論文 - 永野　惇

Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan

Kawakatsu Y., Sakamoto T., Nakayama H., Kaminoyama K., Igarashi K., Yasugi M., Kudoh H., Nagano A.J., Yano K., Kubo N., Notaguchi M., Kimura S.

Horticulture Research （Horticulture Research）  8 （ 1 ）  2021年12月

ISSN  20527276

　概要を見る

Since ancient times, humans have bred several plants that we rely on today. However, little is known about the divergence of most of these plants. In the present study, we investigated the divergence of Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey), a traditional leafy vegetable in Kyoto (Japan), by combining genetic analysis and a survey of ancient literature. Mibuna is considered to have been bred 200 years ago from Mizuna, another traditional leafy vegetable in Kyoto. Mibuna has simple spatulate leaves, whereas Mizuna has characteristic serrated leaves. The quantitative trait loci (QTL) and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate. Interestingly, the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression. Furthermore, we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past. Indeed, the genomic analysis of multiple turnip cultivars showed that one of the cultivars, Murasakihime, has almost the same sequence in the BrTCP15 region as Mibuna. These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

• Access to Document (DOI)

Maintaining higher leaf photosynthesis after heading stage could promote biomass accumulation in rice

Honda S., Ohkubo S., San N.S., Nakkasame A., Tomisawa K., Katsura K., Ookawa T., Nagano A.J., Adachi S.

Scientific Reports （Scientific Reports）  11 （ 1 ）  2021年12月

　概要を見る

Leaf photosynthetic rate changes across the growing season as crop plants age. Most studies of leaf photosynthesis focus on a specific growth stage, leaving the question of which pattern of photosynthetic dynamics maximizes crop productivity unanswered. Here we obtained high-frequency data of canopy leaf CO2 assimilation rate (A) of two elite rice (Oryza sativa) cultivars and 76 inbred lines across the whole growing season. The integrated A value after heading was positively associated with crop growth rate (CGR) from heading to harvest, but that before heading was not. A curve-smoothing analysis of A after heading showed that accumulated A at > 80% of its maximum (A80) was positively correlated with CGR in analyses of all lines mixed and of lines grouped by genetic background, while the maximum A and accumulated A at ≤ 80% were less strongly correlated with CGR. We also found a genomic region (~ 12.2 Mb) that may enhance both A80 and aboveground biomass at harvest. We propose that maintaining a high A after heading, rather than having high maximum A, is a potential target for enhancing rice biomass accumulation.

• Access to Document (DOI)

H3K27me3 demethylases alter HSP22 and HSP17.6C expression in response to recurring heat in Arabidopsis

Yamaguchi N., Matsubara S., Yoshimizu K., Seki M., Hamada K., Kamitani M., Kurita Y., Nomura Y., Nagashima K., Inagaki S., Suzuki T., Gan E.S., To T., Kakutani T., Nagano A.J., Satake A., Ito T.

Nature Communications （Nature Communications）  12 （ 1 ）  2021年12月

　概要を見る

Acclimation to high temperature increases plants’ tolerance of subsequent lethal high temperatures. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6C loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate these HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

• Access to Document (DOI)

Genome editing reveals fitness effects of a gene for sexual dichromatism in Sulawesian fishes

Ansai S., Mochida K., Fujimoto S., Mokodongan D.F., Sumarto B.K.A., Masengi K.W.A., Hadiaty R.K., Nagano A.J., Toyoda A., Naruse K., Yamahira K., Kitano J.

Nature Communications （Nature Communications）  12 （ 1 ）  2021年12月

　概要を見る

Sexual selection drives rapid phenotypic diversification of mating traits. However, we know little about the causative genes underlying divergence in sexually selected traits. Here, we investigate the genetic basis of male mating trait diversification in the medaka fishes (genus Oryzias) from Sulawesi, Indonesia. Using linkage mapping, transcriptome analysis, and genome editing, we identify csf1 as a causative gene for red pectoral fins that are unique to male Oryzias woworae. A cis-regulatory mutation enables androgen-induced expression of csf1 in male fins. csf1-knockout males have reduced red coloration and require longer for mating, suggesting that coloration can contribute to male reproductive success. Contrary to expectations, non-red males are more attractive to a predatory fish than are red males. Our results demonstrate that integrating genomics with genome editing enables us to identify causative genes underlying sexually selected traits and provides a new avenue for testing theories of sexual selection.

• Access to Document (DOI)

Field multi-omics analysis reveals a close association between bacterial communities and mineral properties in the soybean rhizosphere

Yamazaki S., Mardani-korrani H., Kaida R., Ochiai K., Kobayashi M., Nagano A.J., Fujii Y., Sugiyama A., Aoki Y.

Scientific Reports （Scientific Reports）  11 （ 1 ）  2021年12月

　概要を見る

The plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere are different from the outer soil region (bulk soil). They establish characteristic conditions including microbiota, metabolites, and minerals, and they can directly affect plant growth and development. However, comprehensive insights into those characteristic environments, especially the rhizosphere, and molecular mechanisms of their formation are not well understood. In the present study, we investigated the spatiotemporal dynamics of the root-associated environment in actual field conditions by multi-omics analyses (mineral, microbiome, and transcriptome) of soybean plants. Mineral and microbiome analyses demonstrated a characteristic rhizosphere environment in which most of the minerals were highly accumulated and bacterial communities were distinct from those in the bulk soil. Mantel’s test and co-abundance network analysis revealed that characteristic community structures and dominant bacterial taxa in the rhizosphere significantly interact with mineral contents in the rhizosphere, but not in the bulk soil. Our field multi-omics analysis suggests a rhizosphere-specific close association between the microbiota and mineral environment.

• Access to Document (DOI)

Elucidation of Japanese pepper (Zanthoxylum piperitum De Candolle) domestication using RAD-Seq

Premarathne M.D.G.P., Fukutome N., Yamasaki K., Hayakawa F., Nagano A.J., Mizuno H., Ibaragi N., Nagano Y.

Scientific Reports （Scientific Reports）  11 （ 1 ）  2021年12月

　概要を見る

Japanese pepper, Zanthoxylum piperitum, is native to Japan and has four well-known lineages (Asakura, Takahara, Budou, and Arima), which are named after their production area or morphology. Restriction-site associated DNA sequencing (RAD-Seq) was used to analyse 93 accessions from various areas, including these four lineages. Single nucleotide variant analysis was used to classify the plants into eight groups: the Asakura and Arima lineages each had two groups, the Takahara and Budou lineages each had one group, and two additional groups were present. In one Asakura group and two Arima groups, the plants were present in agricultural fields and mountains, thus representing the early stage of domestication of the Japanese pepper. The second Asakura lineage group was closely related to plants present in various areas, and this represents the second stage of domestication of this plant because, after early domestication, genetically related lineages with desirable traits spread to the periphery. These results demonstrate that domestication of Japanese pepper is ongoing. In addition, this study shows that spineless plants are polyphyletic, despite the spineless lineage being considered a subspecies of Japanese pepper.

• Access to Document (DOI)

Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan

Imai R., Tsuda Y., Ebihara A., Matsumoto S., Tezuka A., Nagano A.J., Ootsuki R., Watano Y.

Scientific Reports （Scientific Reports）  11 （ 1 ）  2021年12月

　概要を見る

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

• Access to Document (DOI)

Relationship between gene regulation network structure and prediction accuracy in high dimensional regression

Okinaga Y., Kyogoku D., Kondo S., Nagano A.J., Hirose K.

Scientific Reports （Scientific Reports）  11 （ 1 ）  2021年12月

　概要を見る

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.

• Access to Document (DOI)

Phylogeographic analysis of Saxifraga fortunei complex (Saxifragaceae) reveals multiple origins of morphological and ecological variations in the Japanese Archipelago

Magota K., Sakaguchi S., Lee J.S., Yamamoto M., Takahashi D., Nagano A.J., Setoguchi H.

Molecular Phylogenetics and Evolution （Molecular Phylogenetics and Evolution）  163 2021年10月

ISSN  10557903

　概要を見る

Phenotypic polymorphism within a species is a notable phenomenon in evolutionary biology to understand the process of adaptive speciation and other historical events. The Saxifraga fortunei complex is a widespread herb found in East Asia. It includes several ecotypic taxa corresponding to their habitat environments. The distribution of the various ecotypes in a limited area of the Japanese Archipelago makes the species a suitable model to investigate the impact of population demographic history and natural selection on lineage diversification. Here, Sanger-based sequencing was used to estimate the divergence timeframe between populations of the Eurasian continent and Japan. Genome-wide SNPs obtained by ddRAD sequencing were used to investigate the phylogeographic origins of ecotypic taxa. The phylogenetic analyses revealed the divergence of the Japanese population from the continental population in the late Miocene. Two distinct regional clades of North and South Japan were identified; phenotypic diversification was evident only in the southern clade. The South Japan clades displayed a historical distribution expansion from north to south. The phenotypic variations appeared to have generated during the expansion. The ecotypic boundaries were incongruent with the genetic grouping. We propose that morphological and ecological specialization in Japanese populations was repeatedly generated by local natural selection.

• Access to Document (DOI)

Resource partitioning is not coupled with assortative mating in sympatrically divergent ricefish in a Wallacean ancient lake

Kakioka R., Sutra N., Kobayashi H., Ansai S., Masengi K.W.A., Nagano A.J., Okuda N., Tanaka R., Sato M., Yamahira K.

Journal of Evolutionary Biology （Journal of Evolutionary Biology）  34 （ 7 ） 1133 - 1143 2021年07月

ISSN  1010061X

　概要を見る

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.

• Access to Document (DOI)

A spinach genome assembly with remarkable completeness, and its use for rapid identification of candidate genes for agronomic traits

Hirakawa H., Toyoda A., Itoh T., Suzuki Y., Nagano A.J., Sugiyama S., Onodera Y.

DNA Research （DNA Research）  28 （ 3 ） 1 - 10 2021年06月

ISSN  13402838

　概要を見る

Spinach (Spinacia oleracea) is grown as a nutritious leafy vegetable worldwide. To accelerate spinach breeding efficiency, a high-quality reference genome sequence with great completeness and continuity is needed as a basic infrastructure. Here, we used long-read and linked-read technologies to construct a de novo spinach genome assembly, designated SOL_r1.1, which was comprised of 287 scaffolds (total size: 935.7 Mb; N50 = 11.3 Mb) with a low proportion of undetermined nucleotides (Ns = 0.34%) and with high gene completeness (BUSCO complete 96.9%). A genome-wide survey of resistance gene analogues identified 695 genes encoding nucleotide-binding site domains, receptor-like protein kinases, receptor-like proteins and transmembrane-coiled coil domains. Based on a high-density double-digest restriction-site associated DNA sequencing-based linkage map, the genome assembly was anchored to six pseudomolecules representing ∼73.5% of the whole genome assembly. In addition, we used SOL_r1.1 to identify quantitative trait loci for bolting timing and fruit/seed shape, which harbour biologically plausible candidate genes, such as homologues of the FLOWERING LOCUS T and EPIDERMAL PATTERNING FACTOR-LIKE genes. The new genome assembly, SOL_r1.1, will serve as a useful resource for identifying loci associated with important agronomic traits and for developing molecular markers for spinach breeding/selection programs.

• Access to Document (DOI)

Mitochondrial introgression by ancient admixture between two distant lacustrine fishes in Sulawesi Island

Horoiwa M., Mandagi I.F., Sutra N., Montenegro J., Tantu F.Y., Masengi K.W.A., Nagano A.J., Kusumi J., Yasuda N., Yamahira K.

PLoS ONE （PLoS ONE）  16 （ 6 June ）  2021年06月

　概要を見る

Sulawesi, an island located in a biogeographical transition zone between Indomalaya and Australasia, is famous for its high levels of endemism. Ricefishes (family Adrianichthyidae) are an example of taxa that have uniquely diversified on this island. It was demonstrated that habitat fragmentation due to the Pliocene juxtaposition among tectonic subdivisions of this island was the primary factor that promoted their divergence; however, it is also equally probable that habitat fusions and resultant admixtures between phylogenetically distant species may have frequently occurred. Previous studies revealed that some individuals of Oryzias sarasinorum endemic to a tectonic lake in central Sulawesi have mitochondrial haplotypes that are similar to the haplotypes of O. eversi, which is a phylogenetically related but geologically distant (ca. 190 km apart) adrianichthyid endemic to a small fountain. In this study, we tested if this reflects ancient admixture of O. eversi and O. sarasinorum. Population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that O. eversi and O. sarasinorum are substantially reproductively isolated from each other. Comparison of demographic models revealed that the models assuming ancient admixture from O. eversi to O. sarasinorum was more supported than the models assuming no admixture; this supported the idea that the O. eversi-like mitochondrial haplotype in O. sarasinorum was introgressed from O. eversi. This study is the first to demonstrate ancient admixture of lacustrine or pond organisms in Sulawesi beyond 100 km. The complex geological history of this island enabled such island-wide admixture of lacustrine organisms, which usually experience limited migration.

• Access to Document (DOI)

Speciation along a latitudinal gradient: The origin of the Neotropical cycad sister pair Dioon sonorense–D. vovidesii (Zamiaceae)

Gutiérrez-Ortega J.S., Molina-Freaner F., Martínez J.F., Pérez-Farrera M.A., Vovides A.P., Hernández-López A., Tezuka A., Nagano A.J., Watano Y., Takahashi Y., Murakami M., Kajita T.

Ecology and Evolution （Ecology and Evolution）  11 （ 11 ） 6962 - 6976 2021年06月

　概要を見る

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.

• Access to Document (DOI)

Genetic basis for variation in the number of cephalic pores in a hybrid zone between closely related species of goby, Gymnogobius breunigii and Gymnogobius castaneus

Kakioka R., Kume M., Ishikawa A., Ansai S., Hosoki T.K., Yamasaki Y.Y., Nagano A.J., Toyoda A., Kitano J.

Biological Journal of the Linnean Society （Biological Journal of the Linnean Society）  133 （ 1 ） 143 - 154 2021年05月

ISSN  00244066

　概要を見る

Populations or species exploiting different habitats can differ in sensory perception as a result of divergent adaptation. In bony fish, the water current is perceived via neuromasts, the end organ of the lateral line system. Although fish in different habitats are known to vary in neuromasts, we know little about the genetic basis for such variation. Here, we investigate the genetic basis for variation in supraorbital neuromasts in a hybrid zone between the Japanese gobies Gymnogobius breunigii and Gymnogobius castaneus. The former has supraorbital canal neuromasts with six cephalic pores, whereas the latter has only superficial neuromasts with no canals or pores in the supraorbital region. Our genomic analysis showed that G. breunigii and G. castaneus occur mainly in the lower and mid/upper reaches, respectively. In a river in northern Japan, hybrids were found at the sites between the habitats of the two species. These hybrids exhibited anomalies of cephalic pores. Using this hybrid zone, we conducted genome-wide association studies and identified one locus significantly associated with the number of pores. Genomic cline analysis in the hybrid zone demonstrated that this locus exhibited a higher introgression rate compared with the genomic background, indicating the possibility of adaptive introgression.

• Access to Document (DOI)

Evolutionary effects of geographic and climatic isolation between Rhododendron tsusiophyllum populations on the Izu Islands and mainland Honshu of Japan

Yoichi W., Takahashi M., Nagano A.J., Uehara K., Abe H.

Heredity （Heredity）  126 （ 5 ） 859 - 868 2021年05月

ISSN  0018067X

　概要を見る

Geographic and environmental isolations of islands and the mainland offer excellent opportunity to investigate colonization and survival dynamics of island populations. We inferred and compared evolutionary processes and the demographic history of Rhododendron tsusiophyllum, in the Izu Islands and the much larger island Honshu, treated here as the mainland, using thousands of nuclear SNPs obtained by ddRAD-seq from eight populations of R. tsusiophyllum and three populations of R. tschonoskii as an outgroup. Phylogenetic relationships and their habitats suggest that R. tsusiophyllum had evolved and migrated from cold north to warm south regions. We detected clear genetic divergence among populations in three regions of Honshu and the Izu Islands, suggesting restricted migration between them due to isolated habitats on mountains even in the mainland. The three regions have different changes in effective population size, especially, genetic diversity and population size of the Izu Islands are small compared to the others. Further, habitats of populations in the Izu Islands are warmer than those in Honshu, suggesting that they have undergone adaptive evolution. Our study provides evidences of montane rather than insular isolation on genetic divergence, survival of populations and significance of adaptive evolution for island populations with small population size and low genetic diversity, despite close proximity to mainland populations.

• Access to Document (DOI)

Phylogeographic and demographic modeling analyses of the multiple origins of the rheophytic goldenrod Solidago yokusaiana Makino

Kyan R., Kimura T., Yamashiro T., Fujii S., Sakaguchi S., Ito M., Nagano A.J., Kudoh H., Maki M.

Heredity （Heredity）  126 （ 5 ） 831 - 845 2021年05月

ISSN  0018067X

　概要を見る

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.

• Access to Document (DOI)

Analysis of Genetic Diversity and Population Structure of Orobanche foetida Populations From Tunisia Using RADseq

Boukteb A., Sakaguchi S., Ichihashi Y., Kharrat M., Nagano A.J., Shirasu K., Bouhadida M.

Frontiers in Plant Science （Frontiers in Plant Science）  12 2021年04月

　概要を見る

Orobanche foetida Poiret is a holoparasitic plant that lacks chlorophyll and totally depending on its host for its growth. Orobanche foetida parasitizes host plant roots and extract nutrient and water via a haustorium. Although O. foetida distributes in the Mediterranean region as a wild plant parasite, it parasitizes faba bean causing serious damages which may reach 90% yield losses in Tunisia. Analysis of genetic diversity of the parasite is important to better understand its evolution and spread, remained largely unknown. In this work, we present the first study on genetic diversity and population structure using the robust technique Restriction-site-Associated DNA sequencing (RADseq) for Orobanche spp. We collected 244 samples of O. foetida from 18 faba bean fields in the north of Tunisia including 17 populations from the north-west and one population form the north-east. To overcome the difficulty of SNP discovery in O. foetida genome as a non-model and tetraploid plant, we utilized three different informatics pipelines, namely UNEAK, pyRAD and Stacks. This study showed that genetic differentiation occurred in the Tunisian O. foetida emphasizing the isolation by distance effect. However, no strong population clustering was detected in this work basing on the three data sets and clustering methods used. The present study shed the light on the current distribution and the genetic variation situation of the fetid broomrape in Tunisia, highlighting the importance of understanding the evolution of this parasite and its genetic background. This will aid in developing efficient strategies to control this parasite and its expansion in Tunisia and worldwide.

• Access to Document (DOI)

Inferring historical survivals of climate relicts: the effects of climate changes, geography, and population-specific factors on herbaceous hydrangeas

Sakaguchi S., Asaoka Y., Takahashi D., Isagi Y., Imai R., Nagano A.J., Qiu Y.X., Li P., Lu R., Setoguchi H.

Heredity （Heredity）  126 （ 4 ） 615 - 629 2021年04月

ISSN  0018067X

　概要を見る

Climate relicts hold considerable importance because they have resulted from numerous historical changes. However, there are major interspecific variations among the ways by which they survived climate changes. Therefore, investigating the factors and timing that affected population demographics can expand our understanding of how climate relicts responded to historical environmental changes. Here, we examined herbaceous hydrangeas of genus Deinanthe in East Asia, which show limited distributions and a remarkable disjunction between Japan and central China. Chloroplast genome and restriction site-associated DNA sequencing revealed that speciation event occurred in the late Miocene (ca. 7–9 Mya) in response to global climate change. Two lineages apparently remained not branched until the middle Quaternary, and afterwards started to diverge to regional population groups. The narrow endemic species in central China showed lower genetic diversity (He = 0.082), as its population size rapidly decreased during the Holocene due to isolation in montane refugia. Insular populations in the three Japanese islands (He = 0.137–0.160) showed a genetic structure that was inconsistent with sea barriers, indicating that it was shaped in the glacial period when its range retreated to coastal refugia on the exposed sea floor. Demographic modelling by stairway-plot analysis reconstructed variable responses of Japanese populations: some experienced glacial bottlenecks in refugial isolation, while post-glacial range expansion seemingly exerted founder effects on other populations. Overall, this study demonstrated the involvement of not just one, but multiple factors, such as the interplay between climate changes, geography, and other population-specific factors, that determine the demographics of climate relicts.

• Access to Document (DOI)

Neighbor GWAS: incorporating neighbor genotypic identity into genome-wide association studies of field herbivory

Sato Y., Yamamoto E., Shimizu K.K., Nagano A.J.

Heredity （Heredity）  126 （ 4 ） 597 - 614 2021年04月

ISSN  0018067X

　概要を見る

An increasing number of field studies have shown that the phenotype of an individual plant depends not only on its genotype but also on those of neighboring plants; however, this fact is not taken into consideration in genome-wide association studies (GWAS). Based on the Ising model of ferromagnetism, we incorporated neighbor genotypic identity into a regression model, named “Neighbor GWAS”. Our simulations showed that the effective range of neighbor effects could be estimated using an observed phenotype when the proportion of phenotypic variation explained (PVE) by neighbor effects peaked. The spatial scale of the first nearest neighbors gave the maximum power to detect the causal variants responsible for neighbor effects, unless their effective range was too broad. However, if the effective range of the neighbor effects was broad and minor allele frequencies were low, there was collinearity between the self and neighbor effects. To suppress the false positive detection of neighbor effects, the fixed effect and variance components involved in the neighbor effects should be tested in comparison with a standard GWAS model. We applied neighbor GWAS to field herbivory data from 199 accessions of Arabidopsis thaliana and found that neighbor effects explained 8% more of the PVE of the observed damage than standard GWAS. The neighbor GWAS method provides a novel tool that could facilitate the analysis of complex traits in spatially structured environments and is available as an R package at CRAN (https://cran.rproject.org/package=rNeighborGWAS).

• Access to Document (DOI)

QTL analysis for early growth in an intercross between native Japanese Nagoya and White Plymouth Rock chicken breeds using RAD sequencing-based SNP markers

Essa B.H., Suzuki S., Nagano A.J., Elkholya S.Z., Ishikawa A.

Animal Genetics （Animal Genetics）  52 （ 2 ） 232 - 236 2021年04月

ISSN  02689146

　概要を見る

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.

• Access to Document (DOI)

Iron deficiency-inducible peptide-coding genes OsIMA1 and OsIMA2 positively regulate a major pathway of iron uptake and translocation in rice

Kobayashi T., Nagano A.J., Nishizawa N.K.

Journal of Experimental Botany （Journal of Experimental Botany）  72 （ 6 ） 2196 - 2211 2021年03月

ISSN  00220957

　概要を見る

Under low iron (Fe) availability, plants transcriptionally induce various genes responsible for Fe uptake and translocation to obtain adequate amounts of Fe. Although transcription factors and ubiquitin ligases involved in these Fe deficiency responses have been identified, the mechanisms coordinating these pathways have not been clarified in rice. Recently identified Fe-deficiency-inducible IRON MAN (IMA)/FE UPTAKE-INDUCING PEPTIDE (FEP) positively regulates many Fe-deficiency-inducible genes for Fe uptake in Arabidopsis. Here, we report that the expression of two IMA/FEP genes in rice, OsIMA1 and OsIMA2, is strongly induced under Fe deficiency, positively regulated by the transcription factors IDEF1, OsbHLH058, and OsbHLH059, as well as OsIMA1 and OsIMA2 themselves, and negatively regulated by HRZ ubiquitin ligases. Overexpression of OsIMA1 or OsIMA2 in rice conferred tolerance to Fe deficiency and accumulation of Fe in leaves and seeds. These OsIMA-overexpressing rice exhibited enhanced expression of all of the known Fe-deficiency-inducible genes involved in Fe uptake and translocation, except for OsYSL2, a Fe-nicotianamine transporter gene, in roots but not in leaves. Knockdown of OsIMA1 or OsIMA2 caused minor effects, including repression of some Fe uptake-and translocation-related genes in OsIMA1 knockdown roots. These results indicate that OsIMA1 and OsIMA2 play key roles in enhancing the major pathway of the Fe deficiency response in rice.

• Access to Document (DOI)

Characterization of citrus leaf blotch virus from Nandina domestica ‘Otafukunanten’

Kamitani M., Nagano A.J., Okuno T.

Journal of General Plant Pathology （Journal of General Plant Pathology）  87 （ 2 ） 113 - 116 2021年03月

ISSN  13452630

　概要を見る

Nandina domestica ‘Otafukunanten’ shrubs with narrow-leaf symptoms were diagnosed with citrus leaf blotch virus (CLBV) infection based on RNA-sequencing. The full-length sequence of the CLBV strain isolated from N. domestica was determined, and the amino acid sequence shared 83.7% and 80.2% identity with the replicase polyprotein and coat protein, respectively, of the most closely related CLBV strain. All samples with narrow-leaf symptoms were infected with CLBV and cucumber mosaic virus. To the best of our knowledge, this is the first report of CLBV infection of a plant in the order Ranunculales.

• Access to Document (DOI)

Fluorescent protein-based imaging and tissue-specific RNA-seq analysis of Arabidopsis hydathodes

Yagi H., Nagano A.J., Kim J., Tamura K., Mochizuki N., Nagatani A., Matsushita T., Shimada T.

Journal of Experimental Botany （Journal of Experimental Botany）  72 （ 4 ） 1260 - 1270 2021年02月

ISSN  00220957

　概要を見る

Hydathodes are typically found at leaf teeth in vascular plants and are involved in water release to the outside. Although morphological and physiological analysis of hydathodes has been performed in various plants, little is known about the genes involved in hydathode function. In this study, we performed fluorescent protein-based imaging and tissue-specific RNA-seq analysis in Arabidopsis hydathodes. We used the enhancer trap line E325, which has been reported to express green fluorescent protein (GFP) at its hydathodes. We found that E325-GFP was expressed in small cells found inside the hydathodes (named E cells) that were distributed between the water pores and xylem ends. No fluorescence of the phloem markers pSUC2:GFP and pSEOR1:SEOR1-YFP was observed in the hydathodes. These observations indicate that Arabidopsis hydathodes are composed of three major components: Water pores, xylem ends, and E cells. In addition, we performed transcriptome analysis of the hydathode using the E325-GFP line. Microsamples were collected from GFP-positive or-negative regions of E325 leaf margins with a needle-based device (~130 μm in diameter). RNA-seq was performed with each single microsample using a high-throughput library preparation method called Lasy-Seq. We identified 72 differentially expressed genes. Among them, 68 genes showed significantly higher and four genes showed significantly lower expression in the hydathode. Our results provide new insights into the molecular basis for hydathode physiology and development.

• Access to Document (DOI)

Neighbor QTL: An interval mapping method for quantitative trait loci underlying plant neighborhood effects

Sato Y., Takeda K., Nagano A.J.

G3: Genes, Genomes, Genetics （G3: Genes, Genomes, Genetics）  11 （ 2 ）  2021年02月

　概要を見る

Phenotypes of sessile organisms, such as plants, rely not only on their own genotypes but also on those of neighboring individuals. Previously, we incorporated such neighbor effects into a single-marker regression using the Ising model of ferromagnetism. However, little is known regarding how neighbor effects should be incorporated in quantitative trait locus (QTL) mapping. In this study, we propose a new method for interval QTL mapping of neighbor effects, designated “neighbor QTL,” the algorithm of which includes: (1) obtaining conditional self-genotype probabilities with recombination fraction between flanking markers; (2) calculating conditional neighbor genotypic identity using the self-genotype probabilities; and (3) estimating additive and dominance deviations for neighbor effects. Our simulation using F2 and backcross lines showed that the power to detect neighbor effects increased as the effective range decreased. The neighbor QTL was applied to insect herbivory on Col × Kas recombinant inbred lines of Arabidopsis thaliana. Consistent with previous results, the pilot experiment detected a self-QTL effect on the herbivory at the GLABRA1 locus. Regarding neighbor QTL effects on herbivory, we observed a weak QTL on the top of chromosome 4, at which a weak self-bolting QTL was also identified. The neighbor QTL method is available as an R package (https://cran.r-project.org/package¼rNeighborQTL), providing a novel tool to investigate neighbor effects in QTL studies.

• Access to Document (DOI)

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population

Hirase S., Tezuka A., Nagano A.J., Sato M., Hosoya S., Kikuchi K., Iwasaki W.

Evolution （Evolution）  75 （ 1 ） 176 - 194 2021年01月

ISSN  00143820

　概要を見る

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747-Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA-sequencing, RAD-sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA-sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO-lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage-specific selection on mitochondria-related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.

• Access to Document (DOI)

Geographic and subsequent biotic isolations led to a diversity anomaly of section Heterotropa (genus Asarum: Aristolochiaceae) in insular versus continental regions of the Sino-Japanese Floristic Region

Takahashi D., Sakaguchi S., Feng Y., Isagi Y., Qiu Y.X., Li P., Lu R.S., Lu C.T., Chung S.W., Lin Y.S., Chen Y.C., Nagano A.J., Kawaguchi L., Setoguchi H.

Journal of Biogeography （Journal of Biogeography）  2021年

ISSN  03050270

　概要を見る

Aim: The Sino-Japanese Floristic Region has extremely high species diversity with respect to temperate plants; however, the reasons for this diversity are poorly understood because most studies have only considered geographic isolation caused by climatic oscillations. In some plant groups, high floral trait diversity and uneven species diversity between insular systems and the continental area suggest other factors may have important roles too. The primary purpose of this study is to reveal how abiotic and biotic factors have shaped the species diversity anomaly of Heterotropa between the insular systems and the continental area. Location: The Sino-Japanese Floristic Region. Taxon: Section Heterotropa (genus Asarum; Aristolochiaceae). Methods: Using ddRAD-seq and chloroplast genome data, we built a time-calibrated phylogenetic tree including 79 species. We estimated the patterns of floral traits (flowering time and floral size) evolution using macroevolutionary modelling, and tested the correlation of speciation rate with the trait evolution rates. Finally, we estimated the isolation factors of all taxa pairs and sister-taxa pairs based on distribution range and floral traits. Results: Phylogenetic analysis indicated that Heterotropa was diverged into two clades (continental clade and insular clade) in the Miocene, and the major subclades corresponded to geographic entities. Most rate shifts accelerating floral trait's evolution occurred during the Pleistocene period. Evolution rate of floral traits showed positive correlation with the speciation rate. Large proportion of taxa in the insular clade are distributed allopatrically. Several sister pairs showed floral trait divergence with geographic overlap. Main Conclusions: The diversification of Heterotropa appears to have been triggered by geographic and climatic events, and subsequent repeated floral trait evolution with and without geographic isolation. Furthermore, the high species diversity in the insular systems would have been formed by the repeated range fragmentations and contractions. Our study demonstrates the importance of multidimensional studies to understand the diversification process of temperate plants in the Sino-Japanese Floristic Region.

• Access to Document (DOI)

A recessive gene pepy-1 encoding Pelota confers resistance to begomovirus isolates of PepYLCIV and PepYLCAV in Capsicum annuum

Koeda S., Onouchi M., Mori N., Pohan N.S., Nagano A.J., Kesumawati E.

Theoretical and Applied Genetics （Theoretical and Applied Genetics）  2021年

ISSN  00405752

　概要を見る

Key message: A begomovirus resistance gene pepy-1, which encodes the messenger RNA surveillance factor Pelota, was identified in pepper (C. annuum) through map-based cloning and functional characterization. Abstract: Pepper yellow leaf curl disease caused by begomoviruses seriously affects pepper (Capsicum spp.) production in a number of regions around the world. Ty genes of tomato, which confer resistance to the tomato yellow leaf curl virus, are the only begomovirus resistance genes cloned to date. In this study, we focused on the identification of begomovirus resistance genes in Capsicum annuum. BaPep-5 was identified as a novel source of resistance against pepper yellow leaf curl Indonesia virus (PepYLCIV) and pepper yellow leaf curl Aceh virus (PepYLCAV). A single recessive locus, which we named as pepper yellow leaf curl disease virus resistance 1 (pepy-1), responsible for PepYLCAV resistance in BaPep-5 was identified on chromosome 5 in an F2 population derived from a cross between BaPep-5 and the begomovirus susceptible accession BaPep-4. In the target region spanning 34 kb, a single candidate gene, the messenger RNA surveillance factor Pelota, was identified. Whole-genome resequencing of BaPep-4 and BaPep-5 and comparison of their genomic DNA sequences revealed a single nucleotide polymorphism (A to G) located at the splice site of the 9th intron of CaPelota in BaPep-5, which caused the insertion of the 9th intron into the transcript, resulting in the addition of 28 amino acids to CaPelota protein without causing a frameshift. Virus-induced gene silencing of CaPelota in the begomovirus susceptible pepper No.218 resulted in the gain of resistance against PepYLCIV, a phenotype consistent with BaPep-5. The DNA marker developed in this study will greatly facilitate marker-assisted breeding of begomovirus resistance in peppers.

• Access to Document (DOI)

Mapping of qtls controlling epicotyl length in adzuki bean (Vigna angularis)

Mori M., Maki K., Kawahata T., Kawahara D., Kato Y., Yoshida T., Nagasawa H., Sato H., Nagano A.J., Bethke P.C., Kato K.

Breeding Science （Breeding Science）  71 （ 2 ） 208 - 216 2021年

ISSN  13447610

　概要を見る

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F2 population derived from a cross between the breeding line ‘Tokei1121’ (T1121, long epicotyls) and the cultivar ‘Erimo167’ (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F5 residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.

• Access to Document (DOI)

Selection of transcripts related to low-temperature tolerance using RNA sequencing from F<inf>2</inf>plants between japonica and indica rice (Oryza sativa L.) cultivars

Fukuda A., Hirose T., Hashida Y., Aoki N., Nagano A.J.

Functional Plant Biology （Functional Plant Biology）  2021年

ISSN  14454408

　概要を見る

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.

• Access to Document (DOI)

Quantitative trait loci for growth-related traits in Japanese quail (Coturnix japonica) using restriction-site associated DNA sequencing

Haqani M.I., Nomura S., Nakano M., Goto T., Nagano A.J., Takenouchi A., Nakamura Y., Ishikawa A., Tsudzuki M.

Molecular Genetics and Genomics （Molecular Genetics and Genomics）  2021年

ISSN  16174615

　概要を見る

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.

• Access to Document (DOI)

Mapping of quantitative trait loci controlling egg-quality and-production traits in japanese quail (Coturnix japonica) using restriction-site associated dna sequencing

Haqani M.I., Nomura S., Nakano M., Goto T., Nagano A.J., Takenouchi A., Nakamura Y., Ishikawa A., Tsudzuki M.

Genes （Genes）  12 （ 5 ）  2021年

　概要を見る

This research was conducted to identify quantitative trait loci (QTL) associated with egg-related traits by constructing a genetic linkage map based on single nucleotide polymorphism (SNP) markers using restriction-site associated DNA sequencing (RAD-seq) in Japanese quail. A total of 138 F2 females were produced by full-sib mating of F1 birds derived from an intercross between a male of the large-sized strain with three females of the normal-sized strain. Eggs were investigated at two different stages: the beginning stage of egg-laying and at 12 weeks of age (second stage). Five eggs were analyzed for egg weight, lengths of the long and short axes, egg shell strength and weight, yolk weight and diameter, albumen weight, egg equator thickness, and yolk color (L*, a*, and b* values) at each stage. Moreover, the age at first egg, the cumulative number of eggs laid, and egg production rate were recorded. RAD-seq developed 118 SNP markers and mapped them to 13 linkage groups using the Map Manager QTX b20 software. Markers were spanned on 776.1 cM with an average spacing of 7.4 cM. Nine QTL were identified on chromosomes 2, 4, 6, 10, 12, and Z using the simple interval mapping method in the R/qtl package. The QTL detected affected 10 egg traits of egg weight, lengths of the long and short axes of egg, egg shell strength, yolk diameter and weight, albumen weight, and egg shell weight at the beginning stage, yellowness of the yolk color at the second stage, and age at first egg. This is the first report to perform a quail QTL analysis of egg-related traits using RAD-seq. These results highlight the effectiveness of RAD-seq associated with targeted QTL and the application of marker-assisted selection in the poultry industry, particularly in the Japanese quail.

• Access to Document (DOI)

Characterization of rhizome transcriptome and identification of a rhizomatous ER body in the clonal plant Cardamine leucantha

Araki K.S., Nagano A.J., Nakano R.T., Kitazume T., Yamaguchi K., Hara-Nishimura I., Shigenobu S., Kudoh H.

Scientific Reports （Scientific Reports）  10 （ 1 ）  2020年12月

　概要を見る

The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.

• Access to Document (DOI)

Genomic predictions and genome-wide association studies based on RAD-seq of quality-related metabolites for the genomics-assisted breeding of tea plants

Yamashita H., Uchida T., Tanaka Y., Katai H., Nagano A.J., Morita A., Ikka T.

Scientific Reports （Scientific Reports）  10 （ 1 ）  2020年12月

　概要を見る

Effectively using genomic information greatly accelerates conventional breeding and applying it to long-lived crops promotes the conversion to genomic breeding. Because tea plants are bred using conventional methods, we evaluated the potential of genomic predictions (GPs) and genome-wide association studies (GWASs) for the genetic breeding of tea quality-related metabolites using genome-wide single nucleotide polymorphisms (SNPs) detected from restriction site-associated DNA sequencing of 150 tea accessions. The present GP, based on genome-wide SNPs, and six models produced moderate prediction accuracy values (r) for the levels of most catechins, represented by (−)-epigallocatechin gallate (r = 0.32–0.41) and caffeine (r = 0.44–0.51), but low r values for free amino acids and chlorophylls. Integrated analysis of GWAS and GP detected potential candidate genes for each metabolite using 80–160 top-ranked SNPs that resulted in the maximum cumulative prediction value. Applying GPs and GWASs to tea accession traits will contribute to genomics-assisted tea breeding.

• Access to Document (DOI)

Endoplasmic reticulum-derived bodies enable a single-cell chemical defense in Brassicaceae plants

Yamada K., Goto-Yamada S., Nakazaki A., Kunieda T., Kuwata K., Nagano A.J., Nishimura M., Hara-Nishimura I.

Communications Biology （Communications Biology）  3 （ 1 ）  2020年12月

　概要を見る

Brassicaceae plants have a dual-cell type of chemical defense against herbivory. Here, we show a novel single-cell defense involving endoplasmic reticulum (ER)-derived organelles (ER bodies) and the vacuoles. We identify various glucosinolates as endogenous substrates of the ER-body β-glucosidases BGLU23 and BGLU21. Woodlice strongly prefer to eat seedlings of bglu23 bglu21 or a glucosinolate-deficient mutant over wild-type seedlings, confirming that the β-glucosidases have a role in chemical defense: production of toxic compounds upon organellar damage. Deficiency of the Brassicaceae-specific protein NAI2 prevents ER-body formation, which results in a loss of BGLU23 and a loss of resistance to woodlice. Hence, NAI2 that interacts with BGLU23 is essential for sequestering BGLU23 in ER bodies and preventing its degradation. Artificial expression of NAI2 and BGLU23 in non-Brassicaceae plants results in the formation of ER bodies, indicating that acquisition of NAI2 by Brassicaceae plants is a key step in developing their single-cell defense system.

• Access to Document (DOI)

Multiple waves of freshwater colonization of the three-spined stickleback in the Japanese Archipelago

Kakioka R., Mori S., Kokita T., Hosoki T.K., Nagano A.J., Ishikawa A., Kume M., Toyoda A., Kitano J.

BMC Evolutionary Biology （BMC Evolutionary Biology）  20 （ 1 ）  2020年12月

　概要を見る

Background: The three-spined stickleback (Gasterosteus aculeatus) is a remarkable system to study the genetic mechanisms underlying parallel evolution during the transition from marine to freshwater habitats. Although the majority of previous studies on the parallel evolution of sticklebacks have mainly focused on postglacial freshwater populations in the Pacific Northwest of North America and northern Europe, we recently use Japanese stickleback populations for investigating shared and unique features of adaptation and speciation between geographically distant populations. However, we currently lack a comprehensive phylogeny of the Japanese three-spined sticklebacks, despite the fact that a good phylogeny is essential for any evolutionary and ecological studies. Here, we conducted a phylogenomic analysis of the three-spined stickleback in the Japanese Archipelago. Results: We found that freshwater colonization occurred in multiple waves, each of which may reflect different interglacial isolations. Some of the oldest freshwater populations from the central regions of the mainland of Japan (hariyo populations) were estimated to colonize freshwater approximately 170,000 years ago. The next wave of colonization likely occurred approximately 100,000 years ago. The inferred origins of several human-introduced populations showed that introduction occurred mainly from nearby habitats. We also found a new habitat of the three-spined stickleback sympatric with the Japan Sea stickleback (Gasterosteus nipponicus). Conclusions: These Japanese stickleback systems differ from those in the Pacific Northwest of North America and northern Europe in terms of divergence time and history. Stickleback populations in the Japanese Archipelago offer valuable opportunities to study diverse evolutionary processes in historical and contemporary timescales.

• Access to Document (DOI)

Repressive chromatin modification underpins the long-term expression trend of a perennial flowering gene in nature

Nishio H., Buzas D.M., Nagano A.J., Iwayama K., Ushio M., Kudoh H.

Nature Communications （Nature Communications）  11 （ 1 ）  2020年12月

　概要を見る

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.

• Access to Document (DOI)

Pepper vein yellows virus 9: a novel polerovirus isolated from chili pepper in Indonesia

Koeda S., Homma K., Kamitani M., Nagano A.J., Taniguchi M., Pohan N., Kesumawati E.

Archives of Virology （Archives of Virology）  165 （ 12 ） 3017 - 3021 2020年12月

ISSN  03048608

　概要を見る

In 2017, a leaf sample from a single chili pepper (Capsicum annuum) plant exhibiting yellowing was collected from Aceh province, Indonesia. Total RNA was extracted from this sample, and RNA-Seq analysis was conducted. Putative infecting viruses were detected by mapping the obtained reads to the full-length viral genome sequences available in the GenBank database (7457 sequences) and the de novo-assembled contigs. RNA-Seq analysis detected polerovirus, begomovirus, and amalgavirus sequences, and the polerovirus-like sequences showed strong similarity to those of previously reported pepper vein yellows viruses (PeVYVs). The complete viral genome sequence obtained by RT-PCR had a length of 6023 nt, had the typical genome organization of a polerovirus and showed a high degree of sequence similarity to PeVYV-2 from Israel. Moreover, the predicted amino acid sequence of the P0 protein of the Indonesian isolate was 85.1% to 88.8% identical to those of other PeVYVs. In accordance with the polerovirus species demarcation criteria, this isolate should be assigned to a new polerovirus species, and we propose the name “pepper vein yellows virus 9” (PeVYV-9) for this virus.

• Access to Document (DOI)

Quantitative trait locus analysis in squash (Cucurbita moschata) based on simple sequence repeat markers and restriction site-associated DNA sequencing analysis

Hashimoto T., Kubo N., Nishimura K., Nagano A.J., Sasaki A., Nakamura Y., Mimura Y.

Horticulturae （Horticulturae）  6 （ 4 ） 1 - 12 2020年12月

　概要を見る

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.

• Access to Document (DOI)

Genetic Architecture of Innate Fear Behavior in Chickens

Ishikawa A., Sakaguchi M., Nagano A.J., Suzuki S.

Behavior Genetics （Behavior Genetics）  50 （ 6 ） 411 - 422 2020年11月

ISSN  00018244

　概要を見る

The genetic architecture of innate fear behavior in chickens is poorly understood. Here, we performed quantitative trait loci (QTL) analysis of innate responses to tonic immobility (TI) and open field (OF) fears in 242 newly hatched chicks of an F2 population between the native Japanese Nagoya breed and the White Leghorn breed using 881 single nucleotide polymorphism markers obtained by restriction site-associated DNA sequencing. At genome-wide 5% significance levels, four QTL for TI traits were revealed on chromosomes 1–3 and 24. Two of these loci had sex-specific effects on the traits. For OF traits, three QTL were revealed on chromosomes 2, 4 and 7. The TI and OF QTL identified showed no overlaps in genomic regions and different modes of inheritance. The three TI QTL and one OF QTL exerted antagonistic effects on the traits. The results demonstrated that context-dependent QTL underlie the variations in innate TI and OF behaviors.

• Access to Document (DOI)

Diurnal metabolic regulation of isoflavones and soyasaponins in soybean roots

Matsuda H., Nakayasu M., Aoki Y., Yamazaki S., Nagano A.J., Yazaki K., Sugiyama A.

Plant Direct （Plant Direct）  4 （ 11 ）  2020年11月

　概要を見る

Isoflavones and soyasaponins are major specialized metabolites accumulated in soybean roots and secreted into the rhizosphere. Unlike the biosynthetic pathway, the transporters involved in metabolite secretion remain unknown. The developmental regulation of isoflavone and soyasaponin secretions has been recently reported, but the diurnal regulation of their biosynthesis and secretion still needs to be further studied. To address these challenges, we conducted transcriptome and metabolite analysis using hydroponically grown soybean plants at 6-hr intervals for 48 hr in a 12-hr-light/12-hr-dark condition. Isoflavone and soyasaponin biosynthetic genes showed opposite patterns in the root tissues; that is, the former genes are highly expressed in the daytime, while the latter ones are strongly induced at nighttime. GmMYB176 encoding a transcription factor of isoflavone biosynthesis was upregulated from ZT0 (6:00 a.m.) to ZT6 (12:00 a.m.), followed by the induction of isoflavone biosynthetic genes at ZT6. The isoflavone aglycone content in the roots accordingly increased from ZT6 to ZT18 (0:00 a.m.). The isoflavone aglycone content in root exudates was kept consistent throughout the day, whereas that of glucosides increased at ZT6, which reflected the decreased expression of the gene encoding beta-glucosidase involved in the hydrolysis of apoplast-localized isoflavone conjugates. Co-expression analysis revealed that those isoflavone and soyasaponin biosynthetic genes formed separate clusters, which exhibited a correlation to ABC and MATE transporter genes. In summary, the results in this study indicated the diurnal regulation of isoflavone biosynthesis in soybean roots and the putative transporter genes responsible for isoflavone and soyasaponin transport.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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'.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

• 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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

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

　概要を見る

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.

• Access to Document (DOI)

Simplification of circadian rhythm measurement using species-independent time-indicated genes

Tanigaki Y., Higashi T., Takayama K., Nagano A., Honjo M., Tezuka A., Kamitani M., Fukuda H.

Current Plant Biology （Current Plant Biology）  19 2019年09月

　概要を見る

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.

• Access to Document (DOI)

Analyses of single nucleotide polymorphisms identified by ddRAD-seq reveal genetic structure of tea germplasm and Japanese landraces for tea breeding

Yamashita H., Katai H., Kawaguchi L., Nagano A.J., Nakamura Y., Morita A., Ikka T.

PLoS ONE （PLoS ONE）  14 （ 8 ）  2019年08月

　概要を見る

To obtain genetic information about the germplasm of tea (Camellia sinensis L.) in Japan, 167 accessions including 138 var. sinensis (96 Japanese var. sinensis and 42 exotic var. sinensis) and 29 Assam hybrids were analyzed using single nucleotide polymorphisms (SNPs) markers identified by double-digest restriction-site-associated DNA sequencing (ddRAD-seq) analysis. Approximately 10,000 SNPs were identified by ddRAD-seq and were mapped across the whole genome. The 167 tea accessions were classified into three genetic subgroups: (1) Japanese var. sinensis; (2) Japanese and exotic var. sinensis; (3) Assam hybrids and exotic var. sinensis. Leaf morphology varied widely within each genetic subgroups. The 96 Japanese var. sinensis were classified into four genetic subgroups as follows; two subgroups of Shizuoka (the largest tea production region) landraces, Uji (most ancient tea production region) landraces, and the pedigree of ‘Yabukita’, the leading green tea cultivar in Japan. These results indicated that the SNP markers obtained from ddRAD-seq are a useful tool to investigate the geographical background and breeding history of Japanese tea. This genetic information revealed the ancestral admixture situation of the ‘Yabukita’ pedigree, and showed that the genome structure of ‘Yabukita’ is clearly different from those of other Japanese accessions.

• Access to Document (DOI)

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

Kamitani M., Nagano A.J., Honjo M.N., Kudoh H.

Microbial Ecology （Microbial Ecology）  78 （ 1 ） 113 - 121 2019年07月

ISSN  00953628

　概要を見る

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.

• Access to Document (DOI)

Frequent chloroplast capture among Isodon (Lamiaceae) species in Japan revealed by phylogenies based on variation in chloroplast and nuclear DNA

Ogishima M., Horie S., Kimura T., Yamashiro T., Dohzono I., Kawaguchi L., Nagano A.J., Maki M.

Plant Species Biology （Plant Species Biology）  34 （ 3 ） 127 - 137 2019年07月

ISSN  0913557X

　概要を見る

A recent phylogenetic study based only on chloroplast DNA (cpDNA) variation revealed that populations of an Isodon species are frequently embedded paraphyletically among other Isodon species. This phylogenetic discrepancy between species taxonomy and molecular phylogeny was considered to have resulted from chloroplast DNA captures and/or incomplete lineage sorting. To elucidate which of these factors was mainly responsible for the observed phylogenetic pattern, we performed phylogenetic analyses of multiple populations of Isodon species in Japan using cpDNA variation, three single-copy nuclear genes, and double-digest restriction-site-associated DNA sequencing (ddRAD-seq). Although a species often shared chlorotypes with other species, our phylogenetical analyses based on variation in the three single-copy nuclear genes and the ddRAD-seq data showed that most populations belonging to the same species were monophyletic at the species level, suggesting that chloroplast capture may have frequently occurred between Isodon species. Some populations of an intraspecific taxon were embedded paraphyletically within the species, regardless of the large amount of phylogenetic information in nuclear DNA; this incongruity may have resulted from incomplete lineage sorting.

• Access to Document (DOI)

Construction of a high-density linkage map for bronze loquat using RAD-Seq

Fukuda S., Nagano Y., Matsuguma K., Ishimoto K., Hiehata N., Nagano A., Tezuka A., Yamamoto T.

Scientia Horticulturae （Scientia Horticulturae）  251   59 - 64 2019年06月

ISSN  03044238

　概要を見る

We constructed a high-density genetic linkage map of bronze loquat (Eriobotrya deflexa) for a three-way cross of loquat (Eriobotrya japonica) × (loquat × bronze loquat) by using single nucleotide polymorphism (SNP) markers generated by RAD-Seq, with the combination of simple sequence repeat (SSR) markers, which have been used in the previous study. The map of bronze loquat (‘Taiwan loquat No. 1’) consisted of 960 loci including 793 SNPs, 166 SSRs (98 apple, 8 loquat and 60 pear SSRs), and the loquat canker resistance gene Pse-a. This map covered 17 linkage groups over a total length of 1707.4 cM with an average distance of 1.78 cM between markers. The number of linkage groups corresponded to the basic chromosome number (n = 17). The analysis of the markers that can be anchored to apple draft genome showed that almost all loquat linkage groups were aligned to the apple draft genome. The constructed map will enable us to determine the location of quantitative trait loci and genes of interest, and to analyze genome synteny in the tribe Pyreae, subfamily Spiraeoideae of the family Rosaceae.

• Access to Document (DOI)

A key metabolic gene for recurrent freshwater colonization and radiation in fishes

Ishikawa A., Kabeya N., Ikeya K., Kakioka R., Cech J.N., Osada N., Leal M.C., Inoue J., Kume M., Toyoda A., Tezuka A., Nagano A.J., Yamasaki Y.Y., Suzuki Y., Kokita T., Takahashi H., Lucek K., Marques D., Takehana Y., Naruse K., Mori S., Monroig O., Ladd N., Schubert C.J., Matthews B., Peichel C.L., Seehausen O., Yoshizaki G., Kitano J.

Science （Science）  364 （ 6443 ） 886 - 889 2019年05月

ISSN  00368075

　概要を見る

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential w-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

• Access to Document (DOI)

Genetic properties responsible for the transgressive segregation of days to heading in rice

Koide Y., Sakaguchi S., Uchiyama T., Ota Y., Tezuka A., Nagano A., Ishiguro S., Takamure I., Kishima Y.

G3: Genes, Genomes, Genetics （G3: Genes, Genomes, Genetics）  9 （ 5 ） 1655 - 1662 2019年05月

　概要を見る

Transgressive segregation produces hybrid progeny phenotypes that exceed the parental phenotypes. Unlike heterosis, extreme phenotypes caused by transgressive segregation are heritably stable. We examined transgressive phenotypes of flowering time in rice, and revealed transgressive segregation in F2 populations derived from a cross between parents with similar (proximal) days to heading (DTH). The DTH phenotypes of the A58 · Kitaake F2 progenies were frequently more extreme than those of either parent. These transgressive phenotypes were maintained in the F3 and F4 populations. Both A58 and Kitaake are japonica rice cultivars adapted to Hokkaido, Japan, which is a high-latitude region, and have a short DTH. Among the four known loci required for a short DTH, three loci had common alleles in A58 and Kitaake, implying there is a similar genetic basis for DTH between the two varieties. A genome-wide single nucleotide polymorphism (SNP) analysis based on the F4 population identified five new quantitative trait loci (QTL) associated with transgressive DTH phenotypes. Each of these QTL had different degrees of additive effects on DTH, and two QTL had an epistatic effect on each other. Thus, a genome-wide SNP analysis facilitated the detection of genetic loci associated with extreme DTH phenotypes, and revealed that the transgressive phenotypes were produced by exchanging the complementary alleles of a few minor QTL in the similar parental phenotypes.

• Access to Document (DOI)

Genomic reconstruction of 100 000-year grassland history in a forested country: Population dynamics of specialist forbs

Yamaura Y., Narita A., Kusumoto Y., Nagano A.J., Tezuka A., Okamoto T., Takahara H., Nakamura F., Isagi Y., Lindenmayer D.

Biology Letters （Biology Letters）  15 （ 5 ）  2019年05月

ISSN  17449561

　概要を見る

Grassland ecosystems worldwide have been extensively converted to other land uses and are globally imperiled. Because many grasslands have been maintained by human activities, understanding their origin and history is fundamentally important to better contemporary management. However, existing methods to reconstruct past vegetation can produce contrasting views on grassland history. Here, we inferred demographic histories of 40 populations of four grassland forb species throughout Japan using high-resolution genome sequences and model-flexible demographic simulation based on the site frequency spectrum. Although two species showed a slight decline in population size between 100 000 - 10 000 years ago, our results suggest that population sizes of studied species have been maintained within the range of 0.5 - 2.0 times the most recent estimates for at least 100 000 years across Japan. Our results suggest that greater than 90% declines in Japanese grasslands and subsequent losses of grassland species in the last 100 years are geologically and biologically important and will have substantial consequences for Japanese biota and culture. People have had critical roles in maintaining disturbance-dependent grassland ecosystems and biota in this warm and wet forested country. In these contexts, disturbances associated with forest harvesting and traditional extensive farming have the potential to maintain grassland ecosystems and can provide important opportunities to reconcile resource production and conservation of grassland biodiversity.

• Access to Document (DOI)

Plant trichomes and a single gene GLABRA1 contribute to insect community composition on field-grown Arabidopsis thaliana

Sato Y., Shimizu-Inatsugi R., Yamazaki M., Shimizu K.K., Nagano A.J.

BMC Plant Biology （BMC Plant Biology）  19 （ 1 ）  2019年04月

　概要を見る

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.

• Access to Document (DOI)

Quantitative trait loci mapping for the shear force value in breast muscle of F<inf>2</inf> chickens

Ono T., Kouguchi T., Ishikawa A., Nagano A.J., Takenouchi A., Igawa T., Tsudzuki M.

Poultry Science （Poultry Science）  98 （ 3 ） 1096 - 1101 2019年03月

ISSN  00325791

　概要を見る

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.

• Access to Document (DOI)

Classification of tea (Camellia sinensis) landraces and cultivars in Kyoto, Japan and other regions, based on simple sequence repeat markers and restriction site-associated DNA sequencing analysis

Kubo N., Mimura Y., Matsuda T., Nagano A.J., Hirai N., Higashimoto S., Yoshida H., Uemura N., Fujii T.

Genetic Resources and Crop Evolution （Genetic Resources and Crop Evolution）  66 （ 2 ） 441 - 451 2019年02月

ISSN  09259864

　概要を見る

The tea plant (Camellia sinensis (L.) Kuntze) of Japan is now thought to have originated in China. Actual cultivation of tea plants presumably started in Kyoto, and then spread to other regions of Japan. Tea gardens used to be composed of heterogenic, seed-derived populations—landraces—selected for climatic preferences and cultivation methods. Cultivars were bred from landraces in the modern era. A number of landraces remain in Kyoto Prefecture. However, little is known about their genetic characteristics compared with other landraces. We investigated the relationships of tea landraces and cultivars from Kyoto Prefecture and other regions. A neighbor-joining phylogram was constructed from 113 lines including 68 landraces, 44 cultivars, and one wild relative, based on simple sequence repeat markers. The lines could be classified into four groups (I–IV). In group I, most of the Kyoto landraces were close to other Japanese landraces, supporting the idea of the spread of tea plants from Kyoto to other regions in the country. The remaining lines were included into groups III and IV, apart from group II, which contained Shizuoka lines. Similar results were observed by restriction site-associated DNA sequencing analysis using 44 cultivars. Our data provide valuable information for the classification of tea lines, especially for the relationships of Kyoto lines among Japanese tea varieties.

• Access to Document (DOI)

Annual transcriptome dynamics in natural environments reveals plant seasonal adaptation

Nagano A.J., Kawagoe T., Sugisaka J., Honjo M.N., Iwayama K., Kudoh H.

Nature Plants （Nature Plants）  5 （ 1 ） 74 - 83 2019年01月

　概要を見る

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.

• Access to Document (DOI)

Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum

Koeda S., Sato K., Saito H., Nagano A.J., Yasugi M., Kudoh H., Tanaka Y.

Theoretical and Applied Genetics （Theoretical and Applied Genetics）  132 （ 1 ） 65 - 80 2019年01月

ISSN  00405752

　概要を見る

Key message: A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. Abstract: The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F 2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC–MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

• Access to Document (DOI)

Genetic analysis of Taishu horses on and off Tsushima Island: Implications for conservation

Tezuka A., Takasu M., Tozaki T., Nagano A.J.

Journal of Equine Science （Journal of Equine Science）  30 （ 2 ） 33 - 40 2019年

ISSN  13403516

　概要を見る

Taishu horses are a native Japanese breed, of which only 41 individuals remained on Tsushima Island in 2018. Their genetic diversity is considered lower than that of other Japanese native horse breeds; thus, it needs to be investigated for sustainable conservation of this breed. Historical records revealed that several Taishu individuals were released areas off-Tsushima Island in mid-1980s. At present, Taishu horses living outside of Tsushima Island, hereafter referred to as Non-Tsushima Taishus (NTTs), are tagged. However, the genetic structure of the NTT individuals remains unclear, and such individuals are not included in the current mating plans for Taishu horses. Herein, we examined the genetic structure of 18 NTT individuals by comparing their genomic (SNP) information with that of individuals on Tsushima Island (TT), four other native Japanese breeds, and one Anglo- Arabian breed by using ddRAD-seq. We found that all individuals related to the Taishu can be grouped in one cluster, which was separated from other horse breeds. Patterns of specific and shared SNPs in NTT individuals closely resembled those of TT individuals, suggesting very minor genetic differences. Meanwhile, the heterozygosity of NTT individuals was slightly higher than that of TT individuals, and many NTT individuals were of fertile age, suggesting that the pedigree of NTT individuals would be useful in breed conservation plans for Taishu horses. Based on their genomic information, we also reconstructed the pedigree structures of four NTT individuals with no family information. The inclusion of NTT individuals in future mating plans on Tsushima Island may be an effective and feasible method for conserving the Taishu horse breed in Japan.

• Access to Document (DOI)

Mapping of quantitative trait loci for growth and carcass-related traits in chickens using a restriction-site associated dna sequencing method

Ono T., Ohara K., Ishikawa A., Kouguchi T., Nagano A.J., Takenouchi A., Igawa T., Tsudzuki M.

Journal of Poultry Science （Journal of Poultry Science）  56 （ 3 ） 166 - 176 2019年

ISSN  13467395

　概要を見る

In the present study, quantitative trait loci (QTLs) analysis was performed to identify the chromosomal positions of growth andcarcass-relatedtrait QTLs using 319 F2 chickens obtainedfrom intercrosses of an Oh-Shamo male and four White Plymouth Rock females. Body weight was measured weekly until the birds were 7 weeks old. Carcassrelatedtraits were also measuredat this timepoint. A genetic linkage map was constructedusing 545 single nucleotide polymorphism (SNP) markers that were developedusing a restriction-site associatedDNA sequencing method. The linkage map includedthe 23 autosomes andthe Z chromosome. Using simple interval QTL mapping, we were able to identify 10 significant andsuggestive main-effect QTLs for growth andcarcass-relatedtraits present on chromosomes 1, 2, 3, 5, 8, 19, 24, andZ. These loci explained 5.60 -16.52% of the phenotypic variances. The chromosomal positions of the 10 QTLs overlappedwith those of previously reportedQTLs, whereas the targetedtraits varied. Our QTLs will aidfuture breeding programs in improving growth andmeat yieldof chickens (e.g., via marker-assisted selection), particularly in the Japanese brandchicken industry.

• Access to Document (DOI)

Transcriptional variation in glucosinolate biosynthetic genes and inducible responses to aphid herbivory on field-grown Arabidopsis thaliana

Sato Y., Tezuka A., Kashima M., Deguchi A., Shimizu-Inatsugi R., Yamazaki M., Shimizu K.K., Nagano A.J.

Frontiers in Genetics （Frontiers in Genetics）  10 （ JUL ）  2019年

　概要を見る

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.

• Access to Document (DOI)

New taxa of Rhododendron tschonoskii alliance (Ericaceae) from East Asia

Yoichi W., Minamitani T., Oh S.H., Nagano A.J., Abe H., Yukawa T.

PhytoKeys （PhytoKeys）  134 （ 2019 ） 97 - 114 2019年

ISSN  13142011

　概要を見る

Three new taxa, Rhododendron sohayakiense Y.Watan. & T.Yukawa (Ericaceae), and its two varieties, var. kiusianum Y.Watan., T.Yukawa & T.Minamitani and var. koreanum Y.Watan. & T.Yukawa are described and illustrated from Japan and South Korea. They can be distinguished from each other and from the other members of the R. tschonoskii alliance, i.e. R. tschonoskii, R. tetramerum, R. trinerve and R. tsusiophyllum, through their combination of leaf size, leaf morphologies including lateral nerves on abaxial leaf surface, corolla morphologies including number of corolla lobes, style length and anther form. Phylogenetic inferences based on chloroplast DNA and genome-wide sequences revealed that each of the three new taxa is monophyletic and they further form a clade. Distributions of the three taxa are also clearly separated from each other and also from the other members of the R. tschonoskii alliance.

• Access to Document (DOI)

Broad distribution spectrum from Gaussian to power law appears in stochastic variations in RNA-seq data

Awazu A., Tanabe T., Kamitani M., Tezuka A., Nagano A.J.

Scientific Reports （Scientific Reports）  8 （ 1 ）  2018年12月

　概要を見る

Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions. In many recent transcriptome analyses based on RNA sequencing (RNA-seq), variations in gene expression levels among replicates were assumed to follow a negative binomial distribution, although the physiological basis of this assumption remains unclear. In this study, RNA-seq data were obtained from Arabidopsis thaliana under eight conditions (21-27 replicates), and the characteristics of gene-dependent empirical probability density function (ePDF) profiles of gene expression levels were analyzed. For A. thaliana and Saccharomyces cerevisiae, various types of ePDF of gene expression levels were obtained that were classified as Gaussian, power law-like containing a long tail, or intermediate. These ePDF profiles were well fitted with a Gauss-power mixing distribution function derived from a simple model of a stochastic transcriptional network containing a feedback loop. The fitting function suggested that gene expression levels with long-tailed ePDFs would be strongly influenced by feedback regulation. Furthermore, the features of gene expression levels are correlated with their functions, with the levels of essential genes tending to follow a Gaussian-like ePDF while those of genes encoding nucleic acid-binding proteins and transcription factors exhibit long-tailed ePDF.

• Access to Document (DOI)

ddRAD-seq based phylogeographic study of Sargassum thunbergii (Phaeophyceae, Heterokonta) around Japanese coast

Kobayashi H., Haino Y., Iwasaki T., Tezuka A., Nagano A.J., Shimada S.

Marine Environmental Research （Marine Environmental Research）  140   104 - 113 2018年09月

ISSN  01411136

　概要を見る

Using genome-wide SNP data obtained from high-throughput techniques based on double digest restriction site-associated DNA sequencing (ddRAD-seq), we elucidated the migration history and genetic diversity of the Japanese population of the ecologically important brown seaweed Sargassum thunbergii (Mertens ex Roth) Kuntze. STRUCTURE and NeighborNet analyses showed a clear genetic differentiation among populations of four geographic regions: Kyushu (POP1); Sea of Japan (POP2); Hokkaido and Tohoku (POP3); and Pacific coast from Kyushu to Kanto (POP4). Approximate Bayesian Computation (ABC) analysis indicated that POP4 diverged first, followed by the separation between POP2 (the largest effective population size) and POP3; POP1 was the last to form, shaped by the mixture of POP2 (73%) and POP4 (27%). High genetic diversity was detected in POP1 and POP2, whereas low genetic diversity was detected in POP3 and POP4. These results indicated that S. thunbergii populations of Kyushu and the Sea of Japan might have been maintained as large and stable populations gathered different lineages from China, Korea and/or Japan.

• Access to Document (DOI)

Assessment of genetic diversity in Coho salmon (Oncorhynchus kisutch) populations with no family records using ddRAD-seq

Hosoya S., Kikuchi K., Nagashima H., Onodera J., Sugimoto K., Satoh K., Matsuzaki K., Yasugi M., Nagano A.J., Kumagayi A., Ueda K., Kurokawa T.

BMC Research Notes （BMC Research Notes）  11 （ 1 ）  2018年08月

　概要を見る

Objective: Selective breeding for desirable traits is becoming popular in aquaculture. In Miyagi prefecture, Japan, a selectively bred population of Coho salmon (Oncorhynchus kisutch) has been established with the original, randomly breeding population maintained separately. Since they have been bred without family records, the genetic diversity within these populations remains unknown. In this study, we estimated the genetic diversity and key quantitative genetic parameters such as heritability and genomic breeding value for body size traits by means of genomic best linear unbiased prediction to assess the genetic health of these populations. Results: Ninety-nine and 83 females from the selective and random groups, respectively, were genotyped at 2350 putative SNPs by means of double digest restriction associated DNA sequencing. The genetic diversity in the selectively bred group was low, as were the estimated heritability and prediction accuracy for length and weight (h 2 = 0.26-0.28; accuracy = 0.34), compared to the randomly bred group (h 2 = 0.50-0.60; accuracy = 0.51-0.54). Although the tested sample size was small, these results suggest that further selection is difficult for the selectively bred population, while there is some potential for the randomly bred group, especially with the aid of genomic information.

• Access to Document (DOI)

A first genetic map in the genus Streptocarpus generated with RAD sequencing based SNP markers

Chen Y., Nishii K., Barber S., Hackett C., Kidner C., Gharbi K., Nagano A., Iwamoto A., Möller M.

South African Journal of Botany （South African Journal of Botany）  117   158 - 168 2018年07月

ISSN  02546299

　概要を見る

Streptocarpus shows a great diversity of shoot and flower morphology, but lacks resources for genetic studies. RAD sequencing with NGS technologies provides large amounts of genotyping data quickly and cost effectively and is very useful for genetic map construction. In this study, three approaches for constructing RAD loci were combined to build the first genetic map for Streptocarpus (Gesneriaceae). A backcross population between Streptocarpus grandis and S. rexii was used as the mapping population. The parents are both diploid (2n = 32) and largely autogamous. Streptocarpus grandis is unifoliate, retaining the enlarged macrocotyledon as sole foliar organ which is monocarpic, and S. rexii is rosulate and perennial. 233 backcross plants were genotyped, and the data from 200 or 150 individuals used to generate the maps. The RAD reads were assembled using a de novo approach, aligned on the reference draft genome sequences of S. rexii using BWA or Stampy, and finally all markers were analyzed combined. In the latter, 599 mappable markers were generated which were assigned to 16 linkage groups, with an average inter-marker distance of 2.6 cM. This work provides a valuable genetic resource for further studies in the large and morphologically diverse genus Streptocarpus.

• Access to Document (DOI)

Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

Hanada K., Tezuka A., Nozawa M., Suzuki Y., Sugano S., Nagano A.J., Ito M., Morinaga S.I.

DNA Research （DNA Research）  25 （ 3 ） 327 - 339 2018年06月

ISSN  13402838

　概要を見る

Lineage-specific duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selection pressures immediately after gene duplication is clear in the speciation process. Here, using Illumina DNA-sequencing reads from Arabidopsis halleri, which has multiple closely related species with high-quality genome assemblies (A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups in Brassicaceae. The duplication frequency of retained genes in the Arabidopsis lineage was 10 times higher than the duplication frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss (Physcomitrella patens). The difference of duplication frequencies can be explained by a rapid decay of anciently duplicated genes. To examine the degree of selection pressure on genes duplicated in either the A. halleri-lyrata or the A. halleri lineage, we examined positive and purifying selection in the A. halleri-lyrata and A. halleri lineages throughout the ratios of nonsynonymous to synonymous substitution rates (KA/ KS). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. Interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication compared with immediately after gene duplication.

• Access to Document (DOI)

Auxin contributes to the intraorgan regulation of gene expression in response to shade

Kim S., Mochizuki N., Deguchi A., Nagano A., Suzuki T., Nagatani A.

Plant Physiology （Plant Physiology）  177 （ 2 ） 847 - 862 2018年06月

ISSN  00320889

　概要を見る

Plants sense and respond to light via multiple photoreceptors including phytochrome. The decreased ratio of red to far-red light that occurs under a canopy triggers shade-avoidance responses, which allow plants to compete with neighboring plants. The leaf acts as a photoperceptive organ in this response. In this study, we investigated how the shade stimulus is spatially processed within the cotyledon. We performed transcriptome analysis on microtissue samples collected from vascular and nonvascular regions of Arabidopsis (Arabidopsis thaliana) cotyledons. In addition, we mechanically isolated and analyzed the vascular tissue. More genes were up-regulated by the shade stimulus in vascular tissues than in mesophyll and epidermal tissues. The genes up-regulated in the vasculature were functionally divergent and included many auxin-responsive genes, suggesting that various physiological/developmental processes might be controlled by shade stimulus in the vasculature. We then investigated the spatial regulation of these genes in the vascular tissues. A small vascular region within a cotyledon was irradiated with far-red light, and the response was compared with that when the whole seedling was irradiated with far-red light. Most of the auxin-responsive genes were not fully induced by the local irradiation, suggesting that perception of the shade stimulus requires that a wider area be exposed to far-red light or that a certain position in the mesophyll and epidermis of the cotyledon be irradiated. This result was consistent with a previous report that auxin synthesis genes are up-regulated in the periphery of the cotyledon. Hence, auxin acts as an important intraorgan signaling factor that controls the vascular shade response within the cotyledon.

• Access to Document (DOI)

Phylogeographic testing of alternative histories of single-origin versus parallel evolution of early flowering serpentine populations of Picris hieracioides L. (Asteraceae) in Japan

Sakaguchi S., Horie K., Kimura T., Nagano A., Isagi Y., Ito M.

Ecological Research （Ecological Research）  33 （ 3 ） 537 - 547 2018年05月

ISSN  09123814

　概要を見る

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.

• Access to Document (DOI)

Does genomic variation in a foundation species predict arthropod community structure in a riparian forest?

Kagiya S., Yasugi M., Kudoh H., Nagano A.J., Utsumi S.

Molecular Ecology （Molecular Ecology）  27 （ 5 ） 1284 - 1295 2018年03月

ISSN  09621083

　概要を見る

Understanding how genetic variation within a foundation species determines the structure of associated communities and ecosystem processes has been an emerging frontier in ecology. Previous studies in common gardens identified close links between intraspecific variation and multispecies community structure, and these findings are now being evaluated directly in the complex natural ecosystem. In this study, we examined to what extent genomic variation in a foundation tree species explains the structure of associated arthropod communities in the field, comparing with spatial, temporal and environmental factors. In a continuous mixed forest, arthropods were surveyed on 85 mature alders (Alnus hirsuta) in 2 years. Moreover, we estimated Nei's genetic distance among the alders based on 1,077 single nucleotide polymorphisms obtained from restricted-site-associated DNA sequencing of the alders’ genome. In both years, we detected significant correlations between genetic distance and dissimilarity of arthropod communities. A generalized dissimilarity modelling indicated that the genetic distance of alder populations was the most important predictor to explain the variance of arthropod communities. Among arthropod functional groups, carnivores were consistently correlated with genetic distance of the foundation species in both years. Furthermore, the extent of year-to-year changes in arthropod communities was more similar between more genetically closed alder populations. This study demonstrates that the genetic similarity rule would be primarily prominent in community assembly of plant-associated arthropods under temporally and spatially variable environments in the field.

• Access to Document (DOI)

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

Sakaguchi S., Kimura T., Kyan R., Maki M., Nishino T., Ishikawa N., Nagano A.J., Honjo M.N., Yasugi M., Kudoh H., Li P., Choi H.J., Chernyagina O.A., Ito M.

Annals of Botany （Annals of Botany）  121 （ 3 ） 489 - 500 2018年02月

ISSN  03057364

　概要を見る

• 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.

• Access to Document (DOI)

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

Nagano Y., Mimura T., Kotoda N., Matsumoto R., Nagano A.J., Honjo M.N., Kudoh H., Yamamoto M.

Tree Genetics and Genomes （Tree Genetics and Genomes）  14 （ 1 ）  2018年02月

ISSN  16142942

　概要を見る

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.

• Access to Document (DOI)

Estimation of the circadian phase by oscillatory analysis of the transcriptome in plants

Takeoka M., Higashi T., Honjo M., Tezuka A., Nagano A., Tanigaki Y., Fukuda H.

Environmental Control in Biology （Environmental Control in Biology）  56 （ 2 ） 67 - 72 2018年

ISSN  1880554X

　概要を見る

The phase of the circadian clock (body time) is related to various aspects of physiology and metabolism in plants. Therefore, it is important to evaluate body time to control plant quality and growth. The molecular timetable method, a statistical analysis of the transcriptome, is useful for plant body time estimation. However, it is necessary to verify some assumptions in this method for high resolution estimation in plants under various conditions. To investigate whether the assumptions are valid, we analyzed the period length and expression pattern of genes. We used Arabidopsis thaliana as a model plant, and both lettuce and tomato as model crops. A. thaliana and lettuce were cultivated under conditions of constant light in a closed plant factory, and tomato was cultivated in a sunlight-type plant factory. We found that estimation of the period length of the plant circadian rhythm was possible and that the gene expression pattern correlates well with test-fit cosine curves. Thus, we successfully demonstrated high-resolution estimation of body time in plants using a molecular timetable method.

• Access to Document (DOI)

Growth and environmental change-independent genes associated with clock gene TOC1 in green perilla

Tanigaki Y., Higashi T., Nagano A., Honjo M., Fukuda H.

Environmental Control in Biology （Environmental Control in Biology）  56 （ 4 ） 137 - 142 2018年

ISSN  1880554X

　概要を見る

Secondary metabolism is regulated by circadian rhythm based on the clock genes expression. Transcriptome analysis is an important tool to understand these mechanisms. However, sufficient genetic information for this analysis is lacking for many crops. In order to analyze the influence of independent clock genes from growth and environmental stimulation, we investigated the gene expression profiles in the roots and leaves of green perilla (Perilla frutescens), which is an important crop used as a herbal medicine. Green perilla was grown under light emitting diode and fluorescent light, after 7, 14, and 35 d. We also compared the coding DNA sequences with those in the other plants. From the transcriptomic data, green perilla was observed to have 74 contigs that did not depend on growth and cultivation condition. Some of these 74 genes might be related to the clock genes through their interactions with other genes. Some 74 genes had TATA box-like motif and a GA motif of the genes that are regulatory targets of timing of CAB expression 1 (TOC1). Thus, 74 genes that did not depend on cultivation condition and growth were considered to be under the control of TOC 1, suggesting the importance of TOC1 in circadian rhythms.

• Access to Document (DOI)

Selection of transcripts affecting initial growth rate of rice backcrossed inbred lines using RNA sequencing data

Fukuda A., Hirose T., Aoki N., Kondo S., Yonekura M., Kataoka T., Ohto C., Nagano A.

Frontiers in Plant Science （Frontiers in Plant Science）  871 2018年

　概要を見る

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.

• Access to Document (DOI)