Published papers

Understanding adaptation mechanisms is important in evolutionary biology. Parallel adaptation provides good opportunities to investigate adaptive evolution. To confirm parallel adaptation, it is effective to examine whether the phenotypic similarity has one or multiple origins and to use demographic modeling to consider the gene flow between ecotypes. Solidago yokusaiana is a rheophyte endemic to the Japanese Archipelago that diverged from Solidago virgaurea. This study examined the parallel origins of S. yokusaiana by distinguishing between multiple and single origins and subsequent gene flow. The haplotypes of noncoding chloroplast DNA and genotypes at 14 nuclear simple sequence repeat (nSSR) loci and single-nucleotide polymorphisms (SNPs) revealed by double-digest restriction-associated DNA sequencing (ddRADseq) were used for phylogeographic analysis; the SNPs were also used to model population demographics. Some chloroplast haplotypes were common to S. yokusaiana and its ancestor S. virgaurea. Also, the population genetic structures revealed by nSSR and SNPs did not correspond to the taxonomic species. The demographic modeling supported the multiple origins of S. yokusaiana in at least four districts and rejected a single origin with ongoing gene flow between the two species, implying that S. yokusaiana independently and repeatedly adapted to frequently flooding riversides.

Serpentine, a chemically extreme substrate with a discontinuous distribution, supports a high proportion of endemic plant species. The evolutionary pathway of edaphic endemics in isolated areas may be of parallel origin (convergence), but this can be difficult to detect due to similar sets of phenotypic traits repeatedly selected for by the extreme conditions of serpentine environments. Testing the alternative histories of single origin vs. parallel evolution in serpentine plant populations provides insight into the ability of plants to adapt to extreme serpentine environments and to disperse between isolated serpentine areas. In this study, we focused on the herb Picris hieracioides L. (Asteraceae), which includes early-flowering serpentine populations surrounded by later flowering, non-serpentine populations on Hokkaido Island, Japan. We performed population genetic analysis based on genome-wide SNPs markers and quantified temporal overlap of flowering times under natural conditions. Phylogeographic analysis confirmed the occurrence of two genetic groups, corresponding to the serpentine and non-serpentine races. Although the two races were found to occur in close parapatry over multiple paired soil sites, little evidence for hybridization was detected. The flowering times of the two races showed significant divergence, with flowering peaks separated by more than 1 month. Overall, this study supported the single origin of serpentine Picris populations, and showed the role of divergent flowering time as a strong prezygotic barrier to maintain the edaphic races over short spatial distances.

Background: Genetic variation in plants alters insect abundance and community structure in the field; however, little is known about the importance of a single gene among diverse plant genotypes. In this context, Arabidopsis trichomes provide an excellent system to discern the roles of natural variation and a key gene, GLABRA1, in shaping insect communities. In this study, we transplanted two independent glabrous mutants (gl1-1 and gl1-2) and 17 natural accessions of Arabidopsis thaliana to two localities in Switzerland and Japan. Results: Fifteen insect species inhabited the plant accessions, with the insect community composition significantly attributed to variations among plant accessions. The total abundance of leaf-chewing herbivores was negatively correlated with trichome density at both field sites, while glucosinolates had variable effects on leaf chewers between the sites. Interestingly, there was a parallel tendency for the abundance of leaf chewers to be higher on gl1-1 and gl1-2 than on their different parental accessions, Ler-1 and Col-0, respectively. Furthermore, the loss of function in the GLABRA1 gene significantly decreased the resistance of plants to the two predominant chewers; flea beetles and turnip sawflies. Conclusions: Overall, our results indicate that insect community composition significantly varies among A. thaliana accessions across two distant field sites, with GLABRA1 playing a key role in altering the abundance of leaf-chewing herbivores. Given that such a trichome variation is widely observed in Brassicaceae plants, the present study exemplifies the community-wide effect of a single plant gene on crucifer-feeding insects in the field.

Recent analyses using single nucleotide polymorphism (SNP) are a feasible mean for local collections which potentially possess useful, but not large, genetic variations. Genomic sequences of more than 3000 accessions released by the International Rice Research Institute (IRRI) can be used to characterize various local rice (Oryza sativa) populations. The aim of this study was to develop a method to facilitate genomic characterization of local rice populations. We mainly used 99 indica rice accessions (81 landraces and 18 improved varieties) from the Mekong Delta Development Research Institute (MDI). We obtained 2301 SNPs after a genomic sequencing analysis of the 99 rice accessions and subsequent filtering. Within the IRRI’s dataset, the landraces fell into a cluster consisting of accessions from Southeast Asian countries (Ind3 cluster), and the MDI improved varieties were grouped in a cluster containing IRRI improved varieties (Ind1B cluster). A principal component analysis suggested that geographical location strongly affects phylogenetic relationships, and the MDI landraces were placed into a Vietnam+Cambodia group. To detect the nucleotide diversity within a population, p-value is commonly used. We think that whole genome distribution of p-values representing the nucleotide diversity of each population can be used to characterize local populations. Our simple profiling using low p-value genomic regions was able to reveal regional characteristics of rice genomes and should be useful for identifying local rice populations.

An F2 population of 239 chickens was obtained by an intercross between Nagoya (NAG), a native Japanese breed with low growth, and White Plymouth Rock (WPR), a Western breed with high growth. Using SNP markers obtained by restriction site-associated DNA sequencing, genome-wide QTL analysis was performed and it revealed three QTL for early postnatal growth in the F2 population at genome-wide 5% significance levels. The most highly significant QTL affecting body weights at 2–4 weeks of age and weight gains at 2–3 and 0–4 weeks was located on GGA4 between 34.0 and 65.6 Mb with LOD scores of 3.9–5.9 and it explained 4.9–9.9% of the total variance of the traits. The analysis provided evidence for significant QTL on GGA2 between 105.6 and 125.2 Mb (LOD = 4.6) and on GGA1 between 51.1 and 61.6 Mb (LOD = 4.0) which had effects on body weight at 3 weeks and body weight gain at 0–1 week respectively. These two genomic regions explained 6.6 and 6.9% of the phenotypic F2 variance of the corresponding traits respectively. The allele derived from WPR at all QTL increased the corresponding traits. Neither sex-specific nor epistatic QTL was detected. The results showed that the GGA4 QTL affecting multiple traits is a key locus responsible for early growth in our chicken cross, suggesting that this QTL may make a great contribution to genetic improvement of growth performance of the NAG breed with a low growth rate.

This study aimed to identify quantitative trait loci (QTLs) for growth-related traits by constructing a genetic linkage map based on single nucleotide polymorphism (SNP) markers in Japanese quail. A QTL mapping population of 277 F2 birds was obtained from an intercross between a male of a large-sized strain and three females of a normal-sized strain. Body weight (BW) was measured weekly from hatching to 16 weeks of age. Non-linear regression growth models of Weibull, Logistic, Gompertz, Richards, and Brody were analyzed, and growth curve parameters of Richards was selected as the best model to describe the quail growth curve of the F2 birds. Restriction-site associated DNA sequencing developed 125 SNP markers that were informative between their parental strains. The SNP markers were distributed on 16 linkage groups that spanned 795.9 centiMorgan (cM) with an average marker interval of 7.3 cM. QTL analysis of phenotypic traits revealed four main-effect QTLs. Detected QTLs were located on chromosomes 1 and 3 and were associated with BW from 4 to 16 weeks of age and asymptotic weight of Richards model at genome-wide significant at 1% or 5% level. No QTL was detected for BW from 0 to 3 weeks of age. This is the first report identified QTLs for asymptotic weight of the Richards parameter in Japanese quail. These results highlight that the combination of QTL studies and the RAD-seq method will aid future breeding programs identify genes underlying the QTL and the application of marker-assisted selection in the poultry industry, particularly the Japanese quail.

The shear force value is one of the major traits that determine meat quality. In the present study, we performed QTL analysis for chicken breast muscle shear force value at 7 wk of age using 545 single nucleotide polymorphism (SNP) markers developed via restriction-site associated DNA sequencing (RAD-seq). An F2 resource family was generated by mating Oh-Shamo, a native Japanese chicken breed, and the White Plymouth Rock chicken breed. A total of 215 F2 birds were produced. Simple interval mapping revealed one significant main-effect QTL between 6.28 and 8.10 Mb SNPs on the chromosome Z with a logarithm of odds score of 5.53 at the genome-wide 5% level. At this QTL, the confidence interval, phenotypic variance explained, and additive effect were 26 cM, 12.24%, and -0.31 in males and -0.34 in females, respectively. No QTL with epistatic interaction effects were detected. To our knowledge, this is the first report on a QTL affecting the shear force value in the chicken breast muscle, using SNP markers derived from RAD-seq.

Squash (Cucurbita moschata) displays wide morphological and genetic variations; however, limited information is available regarding the genetic loci of squash that control its agronomic traits. To obtain basic genetic information for C. moschata, an F2 population was prepared derived from a cross between the Vietnamese cultivar ‘Bí Hô Lô TN 6 (TN 6)' and the Japanese cultivar ‘Shishigatani’, and flowering and fruit traits were examined. Overall, the traits showed a continuous distribution in the F2 population, suggesting that they were quantitative traits. A linkage map was constructed based on simple sequence repeat and restriction site-associated DNA (RAD) markers to detect quantitative trait loci (QTLs). Twelve QTLs for flowering and fruit traits, as well as one phenotypic trait locus, were successfully localized on the map. The present QTLs explained the phenotypic variations at a moderate to relatively high level (16.0%-47.3%). RAD markers linked to the QTLs were converted to codominant cleaved amplified polymorphic sequence (CAPS) and derived CAPS markers for the easy detection of alleles. The information reported here provides useful information for understanding the genetics of Cucurbita and other cucurbit species, and for the selection of individuals with ideal traits during the breeding of Cucurbita vegetables.

• Background and Aims The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. • Methods We first compared the performance of phylogenetic analysis based on the plastid matK and trnL–F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. • Key Results Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0–84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. • Conclusions The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

The least absolute shrinkage and selection operator (lasso) and principal component regression (PCR) are popular methods of estimating traits from high-dimensional omics data, such as transcriptomes. The prediction accuracy of these estimation methods is highly dependent on the covariance structure, which is characterized by gene regulation networks. However, the manner in which the structure of a gene regulation network together with the sample size affects prediction accuracy has not yet been sufficiently investigated. In this study, Monte Carlo simulations are conducted to investigate the prediction accuracy for several network structures under various sample sizes. When the gene regulation network is a random graph, a sufficiently large number of observations are required to ensure good prediction accuracy with the lasso. The PCR provided poor prediction accuracy regardless of the sample size. However, a real gene regulation network is likely to exhibit a scale-free structure. In such cases, the simulation indicates that a relatively small number of observations, such as N= 300 , is sufficient to allow the accurate prediction of traits from a transcriptome with the lasso.

Natural environments require organisms to possess robust mechanisms allowing responses to seasonal trends. In Arabidopsis halleri, the flowering regulator AhgFLC shows upregulation and downregulation phases along with long-term past temperature, but the underlying machinery remains elusive. Here, we investigate the seasonal dynamics of histone modifications, H3K27me3 and H3K4me3, at AhgFLC in a natural population. Our advanced modelling and transplant experiments reveal that H3K27me3-mediated chromatin regulation at AhgFLC provides two essential properties. One is the ability to respond to the long-term temperature trends via bidirectional interactions between H3K27me3 and H3K4me3; the other is the ratchet-like character of the AhgFLC system, i.e. reversible in the entire perennial life cycle but irreversible during the upregulation phase. Furthermore, we show that the long-term temperature trends are locally indexed at AhgFLC in the form of histone modifications. Our study provides a more comprehensive understanding of H3K27me3 function at AhgFLC in a complex natural environment.

Sympatric speciation is considered to be difficult without the coupling between ecological traits that allow resource partitioning and reproductive traits that allow assortative mating. Such “magic traits” are known to be involved in most of the compelling examples of sympatric speciation. In this study, we report a possible case of sympatric speciation without magic traits. Three species of ricefish (genus Oryzias) are suggested to have diverged sympatrically within Lake Poso, an ancient lake in Sulawesi. An analysis of genome-wide single-nucleotide polymorphisms showed that these three species are reproductively isolated from each other throughout the lake. Stable isotope analyses revealed that the three species use different food resources, which reflect differences in their feeding morphologies (gill rakers and digestive tracts) and feeding sites. Field and laboratory observations showed that O. nebulosus and O. orthognathus share a mating habitat of cobbles, where they scatter fertilized eggs, whereas this site is never used by O. nigrimas, indicating that assortative mating is partly achieved by spatial isolation. The small, less-adhesive eggs of O. nebulosus and O. orthognathus probably reflect their adaptation to spawning on cobble beaches. Laboratory mating experiments showed strong prezygotic isolation between O. nebulosus and O. orthognathus, which is achieved by strong species recognition presumably by both sexes based on species-specific mating dances and nuptial coloration. In summary, the assortative mating of O. nebulosus and O. orthognathus is probably not coupled to resource partitioning. We discussed how sympatric speciation among these species might have been achieved even without magic traits.

Diel and seasonal oscillations are two major environmental changes in nature. While organisms cope with the former by the well-characterized mechanism of the circadian clock1,2, there is limited information on the molecular mechanisms underlying long-term responses to the latter3–5. Histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone modification, imparts stability and plasticity to gene regulation during developmental transitions6–9. Here we studied the seasonal and diel dynamics of H3K27me3 at the genome-wide level in a natural population of perennial Arabidopsis halleri and compared these dynamics with those of histone H3 lysine 4 trimethylation (H3K4me3), an active histone modification. Chromatin immunoprecipitation sequencing revealed that H3K27me3 exhibits seasonal plasticity and diel stability. Furthermore, we found that the seasonal H3K27me3 oscillation is delayed in phase relative to the H3K4me3 oscillation, particularly for genes associated with environmental memory. Our findings suggest that H3K27me3 monitors past transcriptional activity to create long-term gene expression trends during organismal responses over weeks in natural fluctuating environments.

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.

Seedling growth is an important factor for direct seeding of rice. However, the genetic and transcriptomic factors involved in this process are largely unknown. In this study, transcripts affecting shoot weight were identified in rice (Oryza sativa L.) using RNA sequencing (RNA-Seq) data from 20 backcrossed inbred lines (BILs) and their parental cultivars. The selection frequency of the genes for the regression model was determined using repeated analysis of random subsets of the transcriptome. The qLTG3-1gene, controlling low-temperature germinability, and short grain 1 gene (SG1), known to decrease organ elongation, showed high frequency. The quantitative trait loci (QTLs) analysis performed for BILs revealed that qLTG3-1 was included in the QTLs for shoot weight but SG1 was not. No nucleotide polymorphisms were found in the coding region of SG1 in either of the parental cultivars. Quantitative real-time PCR showed that SG1 expression was negatively correlated with shoot weight for all 104 BILs analyzed in this study. Expression QTL (eQTLs) analysis showed an eQTL for SG1 expression located in the same region as the QTL for shoot weight. However, no eQTLs were detected on the same chromosome as SG1, suggesting that nucleotide polymorphisms around the gene do not affect its expression in analyzed growth stage. Overall, these results indicate that RNA-Seq is a useful tool for identifying transcripts that can be related to seedling growth rate.

At low temperatures (18°C), seedlings of an indica rice (Oryza sativa L.) cultivar Kasalath showed symptoms of chlorosis, although the leaves of a japonica cultivar Arroz da Terra remained green. In this study, transcripts related to the chlorophyll content of rice seedlings grown at 18°C were investigated using RNA-sequencing (RNA-Seq) data for F2 crosses between cultivars Arroz da Terra and Kasalath, as well as their parental cultivars. Differential expression analysis revealed that gene ontology terms related to 'photosynthesis' were significantly enriched in lowly expressed genes at 18°C than at 25°C in Kasalath. However, the gene ontology terms related to 'response to stress' were significantly enriched in highly expressed genes at 18°C than at 25°C in Kasalath. When the F2 plants were grown at 18°C, their chlorophyll contents varied. Transcripts with expression levels related to chlorophyll content were statistically selected using RNA-Seq data from 21 F2 plants. In regression models, frequently selected genes included four photosynthetic and two stress-responsive genes. The expression values of four photosynthetic and two stress-responsive genes in high-frequency selected genes were significantly correlated with chlorophyll content not only in plants analysed using RNA-Seq but also in 95 F2 plants.

The circadian rhythm varies among species, and the distribution of common circadian rhythm-related genes in plants is not yet clearly understood. In this study, we analyzed the transcriptome data from plants of three different species (Solanum lycopersicum, Arabidopsis thaliana, and Lactuca sativa) and their circadian rhythms. Homology of the gene sequences was analyzed. Thirty genes containing time information were found to be common among the three species studied and were used to measure the circadian rhythm. Because 22 of these 30 genes were associated with photosynthesis, we suggest that light control could be used to regulate the circadian rhythm. Currently, a high-cost transcriptome analysis is required for the measurement of circadian rhythm; however, our results showed that it was possible to reduce the number of target genes to 30 and, hence, to reduce the cost of the analysis. Our findings will enable easier estimation of circadian time, which, in turn, will facilitate environmental control for plant growth through better control of circadian time, thus facilitating better crop management practice.

Latitude is correlated with environmental components that determine the distribution of biodiversity. In combination with geographic factors, latitude-associated environmental variables are expected to influence speciation, but empirical evidence on how those factors interplay is scarce. We evaluated the genetic and environmental variation among populations in the pair of sister species Dioon sonorense–D. vovidesii, two cycads distributed along a latitudinal environmental gradient in northwestern Mexico, to reveal their demographic histories and the environmental factors involved in their divergence. Using genome-wide loci data, we determined the species delimitation, estimated the gene flow, and compared multiple demographic scenarios of divergence. Also, we estimated the variation of climatic variables among populations and used ecological niche models to test niche overlap between species. The effect of geographic and environmental variables on the genetic variation among populations was evaluated using linear models. Our results suggest the existence of a widespread ancestral population that split into the two species ~829 ky ago. The geographic delimitation along the environmental gradient occurs in the absence of major geographic barriers, near the 28th parallel north, where a zonation of environmental seasonality exists. The northern species, D. vovidesii, occurs in more seasonal environments but retains the same niche of the southern species, D. sonorense. The genetic variation throughout populations cannot be solely explained by stochastic processes; the latitudinal-associated seasonality has been an additive factor that strengthened the species divergence. This study represents an example of how speciation can be achieved by the effect of the latitude-associated factors on the genetic divergence among populations.

Stacksbinder (available at https://ps.agr.ryukoku.ac.jp/stacksbinder/) is a web-based tool for summarizing the output of software Stacks, which is widely used to analyze restriction site-associated DNA sequencing (RAD-Seq) data. Although summarization is an essential step in RAD-Seq analysis, no specific tools exist for summarizing and visualizing these data. Stacksbinder generates a summary report by using files exported from Stacks. The report is HTML-based and easy to browse in any environment. It consists of plots and tables containing information that can be used to assess the quality of experiments and determine the filtering of single nucleotide polymorphisms.

Recently, increasing attempts have been made to understand how plant genes function in natura. In this context, transcriptional profiles represent plant physiological status in response to environmental stimuli. Herein, we combined high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana grown at a field site in Switzerland. We found that genes with the gene ontology (GO) annotations of “glucosinolate biosynthetic process” and “response to insects” were most significantly enriched, and the expression of these was highly variable among plant accessions. Nearly half of the total expression variation in the glucosinolate biosynthetic genes (AOPs, ESM1, ESP, and TGG1) was explained by among-accession variation. Of these genes, the expression level of AOP3 differed among Col-0 accession individuals depending on the abundance of the mustard aphid (Lipaphis erysimi). We also found that the expression of the major cis-jasmone activated gene CYP81D11 was positively correlated with the number of flea beetles (Phyllotreta striolata and Phyllotreta atra). Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to attack by mustard aphids. The combined results from RNA-Seq and our ecological survey illustrate the feasibility of using field transcriptomics to detect an inducible defense, providing a first step towards an in natura understanding of biotic interactions involving phenotypic plasticity.