研究者詳細 - 永野　惇

論文 - 永野　惇

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Gene regulatory network and its constituent transcription factors that control nitrogen-deficiency responses in rice

Ueda Y., Ohtsuki N., Kadota K., Tezuka A., Nagano A.J., Kadowaki T., Kim Y., Miyao M., Yanagisawa S.

New Phytologist （New Phytologist） 227 （ 5 ） 1434 - 1452 2020年09月

ISSN 0028646X

Increase in the nitrogen (N)-use efficiency and optimization of N response in crop species are urgently needed. Although transcription factor-based genetic engineering is a promising approach for achieving these goals, transcription factors that play key roles in the response to N deficiency have not been studied extensively. Here, we performed RNA-seq analysis of root samples of 20 Asian rice (Oryza sativa) accessions with differential nutrient uptake. Data obtained from plants exposed to N-replete and N-deficient conditions were subjected to coexpression analysis and machine learning-based pathway inference to dissect the gene regulatory network required for the response to N deficiency. Four transcription factors, including members of the G2-like and bZIP families, were predicted to function as key regulators of gene transcription within the network in response to N deficiency. Cotransfection assays validated inferred novel regulatory pathways, and further analyses using genome-edited knockout lines suggested that these transcription factors are important for N-deficiency responses in planta. Many of the N deficiency-responsive genes, including those encoding key regulators within the network, were coordinately regulated by transcription factors belonging to different families. Transcription factors identified in this study could be valuable for the modification of N response and metabolism.

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Niche conservatism promotes speciation in cycads: the case of Dioon merolae (Zamiaceae) in Mexico

Gutiérrez-Ortega J.S., Salinas-Rodríguez M.M., Ito T., Pérez-Farrera M.A., Vovides A.P., Martínez J.F., Molina-Freaner F., Hernández-López A., Kawaguchi L., Nagano A.J., Kajita T., Watano Y., Tsuchimatsu T., Takahashi Y., Murakami M.

New Phytologist （New Phytologist） 227 （ 6 ） 1872 - 1884 2020年09月

ISSN 0028646X

Niche conservatism is the tendency of lineages to retain the same niche as their ancestors. It constrains biological groups and prevents ecological divergence. However, theory predicts that niche conservatism can hinder gene flow, strengthen drift and increase local adaptation: does it mean that it also can facilitate speciation? Why does this happen? We aim to answer these questions. We examined the variation of chloroplast DNA, genome-wide single nucleotide polymorphisms, morphological traits and environmental variables across the Dioon merolae cycad populations. We tested geographical structure, scenarios of demographic history, and niche conservatism between population groups. Lineage divergence is associated with the presence of a geographical barrier consisting of unsuitable habitats for cycads. There is a clear genetic and morphological distinction between the geographical groups, suggesting allopatric divergence. However, even in contrasting available environmental conditions, groups retain their ancestral niche, supporting niche conservatism. Niche conservatism is a process that can promote speciation. In D. merolae, lineage divergence occurred because unsuitable habitats represented a barrier against gene flow, incurring populations to experience isolated demographic histories and disparate environmental conditions. This study explains why cycads, despite their ancient lineage origin and biological stasis, have been able to diversify into modern ecosystems worldwide.

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Seasonal plasticity and diel stability of H3K27me3 in natural fluctuating environments

Nishio H., Nagano A.J., Ito T., Suzuki Y., Kudoh H.

Nature Plants （Nature Plants） 6 （ 9 ） 1091 - 1097 2020年09月

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.

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Genome-wide patterns of divergence and introgression after secondary contact between Pungitius sticklebacks: Speciation genomics of Pungitius

Yamasaki Y.Y., Kakioka R., Takahashi H., Toyoda A., Nagano A.J., Machida Y., Møller P.R., Kitano J.

Philosophical Transactions of the Royal Society B: Biological Sciences （Philosophical Transactions of the Royal Society B: Biological Sciences） 375 （ 1806 ） 2020年08月

ISSN 09628436

Speciation is a continuous process. Although it is known that differential adaptation can initiate divergence even in the face of gene flow, we know relatively little about the mechanisms driving complete reproductive isolation and the genomic patterns of divergence and introgression at the later stages of speciation. Sticklebacks contain many pairs of sympatric species differing in levels of reproductive isolation and divergence history. Nevertheless, most previous studies have focused on young species pairs. Here, we investigated two sympatric stickleback species, Pungitius pungitius and P. sinensis, whose habitats overlap in eastern Hokkaido; these species show hybrid male sterility, suggesting that they may be at a late stage of speciation. Our demographic analysis using whole-genome sequence data showed that these species split 1.73 Ma and came into secondary contact 37 200 years ago after a period of allopatry. This long period of allopatry might have promoted the evolution of intrinsic incompatibility. Although we detected on-going gene flow and signatures of introgression, overall genomic divergence was high, with considerable heterogeneity across the genome. The heterogeneity was significantly associated with variation in recombination rate. This sympatric pair provides new avenues to investigate the late stages of the stickleback speciation continuum. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

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A ddRAD-based population genetics and phylogenetics of an endangered freshwater fish from Japan

Takahashi T., Nagano A.J., Kawaguchi L., Onikura N., Nakajima J., Miyake T., Suzuki N., Kanoh Y., Tsuruta T., Tanimoto T., Yasui Y., Oshima N., Kawamura K.

Conservation Genetics （Conservation Genetics） 21 （ 4 ） 641 - 652 2020年08月

ISSN 15660621

Rhodeus ocellatus kurumeus is a small cyprinid fish endemic to the Japanese archipelago. This fish frequently hybridizes with an exotic subspecies Rhodeus ocellatus ocellatus, and non-introgressed populations of R. o. kurumeus have greatly decreased. Previous studies based on mitochondrial and nuclear DNA identified introgressed populations and inferred phylogenetic relationships, but these approaches may lead to underestimates of introgression or ambiguous results owing to the small number of genetic markers used. The present study applied double digest restriction site-associated DNA (ddRAD) sequencing to assignment tests and phylogenetic inferences. Data matrices can vary in size and content depending on the strategies used to process ddRAD sequences; therefore, we prepared 25 data matrices with various processing strategies. The assignment tests based on the 25 data matrices resulted in similar assignment patterns and provided evidence for introgression in some populations that had been identified as non-introgressed populations in previous studies. The maximum-likelihood (ML) phylogenetic trees differed in robustness and topology among the 25 matrices; interestingly, ML trees with low statistical support reflected geographical distributions of fish better than ML trees with high statistical support. These results indicate that ddRAD sequencing can detect introgression with greater sensitivity than conventional DNA markers, that ddRAD sequencing is useful for phylogenetic inference among closely related populations within a subspecies, and that statistically robust ML trees do not necessarily reflect the true phylogeny. Application of ddRAD data to conservation genetics and evolutionary history of this subspecies are also discussed.

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Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data

Okuyama Y., Goto N., Nagano A.J., Yasugi M., Kokubugata G., Kudoh H., Qi Z., Ito T., Kakishima S., Sugawara T.

Annals of Botany （Annals of Botany） 126 （ 2 ） 245 - 260 2020年08月

ISSN 03057364

• 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.

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Genetic consequences of being a dwarf: Do evolutionary changes in life-history traits influence gene flow patterns in populations of the world's smallest goldenrod?

Sakaguchi S., Nagano A.J., Yasugi M., Kudoh H., Ishikawa N., Ito M.

Annals of Botany （Annals of Botany） 126 （ 1 ） 163 - 177 2020年06月

ISSN 03057364

Background and Aims: Contrasting life-history traits can evolve through generations of dwarf plant ecotypes, yet such phenotypic changes often involve decreased plant size and reproductive allocation, which can configure seed dispersal patterns and, subsequently, population demography. Therefore, evolutionary transitions to dwarfism can represent good study systems to test the roles of life-history traits in population demography by comparing genetic structure between related but phenotypically divergent ecotypes. Methods: In this study, we examined an ecotypic taxon pair of the world's smallest goldenrod (stem height 2.6 cm) in alpine habitats and its closely related lowland taxon (30-40 cm) found on Yakushima Island, Japan. Genetic variation in chloroplast DNA sequences, nuclear microsatellites and genome-wide single-nucleotide polymorphisms were used to investigate 197 samples from 16 populations, to infer the population genetic demography and compare local genetic structure of the ecotypes. Key Results: We found a pronounced level of genetic differentiation among alpine dwarf populations, which were much less geographically isolated than their lowland counterparts. In particular, several neighbouring dwarf populations (located ~500 m apart) harboured completely different sets of chloroplast haplotypes and nuclear genetic clusters. Demographic modelling revealed that the dwarf populations have not exchanged genes at significant levels after population divergence. Conclusions: These lines of evidence suggest that substantial effects of genetic drift have operated on these dwarf populations. The low-growing stature and reduced fecundity (only 3.1 heads per plant) of the dwarf plants may have reduced gene flow and rare long-distance seed dispersal among habitat patches, although the effects of life-history traits require further evaluation using ecological approaches.

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Genetic analysis of body weight in wild populations of medaka fish from different latitudes

Yassumoto T.I., Nakatsukasa M., Nagano A.J., Yasugi M., Yoshimura T., Shinomiya A.

PLoS ONE （PLoS ONE） 15 （ 6 ） 2020年06月

The genetic bases of growth and body weight are of economic and scientific interest, and teleost fish models have proven useful in such investigations. The Oryzias latipes species complex (medaka) is an abundant freshwater fish in Japan and suitable for genetic studies. We compared two wild medaka stocks originating from different latitudes. The Maizuru population from higher latitudes weighed more than the Ginoza population. We investigated the genetic basis of body weight, using quantitative trait locus (QTL) analysis of the F2 offspring of these populations. We detected one statistically significant QTL for body weight on medaka chromosome 4 and identified 12 candidate genes that might be associated with body weight or growth. Nine of these 12 genes had at least one single nucleotide polymorphism that caused amino acid substitutions in protein-coding regions, and we estimated the effects of these substitutions. The present findings might contribute to the marker-assisted selection of economically important aquaculture species.

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Stacksbinder: online tool for visualizing and summarizing Stacks output to aid filtering of SNPs identified using RAD sequencing

Yasugi M., Tezuka A., Nagano A.J.

Conservation Genetics Resources （Conservation Genetics Resources） 12 （ 1 ） 2020年03月

ISSN 18777252

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.

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

Tsujimoto M., Araki K.S., Honjo M.N., Yasugi M., Nagano A.J., Akama S., Hatakeyama M., Shimizu-Inatsugi R., Sese J., Shimizu K.K., Kudoh H.

AoB PLANTS （AoB PLANTS） 12 （ 1 ） 2020年02月

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.

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

Honjo M.N., Emura N., Kawagoe T., Sugisaka J., Kamitani M., Nagano A.J., Kudoh H.

ISME Journal （ISME Journal） 14 （ 2 ） 506 - 518 2020年02月

ISSN 17517362

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.

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Genetic isolation by distance in the yellowfin goby populations revealed by RAD sequencing

Hirase S., Tezuka A., Nagano A., Kikuchi K., Iwasaki W.

Ichthyological Research （Ichthyological Research） 67 （ 1 ） 98 - 104 2020年01月

ISSN 13418998

Population genetic studies of marine species sometimes find no clear patterns of genetic differentiation, but signals of geographic gene flow typified by isolation by distance (IBD) due to high dispersal ability. A previous study of mitochondrial DNA of the yellowfin goby Acanthogobius flavimanus indicated that this species also does not have clearly differentiated population groups around the Japanese archipelago, in contrast with other coastal goby species that have highly differentiated groups between the Sea of Japan and Pacific Ocean populations. Here, to clearly reveal phylogeographic structures of the yellowfin goby around the Japanese archipelago, we conducted large-scale RAD-sequencing analysis of their nuclear genomic SNP loci. As a result, IBD patterns were confirmed across the yellowfin goby populations, and the larval dispersal distance of this species was revealed to be approximately 19 km on average. In addition, different larval dispersal distances were estimated for its Sea of Japan and Pacific Ocean populations, suggesting that different coastal environments and/or ecological features may affect its dispersal rates. These results would provide foundations for ecological and conservation studies of the yellowfin goby and coastal fishes having similar dispersal strategy to this species.

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Genomic and phenotypic consequences of two independent secondary contact zones between allopatric lineages of the anadromous ice goby Leucopsarion petersii

Hirase S., Kokita T., Nagano A.J., Kikuchi K.

Heredity （Heredity） 124 （ 1 ） 223 - 235 2020年01月

ISSN 0018067X

Genetic and phenotypic analyses of independent secondary contact zones between certain pairs of divergent populations offer powerful opportunities to assess whether the consequences vary with different environmental backgrounds. Populations of the ice goby Leucopsarion petersii are distributed throughout the Japanese archipelago and comprise genetically and phenotypically divergent groups in the Japan Sea and the Pacific Ocean. In particular, populations in the Japan Sea have a larger body size and numbers of vertebrae than those in the Pacific Ocean. Herein, we performed integrated analyses of genotypes and phenotypes of two independent secondary contact zones and investigated their consequences. Population genetic analyses revealed asymmetric introgression of the mitochondrial genome of either lineage relative to little admixture of nuclear genomes in both secondary contact zones. On phenotype analyses, vertebral numbers were clearly explained by nuclear genomic ancestry in both secondary contact zones, whereas body size was not, suggesting that a little introgression of nuclear genes regulates body size. Actually, we observed biased introgression of a candidate gene, neuropeptide Y (NPY), which potentially controls body size in the ice goby. Moreover, the body size changes in the introgressed populations possibly affect the introgression patterns of mitochondrial genomes across these zones. Collectively, our results demonstrated that genomic and phenotypic consequences of secondary contact varied in marine variable environments.

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Characterization and quantitative trait locus mapping of late-flowering from a Thai soybean cultivar introduced into a photoperiod-insensitive genetic background

Sun F., Xu M., Park C., Dwiyanti M.S., Nagano A.J., Zhu J., Watanabe S., Kong F., Liu B., Yamada T., Abe J.

PLoS ONE （PLoS ONE） 14 （ 12 ） 2019年12月

The timing of both flowering and maturation determine crop adaptability and productivity. Soybean (Glycine max) is cultivated across a wide range of latitudes. The molecular-genetic mechanisms for flowering in soybean have been determined for photoperiodic responses to long days (LDs), but remain only partially determined for the delay of flowering under short-day conditions, an adaptive trait of cultivars grown in lower latitudes. Here, we characterized the late-flowering (LF) habit introduced from the Thai cultivar K3 into a photoperiod-insensitive genetic background under different photo-thermal conditions, and we analyzed the genetic basis using quantitative trait locus (QTL) mapping. The LF habit resulted from a basic difference in the floral induction activity and from the suppression of flowering, which was caused by red light-enriched LD lengths and higher temperatures, during which FLOWERING LOCUS T (FT) orthologs, FT2a and FT5a, were strongly down-regulated. QTL mapping using gene-specific markers for flowering genes E2, FT2a and FT5a and 829 single nucleotide polymorphisms obtained from restriction-site associated DNA sequencing detected three QTLs controlling the LF habit. Of these, a QTL harboring FT2a exhibited large and stable effects under all the conditions tested. A resequencing analysis detected a nonsynonymous substitution in exon 4 of FT2a from K3, which converted the glycine conserved in FT-like proteins to the aspartic acid conserved in TERMINAL FLOWER 1-like proteins (floral repressors), suggesting a functional depression in the FT2a protein from K3. The effects of the remaining two QTLs, likely corresponding to E2 and FT5a, were environment dependent. Thus, the LF habit from K3 may be caused by the functional depression of FT2a and the down-regulation of two FT genes by red light-enriched LD conditions and high temperatures.

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Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

Kamitani M., Kashima M., Tezuka A., Nagano A.J.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions but its use in large-scale experiments is limited by its high cost and labour requirements. In this study, we have established a high-throughput and cost-effective RNA-Seq library preparation method that does not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62 °C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired-end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, causing over-estimations of the quantity of DNA and unstable tagmentation results. Degradation of RNA before tagmentation was found to be necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq) and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10–30 °C) affected the plant transcriptomes using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

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Identification of quantitative trait loci for increased α-tocopherol biosynthesis in wild soybean using a high-density genetic map

Park C., Dwiyanti M.S., Nagano A.J., Liu B., Yamada T., Abe J.

BMC Plant Biology （BMC Plant Biology） 19 （ 1 ） 2019年11月

Background: Soybean is one of the most important crop sources of tocopherols (Toc). However, the content of α-Toc, an isoform with the highest vitamin E activity in humans, is low in most cultivars. With the aim of broadening genetic variability, we performed quantitative trait locus (QTL) analysis for a high seed α-Toc trait detected in a wild soybean and characterized the sequence polymorphisms and expression profiles of γ-tocopherol methyltransferase (γ-TMT) genes as potential candidates. Results: A recombinant inbred line population was developed from a cross between the low α-Toc breeding line TK780 and the high α-Toc wild accession B04009. The α-Toc content in seeds correlated strongly with the ratio of α-Toc to γ-Toc contents. QTL analysis using a high-density map constructed with 7710 single nucleotide polymorphisms (SNPs) generated by restriction site-associated DNA sequencing detected six QTLs involved in α-Toc biosynthesis. Of these, three in chromosomes (Chr) 9, 11, and 12 produced consistent effects during a 2-year trial. B04009 allele at QTLs in Chr9 and Chr12 and TK780 allele at the QTL in Chr11 each promoted the conversion of γ-Toc to α-Toc, which elevated the seed α-Toc content. SNPs and indels were detected between the parents in three γ-TMT genes (γ-TMT1, γ-TMT2, and γ-TMT3) co-located in the QTLs in Chr9 and Chr12, of which some existed in the cis-regulatory elements associated with seed development and functions. In immature cotyledons, γ-TMT3 was expressed at higher levels in B04009 than TK780, irrespective of two thermal conditions tested, whereas the expression of γ-TMT2 was markedly upregulated under higher temperatures, particularly in B04009. Conclusions: We identified QTLs consistently controlling α-Toc biosynthesis in wild soybean seeds in 2-year trials. The QTL on Chr9 had been previously identified in soybean, whereas the QTLs on Chr11 and Chr12 were novel. Further molecular dissections and characterization of the QTLs may facilitate the use of high α-Toc alleles from wild soybean in soybean breeding and an understanding of the molecular mechanisms underlying α-Toc biosynthesis in soybean seeds.

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Construction of high-resolution RAD-seq based linkage map, anchoring reference genome, and QTL mapping of the sex chromosome in the marine medaka Oryzias melastigma

Lee B.Y., Kim M.S., Choi B.S., Nagano A.J., Au D.W.T., Wu R.S.S., Takehana Y., Lee J.S.

G3: Genes, Genomes, Genetics （G3: Genes, Genomes, Genetics） 9 （ 11 ） 3537 - 3545 2019年11月

Medaka (Oryzias sp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resource, the marine medaka Oryzias melastigma is also applicable for the marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 single nucleotide polymorphism (SNP) markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with SNP markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics.

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Profiling SNP and nucleotide diversity to characterize mekong delta rice landraces in southeast asian populations

Tam N.T., Dwiyanti M.S., Koide Y., Nagano A.J., Ky H., Tin H.Q., Hien N.L., Dung L.V., Kishima Y.

Plant Genome （Plant Genome） 12 （ 3 ） 2019年11月

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.

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High-yielding rice Takanari has superior photosynthetic response to a commercial rice Koshihikari under fluctuating light

Adachi S., Tanaka Y., Miyagi A., Kashima M., Tezuka A., Toya Y., Kobayashi S., Ohkubo S., Shimizu H., Kawai-Yamada M., Sage R.F., Nagano A.J., Yamori W.

Journal of Experimental Botany （Journal of Experimental Botany） 70 （ 19 ） 5287 - 5297 2019年10月

ISSN 00220957

Leaves within crop canopies experience variable light over the course of a day, which greatly affects photosynthesis and crop productivity. Little is known about the mechanisms of the photosynthetic response to fluctuating light and their genetic control. Here, we examined gas exchange, metabolite levels, and chlorophyll fluorescence during the photosynthetic induction response in an Oryza sativa indica cultivar with high yield (Takanari) and a japonica cultivar with lower yield (Koshihikari). Takanari had a faster induction response to sudden increases in light intensity than Koshihikari, as demonstrated by faster increases in net CO2 assimilation rate, stomatal conductance, and electron transport rate. In a simulated light regime that mimicked a typical summer day, the faster induction response in Takanari increased daily CO2 assimilation by 10%. The faster response of Takanari was explained in part by its maintenance of a larger pool of Calvin-Benson cycle metabolites. Together, the rapid responses of electron transport rate, metabolic flux, and stomatal conductance in Takanari contributed to the greater daily carbon gain under fluctuating light typical of natural environments.

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Evidence for sympatric speciation in a Wallacean ancient lake

Sutra N., Kusumi J., Montenegro J., Kobayashi H., Fujimoto S., Masengi K.W.A., Nagano A.J., Toyoda A., Matsunami M., Kimura R., Yamahira K.

Evolution （Evolution） 73 （ 9 ） 1898 - 1915 2019年09月

ISSN 00143820

Sympatric speciation has been demonstrated in few empirical case studies, despite intense searches, because of difficulties in testing the criteria for this mode of speciation. Here, we report a possible case of sympatric speciation in ricefishes of the genus Oryzias on Sulawesi, an island of Wallacea. Three species of Oryzias are known to be endemic to Lake Poso, an ancient tectonic lake in central Sulawesi. Phylogenetic analyses using RAD-seq-derived single nucleotide polymorphisms (SNPs) revealed that these species are monophyletic. We also found that the three species are morphologically distinguishable and clearly separated by population-structure analyses based on the SNPs, suggesting that they are reproductively isolated from each other. A mitochondrial DNA chronogram suggested that their speciation events occurred after formation of the tectonic lake, and existence of a historical allopatric phase was not supported by coalescent-based demographic inference. Demographic inference also suggested introgressive hybridization from an outgroup population. However, differential admixture among the sympatric species was not supported by any statistical tests. These results all concur with criteria necessary to demonstrate sympatric speciation. Ricefishes in this Wallacean lake provide a promising new model system for the study of sympatric speciation.

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永野 惇

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永野　惇

論文 - 永野　惇