Biology subjects

Nagano, A. J.

Publications and source records attributed to Nagano, A. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

An efficient early-pooling protocol for environmental DNA metabarcoding

Environmental DNA (eDNA) metabarcoding, a method that applies high-throughput sequencing and universal primer sets to eDNA analysis, has been a promising approach for efficient, comprehensive biodiversity monitoring. However, significant money-, labor-, and time-costs are still required for performing eDNA metabarcoding. In the present study, we assessed the performance of an "early-pooling" protocol (a protocol based on the 1st PCR indexing) to reduce the experimental costs of the library preparation for eDNA metabarcoding. Specifically, we performed three experiments to test the effects of 1st PCR and 2nd PCR indexing protocols on the community composition revealed by eDNA metabarcoding, of post-1st-PCR exonuclease purification on index-hopping, and of the number of PCR replicates and eDNA template volume on the number of detected OTUs. By analyzing 204 eDNA libraries from 3 natural aquatic ecosystems and 1 mock eDNA sample, we show that (i) the 1st PCR indexing does not cause clear biases in the outcomes of eDNA metabarcoding, (ii) post-1st-PCR exonuclease purification reduces the risk of index-hopping, and (iii) increasing the eDNA template volume can increase the number of detected OTUs and reduce the variations in detected community compositions, as can increasing the number of the 1st PCR replicates. Our results show that an early-pooling protocol with post-1st-PCR exonuclease purification and an increased amount of DNA template will reduce the risk of index-hopping, the costs for consumables and reagents, and the handling time in the library preparation, and that it produces comparable results to a 2nd-PCR-indexing protocol. Therefore, once a target metabarcoding region is determined and a set of indexed-1st-PCR primers is prepared, the early-pooling protocol provides a cost-, labor-, and time-efficient way to process a large number of samples.

ecology↗

bioRxiv2021

Microevolution of Pieris butterfly genes involved in host-plant adaptation along a host-plant community cline

Herbivorous insects have evolved counteradaptations to overcome the chemical defenses of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established,little is known about their microevolutionary dynamics in the field. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralog major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defense system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. P. melete and P. napi both revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defense.

evolutionary biology↗

bioRxiv2021

Fillable and Unfillable Gaps in Rice Transcriptome under Field and Controlled Environments

The differences between plants grown in field and controlled environments have long been recognised; however, few studies have addressed the underlying molecular mechanisms. Here, we show fillable and unfillable gaps in the transcriptomes of rice grown in field and controlled environments by utilising SmartGC, a high-performance growth chamber that reproduces the fluctuating irradiance, temperature, and humidity of field environments. Rice transcriptome dynamics in SmartGC mimicked those in the field, particularly during the morning and evening; those in conventional growth chamber conditions did not. Further analysis revealed that fluctuation of irradiance affects transcriptome dynamics in the morning and evening, while fluctuation of temperature only affects transcriptome dynamics in the morning. We found upregulation of genes related to biotic and abiotic stress, whose expression was affected by environmental factors that cannot be mimicked by SmartGC. Our results accelerate the understanding of plant responses to field environments for both field and laboratory studies.

plant biology↗

bioRxiv2021

Artificial mimicry of seasonal transcriptome dynamics in Arabidopsis thaliana reveals short- and long-term responses to environmental conditions

Plants must respond to various seasonally changing environmental stimuli. In a previous study, seasonally oscillating genes were identified by a massive time-series transcriptome analysis for the wild population of Arabidopsis halleri ssp. gemmifera, a sister species to Arabidopsis thaliana. It was not clear how environmental stimuli shaped the seasonal expression pattern of these seasonally oscillating genes. In this study we show that responses in different time-scales contributed to the formation of seasonal expression patterns for several genes. To analyze the seasonally oscillating genes, we established an experimental system to mimic seasonal expression trends using A. thaliana and a "smart growth chamber mini," a hand-made low-cost small chamber. Arabidopsis thaliana plants were cultured under conditions that mimicked the average monthly temperatures and daylengths under different day-scale incubation. In total, the seasonal trends of 1627 seasonally oscillating genes were mimicked, and they showed varying temporal responses (constant, transient, and incremental) to environmental stimuli. Our results suggest that plants perceive and integrate information regarding environmental stimuli in the field by combining seasonally oscillating genes with different temporal responsiveness.

plant biology↗

bioRxiv2021

Using a two-stage convolutional neural network to rapidly identify tiny herbivorous beetles in the field

Recently, deep convolutional neural networks (CNN) have been adopted to help beginners identify insect species from field images. However, the application of these methods on the identification of tiny congeneric species moving across heterogeneous background remains difficult. To enable rapid and automatic identification in the field, we customized a method involving real-time object detection of two Phyllotreta beetles. We first performed data augmentation using transformations, syntheses, and random erasing of the original images. We then proposed a two-stage method for the detection and identification of small insects based on CNN, where YOLOv4 and EfficientNet were used as a region proposal network and a re-identification method, respectively. Evaluation of the model revealed that one-step object detection by YOLOv4 alone was not precise (Precision = 0.55) when classifying two species of flea beetles and background objects. In contrast, the two-step CNNs improved the precision (Precision = 0.89) with moderate accuracy (F-measure = 0.55) and acceptable speed (ca. 5 frames per second for full HD images) of detection and identification of insect species in the field. Although real-time identification of tiny insects remains a challenge in the field, our method aids in improving small object detection on a heterogeneous background.

ecology↗

bioRxiv2020

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

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.

plant biology↗

bioRxiv2020

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

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 lake. 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 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.

evolutionary biology↗

bioRxiv2020

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

Using current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.

molecular biology↗

bioRxiv2020

Effects of underlying gene-regulation network structure on prediction accuracy in high-dimensional regression

MotivationThe 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. ResultsWhen the gene regulation network was random graph, the simulation indicated that models with high estimation accuracy could be achieved with small sample sizes. However, a real gene regulation network is likely to exhibit a scale-free structure. In such cases, the simulation indicated that a relatively large number of observations is required to accurately predict traits from a transcriptome. Availability and implementationSource code at https://github.com/keihirose/simrnet Contacthirose@imi.kyushu-u.ac.jp

bioinformatics↗

bioRxiv2020

Neighbor QTL: an interval mapping method for quantitative trait loci underlying neighbor effects

Phenotypes of sessile organisms, such as plants, rely not only on their own genotype but also on the genotypes of neighboring individuals. Previously, we incorporated such neighbor effects into a single-marker regression using the Ising model of ferromagnetism. However, little is known about how to incorporate neighbor effects in quantitative trait locus (QTL) mapping. In this study, we propose a new method for interval QTL mapping of neighbor effects, named "Neighbor QTL". The algorithm of neighbor QTL involves the following: (i) obtaining conditional self-genotype probabilities with recombination fraction between flanking markers, (ii) calculating neighbor genotypic identity using the self-genotype probabilities, and (iii) estimating additive and dominance deviation for neighbor effects. Our simulation using F2 and backcross lines showed that the power to detect neighbor effects increased as the effective range became smaller. The neighbor QTL was applied to insect herbivory on Col x Kas recombinant inbred lines of Arabidopsis thaliana. Consistent with previous evidence, the pilot experiment detected a self QTL effect on the herbivory at GLABRA1 locus. We also observed a weak QTL on chromosome 4 regarding neighbor effects on the herbivory. The neighbor QTL method is available as an R package (https://cran.r-project.org/package=rNeighborQTL), providing a novel tool to investigate neighbor effects in QTL studies.

genetics↗

bioRxiv2020

Removal of repressive histone marks creates epigenetic memory of recurring heat in Arabidopsis

Acclimation to high temperature increases plants tolerance of subsequent lethal high temperatures1-3. 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) proteins4-8, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at key HEAT SHOCK PROTEIN (HSP) loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate 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.

plant biology↗

bioRxiv2020

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

The Sino-Japanese Floristic Region is highly diverse 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. Heterotropa (genus Asarum; Aristolochiaceae) diverges here and shows high species diversity in insular systems (63 species) compared to continental areas (25 species). Heterotropa shows low dispersal ability with small distribution ranges, implying diversification by geographic events, and high floral diversity, implying pollinator-mediated diversification. To reveal how abiotic and biotic factors have shaped the diversity anomaly of Heterotropa, we conducted phylogenetic analysis using ddRAD-seq and chloroplast genome datasets including 79 species, estimation of floral trait evolution, and comparison of isolation factors within clades based on distribution range and floral trait analysis. Phylogenetic analysis indicates that Heterotropa originated in mainland China and expanded to the Japanese Archipelago in the Miocene, and the major clades almost correspond to geographic distributions. Floral traits evolved repeatedly in the tip nodes within the clades. Although the major clades include a high proportion of species pairs showing isolation by floral traits, there are no conditional relationships between two isolation factors, indicating that most species pairs with floral trait isolation are distributed allopatrically. The repeated exposure and submergence of land-bridges caused by climatic oscillations would have led to significant population fragmentation in insular systems. Thus, the diversity anomaly of Heterotropa would have resulted from geographic and climatic events during the Miocene, while subsequent repeated floral trait evolution would have followed geographic isolation during the Pleistocene.

plant biology↗

bioRxiv2020

Diurnal metabolic regulation of isoflavones and soyasaponins in soybean roots

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-hour intervals for 48 hours in a 12-h-light/12-h-dark condition. Isoflavone and soyasaponin biosynthetic genes showed opposite patterns in the root tissues; that is, the former genes are highly expressed in daytime, while the latter ones are strongly induced at nighttime. GmMYB176 encoding a transcription factor of isoflavone biosynthesis was upregulated from ZT0 (6:00 am) to ZT6 (12:00 am), 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 am), accompanied by an increase in glucoside levels that peaked at ZT0. 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. As 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.

plant biology↗

bioRxiv2019

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

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 from 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).

genetics↗

bioRxiv2019

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

Recently, increasing attempts have been made to understand how plant genes function in natura studies. To determine whether plant defense genes are activated under multiple biotic stimuli, we combined a high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana growing on the field site of Switzerland. We found that genes with GO annotations "glucosinolate biosynthetic process" and "response to insects" were the most significantly enriched, exhibiting largely variable expression among plant accessions. Nearly half of the total expression variation in glucosinolate biosynthetic genes, AOPs, ESM1, ESP, and TGG1, was explained by among-accession variance. Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to the mustard aphid Lipaphis erysimi. In addition, we also found that the expression of a major cis-jasmone activated gene CYP81D11 was positively correlated with the number of the flea beetles Phyllotreta spp. The combined results from RNA-Seq and insect surveys suggested that plants can activate their defenses even when they are exposed to multiple biotic stimuli in natura.

plant biology↗

bioRxiv2018

High-throughput RNA-Seq analysis on plant response under fluctuating temperature conditions

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions; its use in large-scale experiments is limited by costs and labour. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method that did not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature ([≥]62{degrees}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, and caused over-estimations of the quantity of DNA, resulting in unstable tagmentation results. Degradation of RNA before tagmentation was 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{degrees}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.

plant biology↗

bioRxiv2018

The ability of ddRAD-Seq to estimate genetic diversity and genetic introgression in endangered native livestock

Unplanned crossbreeding between a native livestock and a specific productive breed was one of the main reasons that caused the loss of valuable genetic resources in native livestock. To avoid further loss and damage of genetic resources in the native livestock, introgressed individuals should be distinguished to eliminate them by preventing any further employment in future mating plans. In general, the genetic diversity of native livestock had already decreased and mass elimination of introgressed individuals from the population endangers their existence. To solve this problem, high-resolution markers are required to discriminate between introgressed variation and native variation. Here, we applied ddRAD-Seq markers for native Japanese horse \"Taishu\" that has undergone recent genetic introgression. Genome-wide ddRAD-Seq markers can distinguish five breeds of native Japanese horses and Anglo-Arabian introgressed breeds. We found the signatures of genetic introgression of Anglo-Arabian at only two chromosomes; however, the signatures were separated in their genome suggesting that it might not be the cause of recent introgression. The genetic diversity of Taishu was less than other Japanese breeds and the decreasing genetic diversity is an urgent issue compared to genetic introgression. Although few signatures of recent introgression were detected, a lot of shared SNPs (10% of all SNPs in Taishu) were detected between Taishu and Anglo-Arabian. To avoid misestimation of the presence and degree of introgression in native livestock, information regarding shared SNPs and population genetic approaches need to be assessed by using the large number of genome-wide markers such as ddRAD-Seq.

genomics↗

bioRxiv2018

Prediction of environmental response in field-grown rice using expression-dynamics-QTL

How genetic variations affect gene expression dynamics of field-grown plants remains unclear. Using statistical analysis of large-scale time-series RNA-sequencing of field-grown rice from chromosome segment substitution lines (CSSLs), we identified 1675 expression dynamics quantitative trait loci (edQTLs) leading to polymorphisms in expression dynamics under field conditions. Based on the edQTL and environmental information, we successfully predicted gene expression under environments different from training environments, and in rice cultivars with more complex genotypes than the CSSLs. Overall, edQTL identification helped understanding the genetic architecture of expression dynamics under field conditions, which is difficult to assess with laboratory experiments1.The prediction of expression based on edQTL and environmental information will contribute to crop breeding by increasing the accuracy of trait prediction under diverse conditions.

bioinformatics↗