A chromosome-level genome assembly of a model conifer plant, the Japanese cedar, Cryptomeria japonica D. Don.

BACKGROUND: The Japanese cedar (Cryptomeria japonica D. Don) is one of the most important Japanese forest trees, occupying approximately 44% of artificial forests and planted in East Asia, the Azores Archipelago, and certain islands in the Indian Ocean. Although the huge genome of the species (ca. 9 Gbp) with abundant repeat elements may have represented an obstacle for genetic analysis, this species is easily propagated by cutting, flowered by gibberellic acid, transformed by Agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica recommend it as a model conifer species for which reference genome sequences are necessary. RESULTS: Herein, we report the first chromosome-level assembly of C. japonica (2n = 22) using third-generation selfed progeny (estimated homozygosity rate = 0.96). Young leaf tissue was used to extract high molecular weight DNA (> 50 kb) for HiFi PacBio long-read sequencing and to construct an Hi-C/Omni-C library for Illumina short-read sequencing. The 29× and 26× genome coverage of HiFi and Illumina reads, respectively, for de novo assembly yielded 2,651 contigs (9.1 Gbp, N50 contig size 12.0 Mbp). Hi-C analysis mapped 97% of the nucleotides on 11 chromosomes. The assembly was verified through comparison with a consensus linkage map comprising 7,781 markers. BUSCO analysis identified ∼ 91% conserved genes. CONCLUSIONS: Annotations of genes and comparisons of repeat elements with other Cupressaceae and Pinaceae species provide a fundamental resource for conifer research.

Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica.

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

Two R2R3-MYB transcription factors from Chinese cedar (Cryptomeria fortunei Hooibrenk) are involved in the regulation of secondary cell wall formation.

As the most abundant renewable energy source, wood comprises the secondary cell wall (SCW). SCW biosynthesis involves lignin and cellulose deposition. Increasing studies have illustrated that R2R3-MYB transcription factors (TFs) play pivotal roles in affecting lignin accumulation and SCW formation. Nevertheless, the regulatory roles of R2R3-MYBs are still unresolved in Cryptomeria fortunei Hooibrenk cambium and wood formation. To dissect the potentials of CfMYBs, we successfully cloned and intensively studied the functions of CfMYB4 and CfMYB5 in SCW formation and abiotic stress response. They both contained the conserved MYB domain capable of forming a special structure that could bind to the core motifs of downstream genes. The phylogenetic tree implied that two CfMYBs clustered into different evolutionary branches. They were predominantly expressed in the stem and were localized to the nucleus. Furthermore, CfMYB4 functioned as an activator to enhance lignin and cellulose accumulation, and increase the SCW thickness by elevating the expression levels of SCW-related genes. By contrast, CfMYB5 negatively regulated lignin and cellulose biosynthesis, and decreased SCW formation by reducing the expression of SCW biosynthetic genes. Our data not only highlight the regulatory functions of CfMYBs in lignin deposition but also provide critical insights into the development of strategies for the genetic improvement of Cryptomeria fortunei wood biomass.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

Overexpression of CfICE1 from Cryptomeria fortunei Enhances Cold, Drought and Salt Stress in Poplar.

ICE1, a regulator of the cold-inducible transcriptome and freezing tolerance, is currently widely believed to be involved in plant resistance to cold stress. In this study, CfICE1 from Cryptomeria fortunei was transformed into poplar. Physiological indicators of transgenic, empty vector and wild-type poplar after abiotic stress (cold, drought and salt) were determined. Transgenic lines had a higher chlorophyll content, antioxidant enzyme activity and soluble protein content, as well as a lower malondialdehyde and hydrogen peroxide content. The ultrastructure of the plant was observed by transmission electron microscopy, and after stress, the cell structure of the transgenic line was more complete than that of the wild type. CfICE1 was upregulated in transgenic poplar trees after abiotic stress (cold, drought and salt). The CfICE1 transgenic plants improved plant resistance by regulating the CBF gene of poplar under cold and salt stress. In terms of plant responses to abiotic stress, this study showed that overexpression of CfICE1 improved the cold, drought and salt tolerance of poplars.

Genotype-by-environment interaction and genetic dissection of heartwood color in Cryptomeria japonica based on multiple common gardens and quantitative trait loci mapping.

The heartwood color of a major plantation tree Cryptomeria japonica shows high variability among clones and cultivars, and brighter heartwood has higher value in the usage of non-laminated wood such as in traditional construction, which makes heartwood color an important trait in breeding of this species. However, the genetic basis of the interactions between genetics and the environment on heartwood color has been understudied while these are necessary for effective breeding programs in multiple environmental condition. The objectives of the present study were to evaluate the effects of genetics and environments on heartwood color and how they interact in contrasting environments, and to identify genomic regions controlling heartwood color in C. japonica across multiple environments. Heartwood color in terms of L*a*b* color space and spectral reflectance was measured in common gardens established in three contrasting sites. Quantitative trait loci (QTL) that affect heartwood color were identified using previously constructed highly saturated linkage maps. Results found that heartwood color was largely genetically controlled, and genotype-by-environment interaction explained one-third of the total genetic variance of heartwood color. The effect of the environment was small compared to the effect of genetics, whereas environmental effects largely varied among heartwood color traits. QTL analysis identified a large number of QTLs with small to moderate effects (phenotypic variation explained of 6.6% on average). Some of these QTLs were stably expressed in multiple environments or had pleiotropic effects on heartwood color and moisture content. These results indicated that genetic variation in phenotypic plasticity plays an important role in regulating heartwood color and that the identified QTLs would maximize the breeding efficiency of heartwood color in C. japonica in heterogeneous environments.

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don).

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses.

MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including Cryptomeria fortunei, has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (U6, 5S and 18S) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in C. fortunei under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and U6 were the most stable RGs for studying C. fortunei miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of C. fortunei to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.

Diversification of terpenoid emissions proposes a geographic structure based on climate and pathogen composition in Japanese cedar.

Biogenic volatile organic compounds emitted from plants are important constituents of atmospheric chemistry and play a major role in the resistance of plants against various environmental stresses. However, little is known about how abiotic and biotic environments on a geographic scale relate to diversifications of the emission. Here, we present variations of terpenes stored in and emitted from leaves of a single species in a common garden, using genetically differentiated local populations of Japanese cedar, the most dominant and widely distributed tree species in Japan. Furthermore, we determined the composition of fungal communities in 50 locations, based on the presence or absence of 158 fungal species inhabiting the cedar. The results showed that terpenoids, especially those that are emitted, were highly diversified and geographically structured among the 12 populations. The total amount of stored terpenes was negatively affected by warm and less-snow climates. On the other hand, variations in some emitted terpenoid species among the populations were correlated to antagonistic fungal species inhabiting the Japanese cedar. We propose that the diversification of composition and amount of stored and emitted terpenoids in the tree species is not only structured by climate, but also antagonistic fungal communities through biological interactions.

Construction of a reference transcriptome for the analysis of male sterility in sugi (Cryptomeria japonica D. Don) focusing on MALE STERILITY 1 (MS1).

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. As the genome of this species is 11 Gbps, it is too large to assemble within a short timeframe. Transcriptomics is one approach that can address this deficiency. Here we designed a workflow consisting of three stages to de novo assemble transcriptome using Oases and Trinity. The three transcriptomic stage used were independent assembly, automatic and semi-manual integration, and refinement by filtering out potential contamination. We identified a set of 49,795 cDNA and an equal number of translated proteins. According to the benchmark set by BUSCO, 87.01% of cDNAs identified were complete genes, and 78.47% were complete and single-copy genes. Compared to other full-length cDNA resources collected by Sanger and PacBio sequencers, the extent of the coverage in our dataset was the highest, indicating that these data can be safely used for further studies. When two tissue-specific libraries were compared, there were significant expression differences between male strobili and leaf and bark sets. Moreover, subtle expression difference between male-fertile and sterile libraries were detected. Orthologous genes from other model plants and conifer species were identified. We demonstrated that our transcriptome assembly output (CJ3006NRE) can serve as a reference transcriptome for future functional genomics and evolutionary biology studies.

Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don).

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Genotype and transcriptome effects on somatic embryogenesis in Cryptomeria japonica.

Somatic embryogenesis (SE), which is in vitro regeneration of plant bodies from somatic cells, represents a useful means of clonal propagation and genetic engineering of forest trees. While protocols to obtain calluses and induce regeneration in somatic embryos have been reported for many tree species, the knowledge of molecular mechanisms of SE development is still insufficient to achieve an efficient supply of somatic embryos required for the industrial application. Cryptomeria japonica, a conifer species widely used for plantation forestry in Japan, is one of the tree species waiting for a secure SE protocol; the probability of normal embryo development appears to depend on genotype. To discriminate the embryogenic potential of embryonal masses (EMs) and efficiently obtain normal somatic embryos of C. japonica, we investigated the effects of genotype and transcriptome on the variation in embryogenic potential. Using an induction experiment with 12 EMs each from six genotypes, we showed that embryogenic potential differs between/within genotypes. Comparisons of gene expression profiles among EMs with different embryogenic potentials revealed that 742 differently expressed genes were mainly associated with pattern forming and metabolism. Thus, we suggest that not only genotype but also gene expression profiles can determine success in SE development. Consistent with previous findings for other conifer species, genes encoding leafy cotyledon, wuschel, germin-like proteins, and glutathione-S-transferases are likely to be involved in SE development in C. japonica and indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential.

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

OBJECTIVE: Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. RESULTS: We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

Transcriptome analysis of heat stressed seedlings with or without pre-heat treatment in Cryptomeria japonica.

With global warming as a major environment concern over the coming years, heat tolerance is an important trait for forest tree survival during the predicted future warmer weather conditions. Cryptomeria japonica is a coniferous species widely distributed throughout Japan, and thus, can adapt to a wide range of air temperatures. To elucidate genes involved in heat response in Cryptomeria japonica, transcriptome analysis was conducted for seedlings under heat shock conditions. To test whether heat acclimation affects levels of gene expression, half of the seedlings were pretreated with moderately high temperatures prior to heat shock. De novo assembly of the transcriptome generated 107,924 unigenes and the analysis of differentially expressed genes was conducted using these unigenes. A total of 5217 differentially expressed genes were identified. Most genes upregulated by heat shock, regardless of pre-heat treatment, were conserved to heat response genes of angiosperm species, such as heat shock factors (Hsf) and heat shock proteins (Hsp). Pre-heating of seedlings affected expression levels of several Hsfs and their induction was lower in pre-heated seedlings than in seedlings without pre-heat treatment. This suggests a conserved role of Hsfs in heat response and heat acclimation in seed plants. On the other hand, many unknown genes were upregulated in only seedlings without pre-heat treatment after heat exposure. Notably, expression of gypsy/Ty3 type retrotransposons was dramatically induced. These findings provide valuable information to develop a better understanding of the molecular mechanisms of heat response and acclimation in C. japonica.

Effects of day length- and temperature-regulated genes on annual transcriptome dynamics in Japanese cedar (Cryptomeria japonica D. Don), a gymnosperm indeterminate species.

Seasonal phenomena in plants are primarily affected by day length and temperature. The shoot transcriptomes of trees grown in the field and a controlled-environment chamber were compared to characterize genes that control annual rhythms and the effects of day length- and temperature-regulated genes in the gymnosperm Japanese cedar (Cryptomeria japonica D. Don), which exhibits seasonally indeterminate growth. Annual transcriptome dynamics were clearly demonstrated by principal component analysis using microarray data obtained under field-grown conditions. Analysis of microarray data from trees grown in a controlled chamber identified 2,314 targets exhibiting significantly different expression patterns under short-day (SD) and long-day conditions, and 2,045 targets exhibited significantly different expression patterns at 15°C (LT; low temperature) versus 25°C. Interestingly, although growth was suppressed under both SD and LT conditions, approximately 80% of the SD- and LT-regulated targets differed, suggesting that each factor plays a unique role in the annual cycle. The top 1,000 up-regulated targets in the growth/dormant period in the field coincided with more than 50% of the SD- and LT-regulated targets, and gene co-expression network analysis of the annual transcriptome indicated a close relationship between the SD- and LT-regulated targets. These results indicate that the respective effects of day length and temperature interact to control annual transcriptome dynamics. Well-known upstream genes of signaling pathways responsive to environmental conditions, such as the core clock (LHY/CjLHYb and CCA1/CjLHYa) and PEBP family (MFT) genes, exhibited unique expression patterns in Japanese cedar compared with previous reports in other species, suggesting that these genes control differences in seasonal regulation mechanisms between species. The results of this study provide new insights into seasonal regulation of transcription in Japanese cedar.

Climate sensitivity of Cryptomeria japonica in two contrasting environments: Perspectives from QTL mapping.

Long-lived forest tree species experience a wide range of environmental conditions throughout their lifespan. Evaluation of the underlying growth and development mechanisms of these species is essential to predict tree growth under climate change. This study investigated climate sensitivity to temperature, precipitation, dry periods, and the associated genomic regions in Cryptomeria japonica, Japan's most commercially important tree. We used tree rings and common garden experiments with three clonal replicates planted in two contrasting environments in Kyushu (Kumamoto site) and Honshu (Chiba site), Japan. Tree growth showed a significant negative correlation with the dry period (>4 days) in March of the year of tree-ring formation at the Chiba site. In contrast, temperature and precipitation had little influence on tree growth. Quantitative trait locus (QTL) analysis was performed to investigate climate sensitivity to dry periods at the Chiba site, revealing 13 significant QTLs. One QTL showed a substantially large contribution to the overall climate sensitivity, accounting for 12.4% of the total phenotypic variation. The phenotypic variance explained (PVE) by other QTLs ranged from 0.9% to 2.9%, and the total PVE by all QTLs was 35.6%. These findings indicate that the tree population at the Chiba site could be vulnerable to drought in early spring and that the QTL showing the greatest impact on climate sensitivity may be closely related to genes associated with tolerance or adaptation to drought stress. The QTLs identified in this study could be useful for molecular breeding, forest management, and predicting the growth of C. japonica under a changing climate.

Development of Rice-Seed-Based Oral Allergy Vaccines Containing Hypoallergenic Japanese Cedar Pollen Allergen Derivatives for Immunotherapy.

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.

Cloning and Functional Analysis of Lignin Biosynthesis Genes Cf4CL and CfCCoAOMT in Cryptomeria fortunei.

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.

T-cell activation by transgenic rice seeds expressing the genetically modified Japanese cedar pollen allergens.

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.

Inferring the demographic history of Japanese cedar, Cryptomeria japonica, using amplicon sequencing.

The evolution of a species depends on multiple forces, such as demography and natural selection. To understand the trajectory and driving forces of evolution of a target species, it is first necessary to uncover that species' population history, such as past and present population sizes, subdivision and gene flow, by using appropriate genetic markers. Cryptomeria japonica is a long-lived monoecious conifer species that is distributed in Japan. There are two main lines (omote-sugi and ura-sugi), which are distinguished by apparent differences in morphological traits that may have contributed to their local adaptation. The evolution of these morphological traits seems to be related to past climatic changes in East Asia, but no precise estimate is available for the divergence time of these two lines and the subsequent population dynamics in this species. Here, we analyzed the nucleotide variations at 120 nuclear genes in 94 individuals by using amplicon sequencing in combination with high-throughput sequencing technologies. Our analysis indicated that the population on Yakushima Island, the southern distribution limit of C. japonica in Japan, diverged from the other populations 0.85 million years ago (MYA). The divergence time of the other populations on mainland Japan was estimated to be 0.32 MYA suggesting that the divergence of omote-sugi and ura-sugi might have occurred before the last glacial maximum. Although we found modest levels of gene flow between the present populations, the long-term isolation and environmental heterogeneity caused by climatic changes might have contributed to the differentiation of the lines and their local adaptation.