strategies: the self-incompatibility loci (S-alleles), which determine compatible crosses, successful fertilization, and fruit set, and the Dormancy Associated MADS-box genes (DAMs), which strongly affect dormancy transitions and flowering time. The S-alleles and DAMs in 'Montmorency' and P. fruticosa were manually annotated and support subgenome assignments. Lastly, the hybridization event 'Montmorency' is descended from was estimated to have occurred less than 1.61 million years ago, making sour cherry a relatively recent allotetraploid. The 'Montmorency' genome highlights the evolutionary complexity of the genus Prunus and will inform future breeding strategies for sour cherry, comparative genomics in the Rosaceae, and questions regarding neopolyploidy.

Gene expression profiling of soaked dry beans (Phaseolus vulgaris L.) reveals cell wall modification plays a role in cooking time.

Dry beans (Phaseolus vulgaris L.) are a nutritious food, but their lengthy cooking requirements are barriers to consumption. Presoaking is one strategy to reduce cooking time. Soaking allows hydration to occur prior to cooking, and enzymatic changes to pectic polysaccharides also occur during soaking that shorten the cooking time of beans. Little is known about how gene expression during soaking influences cooking times. The objectives of this study were to (1) identify gene expression patterns that are altered by soaking and (2) compare gene expression in fast-cooking and slow-cooking bean genotypes. RNA was extracted from four bean genotypes at five soaking time points (0, 3, 6, 12, and 18 h) and expression abundances were detected using Quant-seq. Differential gene expression analysis and weighted gene coexpression network analysis were used to identify candidate genes within quantitative trait loci for water uptake and cooking time. Genes related to cell wall growth and development as well as hypoxic stress were differentially expressed between the fast- and slow-cooking beans due to soaking. Candidate genes identified in the slow-cooking beans included enzymes that increase intracellular calcium concentrations and cell wall modification enzymes. The expression of cell wall-strengthening enzymes in the slow-cooking beans may increase their cooking time and ability to resist osmotic stress by preventing cell separation and water uptake in the cotyledon.

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Chromosome-scale genome assemblies and annotations for Poales species Carex cristatella, Carex scoparia, Juncus effusus, and Juncus inflexus.

The majority of sequenced genomes in the monocots are from species belonging to Poaceae, which include many commercially important crops. Here, we expand the number of sequenced genomes from the monocots to include the genomes of 4 related cyperids: Carex cristatella and Carex scoparia from Cyperaceae and Juncus effusus and Juncus inflexus from Juncaceae. The high-quality, chromosome-scale genome sequences from these 4 cyperids were assembled by combining whole-genome shotgun sequencing of Nanopore long reads, Illumina short reads, and Hi-C sequencing data. Some members of the Cyperaceae and Juncaceae are known to possess holocentric chromosomes. We examined the repeat landscapes in our sequenced genomes to search for potential repeats associated with centromeres. Several large satellite repeat families, comprising 3.2-9.5% of our sequenced genomes, showed dispersed distribution of large satellite repeat clusters across all Carex chromosomes, with few instances of these repeats clustering in the same chromosomal regions. In contrast, most large Juncus satellite repeats were clustered in a single location on each chromosome, with sporadic instances of large satellite repeats throughout the Juncus genomes. Recognizable transposable elements account for about 20% of each of the 4 genome assemblies, with the Carex genomes containing more DNA transposons than retrotransposons while the converse is true for the Juncus genomes. These genome sequences and annotations will facilitate better comparative analysis within monocots.

The bowfin genome illuminates the developmental evolution of ray-finned fishes.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin's importance for illuminating vertebrate biology and diversity in the genomic era.

Seeing through the forest: The gaze path to purchase.

Eye tracking studies have analyzed the relationship between visual attention to point of purchase marketing elements (price, signage, etc.) and purchase intention. Our study is the first to investigate the relationship between the gaze sequence in which consumers view a display (including gaze aversion away from products) and the influence of consumer (top down) characteristics on product choice. We conducted an in-lab 3 (display size: large, moderate, small) X 2 (price: sale, non-sale) within-subject experiment with 92 persons. After viewing the displays, subjects completed an online survey to provide demographic data, self-reported and actual product knowledge, and past purchase information. We employed a random forest machine learning approach via R software to analyze all possible three-unit subsequences of gaze fixations. Models comparing multiclass F1-macro score and F1-micro score of product choice were analyzed. Gaze sequence models that included gaze aversion more accurately predicted product choice in a lab setting for more complex displays. Inclusion of consumer characteristics generally improved model predictive F1-macro and F1-micro scores for less complex displays with fewer plant sizes Consumer attributes that helped improve model prediction performance were product expertise, ethnicity, and previous plant purchases.

The Effector Repertoire of the Hop Downy Mildew Pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew (DM), one of the most destructive diseases of cultivated hop that can lead to 100% crop loss in susceptible cultivars. We used the published genome of P. humuli to predict the secretome and effectorome and analyze the transcriptome variation among diverse isolates and during infection of hop leaves. Mining the predicted coding genes of the sequenced isolate OR502AA of P. humuli revealed a secretome of 1,250 genes. We identified 296 RXLR and RXLR-like effector-encoding genes in the secretome. Among the predicted RXLRs, there were several WY-motif-containing effectors that lacked canonical RXLR domains. Transcriptome analysis of sporangia from 12 different isolates collected from various hop cultivars revealed 754 secreted proteins and 201 RXLR effectors that showed transcript evidence across all isolates with reads per kilobase million (RPKM) values > 0. RNA-seq analysis of OR502AA-infected hop leaf samples at different time points after infection revealed highly expressed effectors that may play a relevant role in pathogenicity. Quantitative RT-PCR analysis confirmed the differential expression of selected effectors. We identified a set of P. humuli core effectors that showed transcript evidence in all tested isolates and elevated expression during infection. These effectors are ideal candidates for functional analysis and effector-assisted breeding to develop DM resistant hop cultivars.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

GPRED-GC: a Gene PREDiction model accounting for 5 '- 3' GC gradient.

BACKGROUND: Gene is a key step in genome annotation. Ab initio gene prediction enables gene annotation of new genomes regardless of availability of homologous sequences. There exist a number of ab initio gene prediction tools and they have been widely used for gene annotation for various species. However, existing tools are not optimized for identifying genes with highly variable GC content. In addition, some genes in grass genomes exhibit a sharp 5 '- 3' decreasing GC content gradient, which is not carefully modeled by available gene prediction tools. Thus, there is still room to improve the sensitivity and accuracy for predicting genes with GC gradients. RESULTS: In this work, we designed and implemented a new hidden Markov model (HMM)-based ab initio gene prediction tool, which is optimized for finding genes with highly variable GC contents, such as the genes with negative GC gradients in grass genomes. We tested the tool on three datasets from Arabidopsis thaliana and Oryza sativa. The results showed that our tool can identify genes missed by existing tools due to the highly variable GC contents. CONCLUSIONS: GPRED-GC can effectively predict genes with highly variable GC contents without manual intervention. It provides a useful complementary tool to existing ones such as Augustus for more sensitive gene discovery. The source code is freely available at https://sourceforge.net/projects/gpred-gc/.

Assembly, annotation, and comparison of Macrophomina phaseolina isolates from strawberry and other hosts.

BACKGROUND: Macrophomina phaseolina is a fungal plant pathogen with a broad host range, but one genotype was shown to exhibit host preference/specificity on strawberry. This pathogen lacked a high-quality genome assembly and annotation, and little was known about genomic differences among isolates from different hosts. RESULTS: We used PacBio sequencing and Hi-C scaffolding to provide nearly complete genome assemblies for M. phaseolina isolates representing the strawberry-specific genotype and another genotype recovered from alfalfa. The strawberry isolate had 59 contigs/scaffolds with an N50 of 4.3 Mb. The isolate from alfalfa had an N50 of 5.0 Mb and 14 nuclear contigs with half including telomeres. Both genomes were annotated with MAKER using transcript evidence generated in this study with over 13,000 protein-coding genes predicted. Unique groups of genes for each isolate were identified when compared to closely related fungal species. Structural comparisons between the isolates reveal large-scale rearrangements including chromosomal inversions and translocations. To include isolates representing a range of pathogen genotypes, an additional 30 isolates were sequenced with Illumina, assembled, and compared to the strawberry genotype assembly. Within the limits of comparing Illumina and PacBio assemblies, no conserved structural rearrangements were identified among the isolates from the strawberry genotype compared to those from other hosts, but some candidate genes were identified that were largely present in isolates of the strawberry genotype and absent in other genotypes. CONCLUSIONS: High-quality reference genomes of M. phaseolina have allowed for the identification of structural changes associated with a genotype that has a host preference toward strawberry and will enable future comparative genomics studies. Having more complete assemblies allows for structural rearrangements to be more fully assessed and ensures a greater representation of all the genes. Work with Illumina data from additional isolates suggests that some genes are predominately present in isolates of the strawberry genotype, but additional work is needed to confirm the role of these genes in pathogenesis. Additional work is also needed to complete the scaffolding of smaller contigs identified in the strawberry genotype assembly and to determine if unique genes in the strawberry genotype play a role in pathogenicity.

Allelic Variation in the Chloroplast Division Gene FtsZ2-2 Leads to Natural Variation in Chloroplast Size.

Chloroplast size varies considerably in nature, but the underlying mechanisms are unknown. By exploiting a near-isogenic line population derived from a cross between the Arabidopsis (Arabidopsis thaliana) accessions Cape Verde Islands (Cvi-1), which has larger chloroplasts, and Landsberg erecta (Ler-0), with smaller chloroplasts, we determined that the large-chloroplast phenotype in Cvi-1 is associated with allelic variation in the gene encoding the chloroplast-division protein FtsZ2-2, a tubulin-related cytoskeletal component of the contractile FtsZ ring inside chloroplasts. Sequencing revealed that the Cvi-1 FtsZ2-2 allele encodes a C-terminally truncated protein lacking a region required for FtsZ2-2 interaction with inner-envelope proteins, and functional complementation experiments in a Columbia-0 ftsZ2-2 null mutant confirmed this allele as causal for the increased chloroplast size in Cvi-1. Comparison of FtsZ2-2 coding sequences in the 1001 Genomes database showed that the Cvi-1 allele is rare and identified additional rare loss-of-function alleles, including a natural null allele, in three other accessions, all of which had enlarged-chloroplast phenotypes. The ratio of nonsynonymous to synonymous substitutions was higher among the FtsZ2-2 genes than among the two other FtsZ family members in Arabidopsis, FtsZ2-1, a close paralog of FtsZ2-2, and the functionally distinct FtsZ1-1, indicating more relaxed constraint on the FtsZ2-2 coding sequence than on those of FtsZ2-1 or FtsZ1-1 Our results establish that allelic variation in FtsZ2-2 contributes to natural variation in chloroplast size in Arabidopsis, and they also demonstrate that natural variation in Arabidopsis can be used to decipher the genetic basis of differences in fundamental cell biological traits, such as organelle size.

Evolutionary characteristics of intergenic transcribed regions indicate rare novel genes and widespread noisy transcription in the Poaceae.

Extensive transcriptional activity occurring in intergenic regions of genomes has raised the question whether intergenic transcription represents the activity of novel genes or noisy expression. To address this, we evaluated cross-species and post-duplication sequence and expression conservation of intergenic transcribed regions (ITRs) in four Poaceae species. Among 43,301 ITRs across the four species, 34,460 (80%) are species-specific. ITRs found across species tend to be more divergent in expression and have more recent duplicates compared to annotated genes. To assess if ITRs are functional (under selection), machine learning models were established in Oryza sativa (rice) that could accurately distinguish between phenotype genes and pseudogenes (area under curve-receiver operating characteristic = 0.94). Based on the models, 584 (8%) and 4391 (61%) rice ITRs are classified as likely functional and nonfunctional with high confidence, respectively. ITRs with conserved expression and ancient retained duplicates, features that were not part of the model, are frequently classified as likely-functional, suggesting these characteristics could serve as pragmatic rules of thumb for identifying candidate sequences likely to be under selection. This study also provides a framework to identify novel genes using comparative transcriptomic data to improve genome annotation that is fundamental for connecting genotype to phenotype in crop and model systems.

Transcriptional Regulation of the Glucose-6-Phosphate/Phosphate Translocator 2 Is Related to Carbon Exchange Across the Chloroplast Envelope.

The exchange of reduced carbon across the inner chloroplast envelope has a large impact on photosynthesis and growth. Under steady-state conditions it is thought that glucose 6-phosphate (G6P) does not cross the chloroplast membrane. However, growth at high CO2, or disruption of starch metabolism can result in the GPT2 gene for a G6P/Pi translocator to be expressed presumably allowing G6P exchange across the chloroplast envelope. We found that after an increase in light, the transcript for GPT2 transiently increases several 100-fold within 2 h in both the Col-0 and WS ecotypes of Arabidopsis thaliana. The increase in transcript for GPT2 is preceded by an increase in transcript for many transcription factors including Redox Responsive Transcription Factor 1 (RRTF1). The increase in GPT2 transcript after exposure to high light is suppressed in a mutant lacking the RRTF1 transcription factor. The GPT2 response was also suppressed in a mutant with a T-DNA insert in the gene for the triose-phosphate/Pi translocator (TPT). However, plants lacking TPT still had a robust rise in RRTF1 transcript in response to high light. From this, we conclude that both RRTF1 (and possibly other transcription factors) and high amounts of cytosolic triose phosphate are required for induction of the expression of GPT2. We hypothesize that transient GPT2 expression and subsequent translation is adaptive, allowing G6P to move into the chloroplast from the cytosol. The imported G6P can be used for starch synthesis or may flow directly into the Calvin-Benson cycle via an alternative pathway (the G6P shunt), which could be important for regulating and stabilizing photosynthetic electron transport and carbon metabolism.

Corrigendum: Transcriptomics Identifies Modules of Differentially Expressed Genes and Novel Cyclotides in Viola pubescens.

[This corrects the article DOI: 10.3389/fpls.2019.00156.].

Transcriptomics Identifies Modules of Differentially Expressed Genes and Novel Cyclotides in Viola pubescens.

Viola is a large genus with worldwide distribution and many traits not currently exemplified in model plants including unique breeding systems and the production of cyclotides. Here we report de novo genome assembly and transcriptomic analyses of the non-model species Viola pubescens using short-read DNA sequencing data and RNA-Seq from eight diverse tissues. First, V. pubescens genome size was estimated through flow cytometry, resulting in an approximate haploid genome of 455 Mbp. Next, the draft V. pubescens genome was sequenced and assembled resulting in 264,035,065 read pairs and 161,038 contigs with an N50 length of 3,455 base pairs (bp). RNA-Seq data were then assembled into tissue-specific transcripts. Together, the DNA and transcript data generated 38,081 ab initio gene models which were functionally annotated based on homology to Arabidopsis thaliana genes and Pfam domains. Gene expression was visualized for each tissue via principal component analysis and hierarchical clustering, and gene co-expression analysis identified 20 modules of tissue-specific transcriptional networks. Some of these modules highlight genetic differences between chasmogamous and cleistogamous flowers and may provide insight into V. pubescens' mixed breeding system. Orthologous clustering with the proteomes of A. thaliana and Populus trichocarpa revealed 8,531 sequences unique to V. pubescens, including 81 novel cyclotide precursor sequences. Cyclotides are plant peptides characterized by a stable, cyclic cystine knot motif, making them strong candidates for drug scaffolding and protein engineering. Analysis of the RNA-Seq data for these cyclotide transcripts revealed diverse expression patterns both between transcripts and tissues. The diversity of these cyclotides was also highlighted in a maximum likelihood protein cladogram containing V. pubescens cyclotides and published cyclotide sequences from other Violaceae and Rubiaceae species. Collectively, this work provides the most comprehensive sequence resource for Viola, offers valuable transcriptomic insight into V. pubescens, and will facilitate future functional genomics research in Viola and other diverse plant groups.

Author Correction: Origin and evolution of the octoploid strawberry genome.

In the version of this article originally published, author Joshua R. Puzey was incorrectly listed as having affiliation 7 (School of Plant Sciences, University of Arizona, Tucson, AZ, USA); affiliation 6 (Department of Biology, College of William and Mary, Williamsburg, VA, USA) is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.