Investigation of regulatory divergence between homoeologs in the recently formed allopolyploids, Tragopogon mirus and T. miscellus (Asteraceae).

Polyploidy is an important evolutionary process throughout eukaryotes, particularly in flowering plants. Duplicated gene pairs (homoeologs) in allopolyploids provide additional genetic resources for changes in molecular, biochemical, and physiological mechanisms that result in evolutionary novelty. Therefore, understanding how divergent genomes and their regulatory networks reconcile is vital for unraveling the role of polyploidy in plant evolution. Here, we compared the leaf transcriptomes of recently formed natural allotetraploids (Tragopogon mirus and T. miscellus) and their diploid parents (T. porrifolius X T. dubius and T. pratensis X T. dubius, respectively). Analysis of 35 400 expressed loci showed a significantly higher level of transcriptomic additivity compared to old polyploids; only 22% were non-additively expressed in the polyploids, with 5.9% exhibiting transgressive expression (lower or higher expression in the polyploids than in the diploid parents). Among approximately 7400 common orthologous regions (COREs), most loci in both allopolyploids exhibited expression patterns that were vertically inherited from their diploid parents. However, 18% and 20.3% of the loci showed novel expression bias patterns in T. mirus and T. miscellus, respectively. The expression changes of 1500 COREs were explained by cis-regulatory divergence (the condition in which the two parental subgenomes do not interact) between the diploid parents, whereas only about 423 and 461 of the gene expression changes represent trans-effects (the two parental subgenomes interact) in T. mirus and T. miscellus, respectively. The low degree of both non-additivity and trans-effects on gene expression may present the ongoing evolutionary processes of the newly formed Tragopogon polyploids (~80-90 years).

Physiological and Molecular Modulations to Drought Stress in the Brassica Species.

Climate change, particularly drought stress, significantly impacts plant growth and development, necessitating the development of resilient crops. This study investigated physiological and molecular modulations to drought stress between diploid parent species and their polyploid progeny in the Brassica species. While no significant phenotypic differences were observed among the six species, drought stress reduced growth parameters by 2.4% and increased oxidative stress markers by 1.4-fold. Drought also triggered the expression of genes related to stress responses and led to the accumulation of specific metabolites. We also conducted the first study of perfluorooctane sulfonic acid (PFOS) levels in leaves as a drought indicator. Lower levels of PFOS accumulation were linked to plants taking in less water under drought conditions. Both diploid and polyploid species responded to drought stress similarly, but there was a wide range of variation in their responses. In particular, responses were less variable in polyploid species than in diploid species. This suggests that their additional genomic components acquired through polyploidy may improve their flexibility to modulate stress responses. Despite the hybrid vigor common in polyploid species, Brassica polyploids demonstrated intermediate responses to drought stress. Overall, this study lays the framework for future omics-level research, including transcriptome and proteomic studies, to deepen our understanding of drought tolerance mechanisms in Brassica species.

Nonadditive gene expression in polyploids.

Allopolyploidy involves hybridization and duplication of divergent parental genomes and provides new avenues for gene expression. The expression levels of duplicated genes in polyploids can show deviation from parental additivity (the arithmetic average of the parental expression levels). Nonadditive expression has been widely observed in diverse polyploids and comprises at least three possible scenarios: (a) The total gene expression level in a polyploid is similar to that of one of its parents (expression-level dominance); (b) total gene expression is lower or higher than in both parents (transgressive expression); and (c) the relative contribution of the parental copies (homeologs) to the total gene expression is unequal (homeolog expression bias). Several factors may result in expression nonadditivity in polyploids, including maternal-paternal influence, gene dosage balance, cis- and/or trans-regulatory networks, and epigenetic regulation. As our understanding of nonadditive gene expression in polyploids remains limited, a new generation of investigators should explore additional phenomena (i.e., alternative splicing) and use other high-throughput "omics" technologies to measure the impact of nonadditive expression on phenotype, proteome, and metabolome.

Molecular changes in Mesembryanthemum crystallinum guard cells underlying the C3 to CAM transition.

ABSTARCT: KEY MESSAGE: The timing and transcriptomic changes during the C3 to CAM transition of common ice plant support the notion that guard cells themselves can shift from C3 to CAM. Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO2 uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C3 photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C3 to CAM remain unknown. Here we determined the transition time from C3 to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3'-mRNA sequencing was conducted in isolated stomatal guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can shift from C3 to CAM. This study provides important information towards introducing CAM stomatal behavior into C3 crops to enhance water use efficiency.

Secretome Analysis of Inductive Signals for BM-MSC Transdifferentiation into Salivary Gland Progenitors.

Severe dry mouth in patients with Sjögren's Syndrome, or radiation therapy for patients with head and neck cancer, significantly compromises their oral health and quality of life. The current clinical management of xerostomia is limited to palliative care as there are no clinically-proven treatments available. Previously, our studies demonstrated that mouse bone marrow-derived mesenchymal stem cells (mMSCs) can differentiate into salivary progenitors when co-cultured with primary salivary epithelial cells. Transcription factors that were upregulated in co-cultured mMSCs were identified concomitantly with morphological changes and the expression of acinar cell markers, such as α-amylase (AMY1), muscarinic-type-3-receptor(M3R), aquaporin-5(AQP5), and a ductal cell marker known as cytokeratin 19(CK19). In the present study, we further explored inductive molecules in the conditioned media that led to mMSC reprogramming by high-throughput liquid chromatography with tandem mass spectrometry and systems biology. Our approach identified ten differentially expressed proteins based on their putative roles in salivary gland embryogenesis and development. Additionally, systems biology analysis revealed six candidate proteins, namely insulin-like growth factor binding protein-7 (IGFBP7), cysteine-rich, angiogenetic inducer, 61(CYR61), agrin(AGRN), laminin, beta 2 (LAMB2), follistatin-like 1(FSTL1), and fibronectin 1(FN1), for their potential contribution to mMSC transdifferentiation during co-culture. To our knowledge, our study is the first in the field to identify soluble inductive molecules that drive mMSC into salivary progenitors, which crosses lineage boundaries.

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

Nitrogen starvation-induced accumulation of triacylglycerol in the green algae: evidence for a role for ROC40, a transcription factor involved in circadian rhythm.

Microalgal triacylglycerol (TAG), a promising source of biofuel, is induced upon nitrogen starvation (-N), but the proteins and genes involved in this process are poorly known. We performed isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics to identify Chlorella proteins with modulated expression under short-term -N. Out of 1736 soluble proteins and 2187 membrane-associated proteins identified, 288 and 56, respectively, were differentially expressed under -N. Gene expression analysis on select genes confirmed the same direction of mRNA modulation for most proteins. The MYB-related transcription factor ROC40 was the most induced protein, with a 9.6-fold increase upon -N. In a previously generated Chlamydomonas mutant, gravimetric measurements of crude total lipids revealed that roc40 was impaired in its ability to increase the accumulation of TAG upon -N, and this phenotype was complemented when wild-type Roc40 was expressed. Results from radiotracer experiments were consistent with the roc40 mutant being comparable to the wild type in recycling membrane lipids to TAG but being impaired in additional de novo synthesis of TAG during -N stress. In this study we provide evidence to support the hypothesis that transcription factor ROC40 has a role in -N-induced lipid accumulation, and uncover multiple previously unknown proteins modulated by short-term -N in green algae.

Comparative evolutionary and developmental dynamics of the cotton (Gossypium hirsutum) fiber transcriptome.

The single-celled cotton (Gossypium hirsutum) fiber provides an excellent model to investigate how human selection affects phenotypic evolution. To gain insight into the evolutionary genomics of cotton domestication, we conducted comparative transcriptome profiling of developing cotton fibers using RNA-Seq. Analysis of single-celled fiber transcriptomes from four wild and five domesticated accessions from two developmental time points revealed that at least one-third and likely one-half of the genes in the genome are expressed at any one stage during cotton fiber development. Among these, ~5,000 genes are differentially expressed during primary and secondary cell wall synthesis between wild and domesticated cottons, with a biased distribution among chromosomes. Transcriptome data implicate a number of biological processes affected by human selection, and suggest that the domestication process has prolonged the duration of fiber elongation in modern cultivated forms. Functional analysis suggested that wild cottons allocate greater resources to stress response pathways, while domestication led to reprogrammed resource allocation toward increased fiber growth, possibly through modulating stress-response networks. This first global transcriptomic analysis using multiple accessions of wild and domesticated cottons is an important step toward a more comprehensive systems perspective on cotton fiber evolution. The understanding that human selection over the past 5,000+ years has dramatically re-wired the cotton fiber transcriptome sets the stage for a deeper understanding of the genetic architecture underlying cotton fiber synthesis and phenotypic evolution.

Genome-wide identification and homeolog-specific expression analysis of the SnRK2 genes in Brassica napus guard cells.

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) proteins constitute a small plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and plant responses to biotic and abiotic stresses. Although SnRK2s have been well-studied in Arabidopsis thaliana, little is known about SnRK2s in Brassica napus. Here we identified 30 putative sequences encoding 10 SnRK2 proteins in the B. napus genome and the expression profiles of a subset of 14 SnRK2 genes in guard cells of B. napus. In agreement with its polyploid origin, B. napus maintains both homeologs from its diploid parents. The results of quantitative real-time PCR (qRT-PCR) and reanalysis of RNA-Seq data showed that certain BnSnRK2 genes were commonly expressed in leaf tissues in different varieties of B. napus. In particular, qRT-PCR results showed that 12 of the 14 BnSnRK2s responded to drought stress in leaves and in ABA-treated guard cells. Among them, BnSnRK2.4 and BnSnRK2.6 were of interest because of their robust responsiveness to ABA treatment and drought stress. Notably, BnSnRK2 genes exhibited up-regulation of different homeologs, particularly in response to abiotic stress. The homeolog expression bias in BnSnRK2 genes suggests that parental origin of genes might be responsible for efficient regulation of stress responses in polyploids. This work has laid a foundation for future functional characterization of the different BnSnKR2 homeologs in B. napus and its parents, especially their functions in guard cell signaling and stress responses.

Comparative Proteomic Analysis of Brassica napus in Response to Drought Stress.

Drought is one of the most widespread stresses leading to retardation of plant growth and development. We examined proteome changes of an important oil seed crop, canola (Brassica napus L.), under drought stress over a 14-day period. Using iTRAQ LC-MS/MS, we identified 1976 proteins expressed during drought stress. Among them, 417 proteins showed significant changes in abundance, and 136, 244, 286, and 213 proteins were differentially expressed in the third, seventh, 10th, and 14th day of stress, respectively. Functional analysis indicated that the number of proteins associated with metabolism, protein folding and degradation, and signaling decreased, while those related to energy (photosynthesis), protein synthesis, and stress and defense increased in response to drought stress. The seventh and 10th-day profiles were similar to each other but with more post-translational modifications (PTMs) at day 10. Interestingly, 181 proteins underwent PTMs; 49 of them were differentially changed in drought-stressed plants, and 33 were observed at the 10th day. Comparison of protein expression changes with those of gene transcription showed a positive correlation in B. napus, although different patterns between transcripts and proteins were observed at each time point. Under drought stress, most protein abundance changes may be attributed to gene transcription, and PTMs clearly contribute to protein diversity and functions.

Molecular reprogramming of Arabidopsis in response to perturbation of jasmonate signaling.

Jasmonates (JAs) are important phytohormones that regulate a wide range of plant processes including growth, development, senescence, and defense. Jasmonate ZIM-domain (JAZ) proteins are repressors in JA signaling. In Arabidopsis thaliana, 12 JAZ encoding genes were identified, but only a few have been studied in detail. In this study, we focused on characterizing the molecular networks involving JAZ2 and JAZ7. To understand the phenotypes and elucidate the regulatory functions of JAZ2 and JAZ7, shoot and root tissues from wild type (WT), jaz2, and jaz7 were harvested for RNA sequencing and metabolomics. Distinct changes of transcripts and metabolites in JA biosynthesis, primary and specialized metabolism, and oxidative stress were observed among the three genotypes. In particular, many defense or stress-associated metabolites and specialized metabolites were increased in response to methyl jasmonate (MeJA) treatment. Most importantly, these changes were subjected to quantitative modulation by the JAZ proteins at both transcriptional and metabolic levels, the degree of which may control resource allocation between growth and defense. This study not only reveals MeJA-induced molecular reprogramming but also demonstrates the functions of JAZ proteins as key regulators in fine-tuning JA signal transduction.

Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.).

Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC-MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30% of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.

Quantitative proteomics of tomato defense against Pseudomonas syringae infection.

Genetic and microarray analyses have provided useful information in the area of plant and pathogen interactions. Pseudomonas syringae pv. tomato DC3000 (Pst) causes bacterial speck disease in tomato. Previous studies have shown that changes in response to pathogen infection at transcript level are variable at different time points. This study provides information not only on proteomic changes between a resistant and a susceptible genotype, but also information on changes between an early and a late time point. Using the iTRAQ quantitative proteomics approach, we have identified 2369 proteins in tomato leaves, and 477 of them were determined to be responsive to Pst inoculation. Unique and differential proteins after each comparison were further analyzed to provide information about protein changes and the potential functions they play in the pathogen response. This information is applicable not only to tomato proteomics, but also adds to the repertoire of proteins now available for crop proteomic analysis and how they change in response to pathogen infection.

The cytonuclear dimension of allopolyploid evolution: an example from cotton using rubisco.

During allopolyploid speciation, two divergent nuclear genomes merge, yet only one (usually the maternal) of the two sets of progenitor organellar genomes is maintained. Rubisco (1,5-bisphosphate carboxylase/oxygenase) is composed of nuclear-encoded small subunits (SSUs) and plastome-encoded large subunits (LSUs), providing an ideal system to explore the evolutionary process of cytonuclear accommodation. Here, we take initial steps in this direction, using Gossypium allopolyploids as our model. SSU copies from divergent (5-10 My) progenitor diploids ("A" and "D" genomes) were combined at the time of polyploid formation 1-2 Ma, with the LSU encoded by the maternal A-genome parent. LSU genes from A- and D-genome diploids and AD-genome allopolyploids were sequenced, revealing several nonsynonymous substitutions and suggesting the possibility of differential selection on the nuclear-encoded rbcS partner following allopolyploid formation. Sequence data for the rbcS gene family revealed nonreciprocal homoeologous recombination between A- and D-rbcS homoeologs in all polyploid species but not in a synthetic intergenomic F1 hybrid, demonstrating "gene conversion" during allopolyploid evolution. All progenitor rbcS genes are retained and expressed in the five extant allopolyploid species, but analysis of the leaf transcriptome showed that A-homoeologs are preferentially expressed in both the allopolyploid and hybrid, consistent with the maternal origin of rbcL. Although rbcS genes from both progenitor genomes are expressed, some appear to have experienced mutations that may represent cytonuclear coevolution.

Proteomic profiling of developing cotton fibers from wild and domesticated Gossypium barbadense.

Pima cotton (Gossypium barbadense) is widely cultivated because of its long, strong seed trichomes ('fibers') used for premium textiles. These agronomically advanced fibers were derived following domestication and thousands of years of human-mediated crop improvement. To gain an insight into fiber development and evolution, we conducted comparative proteomic and transcriptomic profiling of developing fiber from an elite cultivar and a wild accession. Analyses using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology identified 1317 proteins in fiber. Of these, 205 were differentially expressed across developmental stages, and 190 showed differential expression between wild and cultivated forms, 14.4% of the proteome sampled. Human selection may have shifted the timing of developmental modules, such that some occur earlier in domesticated than in wild cotton. A novel approach was used to detect possible biased expression of homoeologous copies of proteins. Results indicate a significant partitioning of duplicate gene expression at the protein level, but an approximately equal degree of bias for each of the two constituent genomes of allopolyploid cotton. Our results demonstrate the power of complementary transcriptomic and proteomic approaches for the study of the domestication process. They also provide a rich database for mining for functional analyses of cotton improvement or evolution.

Conservation and canalization of gene expression during angiosperm diversification accompany the origin and evolution of the flower.

The origin and rapid diversification of the angiosperms (Darwin's "Abominable Mystery") has engaged generations of researchers. Here, we examine the floral genetic programs of phylogenetically pivotal angiosperms (water lily, avocado, California poppy, and Arabidopsis) and a nonflowering seed plant (a cycad) to obtain insight into the origin and subsequent evolution of the flower. Transcriptional cascades with broadly overlapping spatial domains, resembling the hypothesized ancestral gymnosperm program, are deployed across morphologically intergrading organs in water lily and avocado flowers. In contrast, spatially discrete transcriptional programs in distinct floral organs characterize the more recently derived angiosperm lineages represented by California poppy and Arabidopsis. Deep evolutionary conservation in the genetic programs of putatively homologous floral organs traces to those operating in gymnosperm reproductive cones. Female gymnosperm cones and angiosperm carpels share conserved genetic features, which may be associated with the ovule developmental program common to both organs. However, male gymnosperm cones share genetic features with both perianth (sterile attractive and protective) organs and stamens, supporting the evolutionary origin of the floral perianth from the male genetic program of seed plants.

Carry-over effects of dry period heat stress on the mammary gland proteome and phosphoproteome in the subsequent lactation of dairy cows.

Exposure to heat stress during a cow's dry period disrupts mammary gland remodeling, impairing mammary function and milk production during the subsequent lactation. Yet, proteomic changes in the mammary gland underlying these effects are not yet known. We investigated alterations in the mammary proteome and phosphoproteome during lactation as a result of dry period heat stress using an isobaric tag for relative and absolute quantitation (iTRAQ)-based approach. Cows were cooled (CL; n = 12) with fans and water soakers in a free stall setting or were heat stressed through lack of access to cooling devices (HT; n = 12) during the entire dry period (approximately 46 days). All cows were cooled postpartum. Mammary biopsies were harvested from a subset of cows (n = 4 per treatment) at 14, 42, and 84 days in milk. Overall, 251 proteins and 224 phosphorylated proteins were differentially abundant in the lactating mammary gland of HT compared to CL cows. Top functions of differentially abundant proteins and phosphoproteins affected were related to immune function and inflammation, amino acid metabolism, reactive oxygen species production and metabolism, tissue remodeling, and cell stress response. Patterns of protein expression and phosphorylation are indicative of increased oxidative stress, mammary gland restructuring, and immune dysregulation due to prior exposure to dry period heat stress. This study provides insights into the molecular underpinnings of disrupted mammary function and health during lactation arising from prior exposure to dry period heat stress, which might have led to lower milk yields.

Evolutionary trends in the floral transcriptome: insights from one of the basalmost angiosperms, the water lily Nuphar advena (Nymphaeaceae).

Current understanding of floral developmental genetics comes primarily from the core eudicot model Arabidopsis thaliana. Here, we explore the floral transcriptome of the basal angiosperm, Nuphar advena (water lily), for insights into the ancestral developmental program of flowers. We identify several thousand Nuphar genes with significantly upregulated floral expression, including homologs of the well-known ABCE floral regulators, deployed in broadly overlapping transcriptional programs across floral organ categories. Strong similarities in the expression profiles of different organ categories in Nuphar flowers are shared with the magnoliid Persea americana (avocado), in contrast to the largely organ-specific transcriptional cascades evident in Arabidopsis, supporting the inference that this is the ancestral condition in angiosperms. In contrast to most eudicots, floral organs are weakly differentiated in Nuphar and Persea, with staminodial intermediates between stamens and perianth in Nuphar, and between stamens and carpels in Persea. Consequently, the predominantly organ-specific transcriptional programs that characterize Arabidopsis flowers (and perhaps other eudicots) are derived, and correlate with a shift towards morphologically distinct floral organs, including differentiated sepals and petals, and a perianth distinct from stamens and carpels. Our findings suggest that the genetic regulation of more spatially discrete transcriptional programs underlies the evolution of floral morphology.

Phylogenomics With Hyb-Seq Unravels Korean Hosta Evolution.

The genus Hosta (Agavoideae and Asparagaceae) is one of the most popular landscaping and ornamental plants native to temperate East Asia. Their popularity has led to extensive hybridization to develop various cultivars. However, their long history of hybridization, cultivation, and selection has brought about taxonomic confusion in the Hosta species delimitation along with their indistinguishable morphology. Here, we conducted the first broad phylogenetic analyses of Hosta species based on the most comprehensive genomic data set to date. To do so, we captured 246 nuclear gene sequences and plastomes from 55 accessions of Korean Hosta species using the Hyb-Seq method. As a result, this study provides the following novel and significant findings: (1) phylogenetic analyses of the captured sequences retrieved six species of Hosta in South Korea compared to five to eleven species based on the previous studies, (2) their phylogenetic relationships suggested that the large genome size was ancestral and the diversification of Korean Hosta species was accompanied by decreases in genome sizes, (3) comparison between nuclear genes and plastome revealed several introgressive hybridization events between Hosta species, and (4) divergence times estimated here showed that Hosta diverged 35.59 million years ago, while Korean Hosta species rapidly diversified during the late Miocene. Last, we explored whether these genomic data could be used to infer the origin of cultivars. In summary, this study provides the most comprehensive genomic resources to be used in phylogenetic, population, and conservation studies of Hosta, as well as for unraveling the origin of many cultivars.

Complete Plastome of Three Korean Asarum (Aristolochiaceae): Confirmation Tripartite Structure within Korean Asarum and Comparative Analyses.

The genus Asarum (Aristolochiaceae) is a well-known resource of medicinal and ornamental plants. However, the taxonomy of Korean Asarum is ambiguous due to their considerable morphological variations. Previously, a unique plastome structure has been reported from this genus. Therefore, we investigated the structural change in the plastomes within three Korean Asarum species and inferred their phylogenetic relationships. The plastome sizes of Asarum species assembled here range from 190,168 to 193,356 bp, which are longer than a typical plastome size (160 kb). This is due to the incorporation and duplication of the small single copy into the inverted repeat, which resulted in a unique tripartite structure. We first verified this unique structure using the Illumina Miseq and Oxford Nanopore MinION platforms. We also investigated the phylogeny of 26 Aristolochiaceae species based on 79 plastid protein-coding genes, which supports the monophyly of Korean Asarum species. Although the 79 plastid protein-coding gene data set showed some limitations in supporting the previous classification, it exhibits its effectiveness in delineating some sections and species. Thus, it can serve as an effective tool for resolving species-level phylogeny in Aristolochiaceae. Last, we evaluated variable sites and simple sequence repeats in the plastome as potential molecular markers for species delimitation.