Effects of fruit dimorphism on genetic structure and gene flow in the coastal shrub Scaevola taccada.

BACKGROUND AND AIMS: Plant propagules often possess specialized morphologies that facilitate dispersal across specific landscapes. In the fruit dimorphism of a coastal shrub, Scaevola taccada, individual plants produce either cork-morph or pulp-morph fruits. The former is buoyant and common on sandy beaches, whereas the latter does not float, is bird-dispersed, and is common on elevated sites such as slopes on sea cliffs and behind rocky shores. We hypothesized that beach populations bridge the heterogeneous landscapes by serving as a source of both fruit types, while dispersal is biased for the pulp morph on elevated sites within the islands and for the cork morph between beaches of different islands. Based on this hypothesis, we predicted that populations in elevated sites would diverge genetically over time due to isolation by distance, whereas beach populations would maintain high genetic similarity via current gene flow. METHODS: The genetic structure and gene flow in S. taccada were evaluated by investigating genome-wide single nucleotide polymorphisms in plants from 17 sampling sites on six islands (belonging to the Ryukyu, Daito and Ogasawara Islands) in Japan. KEY RESULTS: Geographical isolation was detected among the three distant island groups. Analyses within the Ryukyu Islands suggested that sandy beach populations were characterized by genetic admixture, whereas populations in elevated sites were relatively isolated between the islands. Pairwise FST values between islands were lowest between sandy beaches, intermediate between sandy beaches and elevated sites, and highest between elevated sites. CONCLUSIONS: Dispersal across the ocean by cork morphs is sufficiently frequent to prevent genetic divergence between beaches of different islands. Stronger genetic isolation of elevated sites between islands suggests that bird dispersal by pulp morphs is restricted mainly within islands. These contrasting patterns of gene flow realized by fruit dimorphism provide evidence that fruit characteristics can strongly mediate genetic structure.

Altered stomatal patterning accompanies a trichome dimorphism in a natural population of Arabidopsis.

Trichomes are large epidermal cells on the surface of leaves that are thought to deter herbivores, yet the presence of trichomes can also negatively impact plant growth and reproduction. Stomatal guard cells and trichomes have shared developmental origins, and experimental manipulation of trichome formation can lead to changes in stomatal density. The influence of trichome formation upon stomatal development in natural populations of plants is currently unknown. Here, we show that a natural population of Arabidopsis halleri that includes hairy (trichome-bearing) and glabrous (no trichomes) morphs has differences in stomatal density that are associated with this trichome dimorphism. We found that glabrous morphs had significantly greater stomatal density and stomatal index than hairy morphs. One interpretation is that this arises from a trade-off between the proportions of cells that have trichome and guard cell fates during leaf development. The differences in stomatal density between the two morphs might have impacts upon environmental adaptation, in addition to herbivory deterrence caused by trichome development.

Genet assignment and population structure analysis in a clonal forest-floor herb, Cardamine leucantha, using RAD-seq.

To study the genetic structure of clonal plant populations, genotyping and genet detection using genetic markers are necessary to assign ramets to corresponding genets. Assignment is difficult as it involves setting a robust threshold of genetic distance for genet distinction as neighbouring genets in a plant population are often genetically related. Here, we used restriction site-associated DNA sequencing (RAD-seq) for a rhizomatous clonal herb, Cardamine leucantha [Brassicaceae] to accurately determine genet structure in a natural population. We determined a draft genome sequence of this species for the first time, which resulted in 66 617 scaffolds with N50 = 6086 bp and an estimated genome size of approximately 253 Mbp. Using genetic distances based on the RAD-seq analysis, we successfully distinguished ramets that belonged to distinct genets even from a half-sib family. We applied these methods to 372 samples of C. leucantha collected at 1-m interval grids within a 20 × 20 m plot in a natural population in Hokkaido, Japan. From these samples, we identified 61 genets with high inequality in terms of genet size and patchy distribution. Spatial autocorrelation analyses indicated significant aggregation within 7 and 4 m at ramet and genet levels, respectively. An analysis of parallel DNA microsatellite loci (simple sequence repeats) suggested that RAD-seq can provide data that allows robust genet assignment. It remains unclear whether the large genets identified here became dominant stochastically or deterministically. Precise identification of genets will assist further study and characterization of dominant genets.

Seasonality of interactions between a plant virus and its host during persistent infection in a natural environment.

Persistent infection, wherein a pathogen is continually present in a host individual, is widespread in virus-host systems. However, little is known regarding how seasonal environments alter virus-host interaction during such metastability. We observed a lineage-to-lineage infection of the host plant Arabidopsis halleri with Turnip mosaic virus for 3 years without severe damage. Virus dynamics and virus-host interactions within hosts were highly season dependent. Virus accumulation in the newly formed leaves was temperature dependent and was suppressed during winter. Transcriptome analyses suggested that distinct defence mechanisms, i.e. salicylic acid (SA)-dependent resistance and RNA silencing, were predominant during spring and autumn, respectively. Transcriptomic difference between infected and uninfected plants other than defence genes appeared transiently only during autumn in upper leaves. However, the virus preserved in the lower leaves is transferred to the clonal offspring of the host plants during spring. In the linage-to-linage infection of the A. halleri-TuMV system, both host clonal reproduction and virus transmission into new clonal rosettes are secured during the winter-spring transition. How virus and host overwinter turned out to be critical for understanding a long-term virus-host interaction within hosts under temperate climates, and more generally, understanding seasonality provides new insight into ecology of plant viruses.

Arabidopsis halleri: a perennial model system for studying population differentiation and local adaptation.

Local adaptation is assumed to occur when populations differ in a phenotypic trait or a set of traits, and such variation has a genetic basis. Here, we introduce Arabidopsis halleri and its life history as a perennial model system to study population differentiation and local adaptation. Studies on altitudinal adaptation have been conducted in two regions: Mt. Ibuki in Japan and the European Alps. Several studies have demonstrated altitudinal adaptation in ultraviolet-B (UV-B) tolerance, leaf water repellency against spring frost and anti-herbivore defences. Studies on population differentiation in A. halleri have also focused on metal hyperaccumulation and tolerance to heavy metal contamination. In these study systems, genome scans to identify candidate genes under selection have been applied. Lastly, we briefly discuss how RNA-Seq can broaden phenotypic space and serve as a link to underlying mechanisms. In conclusion, A. halleri provides us with opportunities to study population differentiation and local adaptation, and relate these to the genetic systems underlying target functional traits.

Seasonal Stability and Dynamics of DNA Methylation in Plants in a Natural Environment.

: DNA methylation has been considered a stable epigenetic mark but may respond to fluctuating environments. However, it is unclear how they behave in natural environments. Here, we analyzed seasonal patterns of genome-wide DNA methylation in a single clone from a natural population of the perennial Arabidopsishalleri. The genome-wide pattern of DNA methylation was primarily stable, and most of the repetitive regions were methylated across the year. Although the proportion was small, we detected seasonally methylated cytosines (SeMCs) in the genome. SeMCs in the CHH context were detected predominantly at repetitive sequences in intergenic regions. In contrast, gene-body CG methylation (gbM) itself was generally stable across seasons, but the levels of gbM were positively associated with seasonal stability of RNA expression of the genes. These results suggest the existence of two distinct aspects of DNA methylation in natural environments: sources of epigenetic variation and epigenetic marks for stable gene expression.

Publisher Correction: Annual transcriptome dynamics in natural environments reveals plant seasonal adaptation.

In Fig. 3b of the version of this Article originally published, a number of arrows indicating repression of downstream processes were mistakenly formatted as arrows indicating activation of downstream processes. This has now been amended in all versions of the Article.

Annual transcriptome dynamics in natural environments reveals plant seasonal adaptation.

As most organisms have evolved in seasonal environments, their environmental responses should be adapted to seasonal transitions. Here we show that the combination of temperature and day length shapes the seasonal dynamics of the transcriptome and adaptation to seasonal environments in a natural habitat of a perennial plant Arabidopsis halleri subsp. gemmifera. Weekly transcriptomes for two years and bihourly diurnal transcriptomes on the four equinoxes/solstices, identified 2,879 and 7,185 seasonally- and diurnally-oscillating genes, respectively. Dominance of annual temperature changes for defining seasonal oscillations of gene expressions was indicated by controlled environment experiments manipulating the natural 1.5-month lag of temperature behind day length. We found that plants have higher fitness in 'natural' chambers than in 'unnatural' chambers simulating in-phase and anti-phase oscillations between temperature and day length. Seasonal temperature responses were disturbed in unnatural chambers. Our results demonstrate how plants use multiple types of environmental information to adapt to seasonal environments.

A Survey on Plant Viruses in Natural Brassicaceae Communities Using RNA-Seq.

Studies on plant viruses are biased towards crop diseases and little is known about viruses in natural vegetation. We conducted extensive surveys of plant viruses in wild Brassicaceae plants occurring in three local plant communities in central Japan. We applied RNA-Seq with selective depletion of rRNA, which allowed us to detect infections of all genome-reported viruses simultaneously. Infections of Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV), Brassica yellows virus, Pelargonium zonate spot virus, and Arabidopsis halleri partitivirus 1 were detected from the two perennial species, Arabidopsis halleri subsp. gemmifera and Rorippa indica. De novo assembly further detected partial sequences of a putative novel virus in Arabis fragellosa. Virus species composition and infection rate differed depending on site and plant species. Viruses were most frequently detected from the perennial clonal plant, A. halleri, in which a high clonal transmission rate of viruses across multiple years was confirmed. Phylogenetic analysis of TuMV and CMV showed that virus strains from wild Brassicaceae were included as a major clade of these viruses with other reported strains from crop plants, suggesting that viruses were shared among wild plants and crops. Our studies indicated that distribution of viruses in natural plant populations are determined by the combinations of life histories of viruses and hosts. Revealing viral distribution in the natural plant communities improves our knowledge on the ecology of plant viruses.

The Long-Term "In Natura" Study Sites of Arabidopsis halleri for Plant Transcription and Epigenetic Modification Analyses in Natural Environments.

The majority of organismal phenomena show functional significance in the context of natural environments. However, we know little about how dynamic gene expression is controlled under natural complex conditions. One of the most attractive challenges in current biology is to understand organismal functions in natural environments. We established and have developed long-term "in natura" study sites of Arabidopsis halleri to evaluate precise control of gene expression in natural environments. At the sites, we monitored meteorological factors, recorded plant growth and phenology, and collected RNA and chromatin samples to investigate dynamics of transcription and epigenetic modifications. Here, we introduce the in natura study sites, especially with the emphasis on methodologies for setting up study sites in natural plant populations and collecting samples used in transcriptomics and epigenetics in natural environments. Although the methods introduced here need to be modified depending on situations of one's study systems, our case can be a model for planning new in natura studies.

Genetic differentiation in cauline-leaf-specific wettability of a rosette-forming perennial Arabidopsis from two contrasting montane habitats.

Background and Aims: An altitudinal gradient of leaf wettability is often observed between and within species. To understand its functional significance, positional variation of leaf surfaces within plants should be taken into account. In rosette-forming plants, rosette leaves are near the ground and their adaxial surfaces are exposed, whereas cauline leaves are lifted from the ground throughout the reproductive season, and their abaxial surfaces are more exposed. Here, we investigated leaf wettability of cauline and rosette leaves of Arabidopsis halleri subsp. gemmifera growing in contrasting montane habitats along an altitudinal gradient at Mt Ibuki, Japan. Methods: We conducted field investigations and a growth chamber experiment to determine whether field-observed variation in leaf wettability was caused by genetic differentiation. We further performed gene expression analysis of a wax-related gene, i.e. AhgCER1, a homologue of A. thaliana ECERIFERUM1 (CER1) that may be involved in differentiation of leaf wettability. Key Results: We found cauline-leaf specific genetic differentiation in leaf wettability between contrasting montane habitats. Cauline leaves of semi-alpine plants, especially on abaxial surfaces, were non-wettable. Cauline leaves of low-altitudinal understorey plants were wettable, and rosette leaves were also wettable in both habitats. AhgCER1 expression corresponded to observed leaf wettability patterns. Conclusions: Low wettability of cauline leaves is hypothesized to keep exposed surfaces dry when they are wrapping flowering buds in early spring, and presumably protects flowering buds from frost damage. The genetic system that controls wax content, specifically for cauline leaves, should be involved in the observed genetic differentiation, and AhgCER1 control is a strong candidate for the underlying genetic mechanism.

Phylogeographic analysis of the East Asian goldenrod (Solidago virgaurea complex, Asteraceae) reveals hidden ecological diversification with recurrent formation of ecotypes.

Background and Aims: The processes and mechanisms underlying lineage diversification are major topics in evolutionary biology. Eurasian goldenrod species of the Solidago virgaurea complex show remarkable morphological and ecological diversity in the Japanese Archipelago, with ecotypic taxa well adapted to specific environments (climate, edaphic conditions and disturbance regimes). The species complex is a suitable model to investigate the evolutionary processes of actively speciating plant groups, due to its ability to evolve in relation to environmental adaptation and its historical population dynamics. Methods: Two chloroplast markers, 18 nuclear microsatellite markers and ddRAD-sequencing were used to infer population genetic demography of S. virgaurea complex with its related species/genera. Key Results: Our analysis showed that populations in Japan form an evolutionary unit, which was genetically diverged from adjacent continental populations. The phylogenetic structure within the archipelago strongly corresponds to the geography, but interestingly there is no concordance between genetic structure and ecotypic boundaries; neighbouring populations of distinct ecotypes share a genetic background. Conclusions: We propose that the traits specific to the ecotypic entities are maintained by natural selection or are very recently generated and have little effect on the genomes, making genome-wide genetic markers unsuitable for detecting ecotypic differentiation. Furthermore, some sporadically distributed taxa (found as rheophytes and alpine plants) were repeatedly generated from a more widespread taxon in geographically distant areas by means of selection. Overall, this study showed that the goldenrod complex has a high ability to evolve, enabling rapid ecological diversification over a recent timeframe.

The population genomic signature of environmental association and gene flow in an ecologically divergent tree species Metrosideros polymorpha (Myrtaceae).

Genomewide markers enable us to study genetic differentiation within a species and the factors underlying it at a much higher resolution than before, which advances our understanding of adaptation in organisms. We investigated genomic divergence in Metrosideros polymorpha, a woody species that occupies a wide range of ecological habitats across the Hawaiian Islands and shows remarkable phenotypic variation. Using 1659 single nucleotide polymorphism (SNP) markers annotated with the genome assembly, we examined the population genetic structure and demographic history of nine populations across five elevations and two ages of substrates on Mauna Loa, the island of Hawaii. The nine populations were differentiated into two genetic clusters distributed on the lower and higher elevations and were largely admixed on the middle elevation. Demographic modelling revealed that the two genetic clusters have been maintained in the face of gene flow, and the effective population size of the high-altitude cluster was much smaller. A FST -based outlier search among the 1659 SNPs revealed that 34 SNPs (2.05%) were likely to be under divergent selection and the allele frequencies of 21 of them were associated with environmental changes along elevations, such as temperature and precipitation. This study shows a genomic mosaic of M. polymorpha, in which contrasting divergence patterns were found. While most genomic polymorphisms were shared among populations, a small fraction of the genome was significantly differentiated between populations in diverse environments and could be responsible for the dramatic adaptation to a wide range of environments.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Circadian Oscillation of the Lettuce Transcriptome under Constant Light and Light-Dark Conditions.

Although, the circadian clock is a universal biological system in plants and it orchestrates important role of plant production such as photosynthesis, floral induction and growth, there are few such studies on cultivated species. Lettuce is one major cultivated species for both open culture and plant factories and there is little information concerning its circadian clock system. In addition, most of the relevant genes have not been identified. In this study, we detected circadian oscillation in the lettuce transcriptome using time-course RNA sequencing (RNA-Seq) data. Constant light (LL) and light-dark (LD) conditions were used to detect circadian oscillation because the circadian clock has some basic properties: one is self-sustaining oscillation under constant light and another is entrainment to environmental cycles such as light and temperature. In the results, 215 contigs were detected as common oscillating contigs under both LL and LD conditions. The 215 common oscillating contigs included clock gene-like contigs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)-like, TOC1 (TIMING OF CAB EXPRESSION 1)-like and LHY (LATE ELONGATED HYPOCOTYL)-like, and their expression patterns were similar to those of Arabidopsis. Functional enrichment analysis by GO (gene ontology) Slim and GO Fat showed that the GO terms of response to light stimulus, response to stress, photosynthesis and circadian rhythms were enriched in the 215 common oscillating contigs and these terms were actually regulated by circadian clocks in plants. The 215 common oscillating contigs can be used to evaluate whether the gene expression pattern related to photosynthesis and optical response performs normally in lettuce.

RNA-Seq reveals virus-virus and virus-plant interactions in nature.

As research on plant viruses has focused mainly on crop diseases, little is known about these viruses in natural environments. To understand the ecology of viruses in natural systems, comprehensive information on virus-virus and virus-host interactions is required. We applied RNA-Seq to plants from a natural population of Arabidopsis halleri subsp. gemmifera to simultaneously determine the presence/absence of all sequence-reported viruses, identify novel viruses and quantify the host transcriptome. By introducing the criteria of read number and genome coverage, we detected infections by Turnip mosaic virus (TuMV), Cucumber mosaic virus and Brassica yellows virus Active TuMV replication was observed by ultramicroscopy. De novo assembly further identified a novel partitivirus, Arabidopsis halleri partitivirus 1 Interestingly, virus reads reached a maximum level that was equivalent to that of the host's total mRNA, although asymptomatic infection was common. AhgAGO2, a key gene in host defence systems, was upregulated in TuMV-infected plants. Multiple infection was frequent in TuMV-infected leaves, suggesting that TuMV facilitates multiple infection, probably by suppressing host RNA silencing. Revealing hidden plant-virus interactions in nature can enhance our understanding of biological interactions and may have agricultural applications.

Detection of Diurnal Variation of Tomato Transcriptome through the Molecular Timetable Method in a Sunlight-Type Plant Factory.

The timing of measurement during plant growth is important because many genes are expressed periodically and orchestrate physiological events. Their periodicity is generated by environmental fluctuations as external factors and the circadian clock as the internal factor. The circadian clock orchestrates physiological events such as photosynthesis or flowering and it enables enhanced growth and herbivory resistance. These characteristics have possible applications for agriculture. In this study, we demonstrated the diurnal variation of the transcriptome in tomato (Solanum lycopersicum) leaves through molecular timetable method in a sunlight-type plant factory. Molecular timetable methods have been developed to detect periodic genes and estimate individual internal body time from these expression profiles in mammals. We sampled tomato leaves every 2 h for 2 days and acquired time-course transcriptome data by RNA-Seq. Many genes were expressed periodically and these expressions were stable across the 1st and 2nd days of measurement. We selected 143 time-indicating genes whose expression indicated periodically, and estimated internal time in the plant from these expression profiles. The estimated internal time was generally the same as the external environment time; however, there was a difference of more than 1 h between the two for some sampling points. Furthermore, the stress-responsive genes also showed weakly periodic expression, implying that they were usually expressed periodically, regulated by light-dark cycles as an external factor or the circadian clock as the internal factor, and could be particularly expressed when the plant experiences some specific stress under agricultural situations. This study suggests that circadian clock mediate the optimization for fluctuating environments in the field and it has possibilities to enhance resistibility to stress and floral induction by controlling circadian clock through light supplement and temperature control.

Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses.

RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the -1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G(1-2)A(6-7) motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.

RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties.

We analyzed the reduced-representation genome sequences of Citrus species by double-digest restriction site-associated DNA sequencing (ddRAD-Seq) using 44 accessions, including typical and minor accessions, such as Bhutanese varieties. The results of this analysis using typical accessions were consistent with previous reports that citron, papeda, pummelo, and mandarin are ancestral species, and that most Citrus species are derivatives or hybrids of these four species. Citrus varieties often reproduce asexually and heterozygosity is highly conserved within each variety. Because this approach could readily detect conservation of heterozygosity, it was able to discriminate citrus varieties such as satsuma mandarin from closely related species. Thus, this method provides an inexpensive way to protect citrus varieties from unintended introduction and to prevent the provision of incorrect nursery stocks to customers. One Citrus variety in Bhutan was morphologically similar to Mexican lime and was designated as Himalayan lime. The current analysis confirmed the previous proposition that Mexican lime is a hybrid between papeda and citron, and also suggested that Himalayan lime is a probable hybrid between mandarin and citron. In addition to Himalayan lime, current analysis suggested that several accessions were formed by previously undescribed combinations.