Different population size change and migration histories created genetic diversity of three oaks in Tokai region, central Japan.

To understand genetic diversity in focal species, it is important to consider the possibility of speciation with gene flow, especially in species with porous genomes such as oaks. We studied genetic diversity and structure in three oaks, Quercus mongolica var. mongolicoides (QM), Q. mongolica var. crispula (QC) and Q. serrata (QS), growing in the Tokai region, central Japan. QM is semi-endemic to the region while the others are common taxa. We also conducted demographic modeling to infer their population size change and migration histories using an approximate Bayesian computation (ABC) approach. The three taxa showed distinct genetic structures but there was genetic admixture among the taxa, especially between QM and QC. ABC analysis of population size change revealed that the population size of QM was stable during and after the last glacial period, while QC and QS showed population expansion after the last glacial maximum. ABC analysis of population divergence and migration revealed that continuous gene flow between QM and QC after their divergence was supported, while between QM and QS, and between QC and QS, secondary contact after sufficient isolation was supported. These historical migration patterns among the three taxa indicate that QM and QC are currently in the early stage or gray zone of speciation, whereas speciation of the other two taxon pairs is considered to have almost been established. Observed gene flow patterns and strength between QM and QC, and between QM and QS, were explained by both flowering patterns and historical distributions, but those between QC and QS were not.

Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Large contribution of clonal reproduction to the distribution of deciduous liana species (Wisteria floribunda) in an old-growth cool temperate forest: evidence from genetic analysis.

Background and Aims: Extensive clonal (vegetative) reproduction in lianas is a common and important life history strategy for regeneration and colonization success. However, few studies have evaluated the contribution of clonal reproduction to stand-level distribution of lianas in their natural habitat using genetic tools. The objectives of the present study were to investigate (1) the contribution of clonal reproduction to the distribution of Wisteria floribunda, (2) the size of clonal patches and (3) how the distribution patterns of W. floribunda clones are affected by micro-topography. Methods: The contribution of clonal reproduction to the distribution of the deciduous liana species W. floribunda was evaluated using genetic analysis across a 6-ha plot of an old-growth temperate forest in Japan and preference in landform between clonal ramets and non-clonal ramets was assessed. Key Results: Of the 391 ramets sampled, clonal reproduction contributed to 71 and 62 % of the total abundance and basal area, respectively, or 57 and 31 % when the largest ramet within a genet was excluded. The large contribution of clonal reproduction to the density and basal area of W. floribunda was consistent with previous observational studies. The largest genet included a patch size of 0.47 ha and ranged over 180 m. Preferred landforms of clonal and non-clonal ramets were significantly different when evaluated by both abundance and basal area. Non-clonal ramets distributed more on lower part of the slope than other landforms in comparison with clonal ramets and trees, possibly reflecting the limitation of clonal growth by stolons. Conclusions: Using genetic analysis, the present study found evidence of a large contribution of clonal reproduction on the distribution of W. floribunda in its natural habitat. The results indicate that clonal reproduction plays an important role not only in the formation of populations but also in determining the distribution patterns of liana species.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don.

BACKGROUND: High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. RESULTS: We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. CONCLUSIONS: Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers.

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.

Tree dynamic response and survival in a category-5 tropical cyclone: The case of super typhoon Trami.

In the future with climate change, we expect more forest and tree damage due to the increasing strength and changing trajectories of tropical cyclones (TCs). However, to date, we have limited information to estimate likely damage levels, and nobody has ever measured exactly how forest trees behave mechanically during a TC. In 2018, a category-5 TC destroyed trees in our ongoing research plots, in which we were measuring tree movement and wind speed in two different tree spacing plots. We found damaged trees in only the wider spaced plot. Here, we present how trees dynamically respond to strong winds during a TC. Sustained strong winds obviously trigger the damage to trees and forests but inter-tree spacing is also a key factor because the level of support from neighboring trees modifies the effective "stiffness" against the wind both at the single tree and whole forest stand level.

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.

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.

Genetic diversity and structure of natural fragmented Chamaecyparis obtusa populations as revealed by microsatellite markers.

The genetic diversity and population structure of hinoki (Chamaecyparis obtusa) in Japan were investigated by examining the distribution of alleles at 13 microsatellite loci in 25 natural populations from Iwaki in northern Japan to Yakushima Island in southern Japan. On average, 26.9 alleles per locus were identified across all populations and 4.0% of the genetic variation was retained among populations (G(ST) = 0.040). According to linkage disequilibrium analysis, estimates of effective population size and detected evidence of bottleneck events, the genetic diversity of some populations may have declined as a result of fragmentation and/or over-exploitation. The central populations located in the Chubu district appear to have relatively large effective population sizes, while marginal populations, such as the Yakushima, Kobayashi and Iwaki populations, have smaller effective population sizes and are isolated from the other populations. Microsatellite analysis revealed the genetic uniqueness of the Yakushima population. Although genetic differentiation between populations was low, we detected a gradual cline in the genetic structure and found that locus Cos2619 may be non-neutral with respect to natural selection.

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 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.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

A revision of the genus Calcigorgia (Cnidaria, Octocorallia, Acanthogorgiidae) with the description of three new species.

Octocorals of the Acanthogorgiid genus Calcigorgia have been examined, from Japan, Sea of Okhotsk, and Bering Sea. The four known species are re-described and scanning electron microscopy (SEM) images of sclerites presented. Three other species are described and depicted, bringing the total number of Calcigorgia species to seven. Calcigorgia simushiri, Dautova 2018 is synonymized with C. spiculifera Broch, 1935. A neotype for C. spiculifera has been designated.

Fine mapping of the male-sterile genes (MS1, MS2, MS3, and MS4) and development of SNP markers for marker-assisted selection in Japanese cedar (Cryptomeria japonica D. Don).

Pollinosis caused by Japanese cedar (Cryptomeria japonica) is a widespread social problem in Japan. To date, 23 male-sterile C. japonica trees have been selected in Japan to address pollinosis, from which four male-sterility loci (MS1, MS2, MS3, and MS4) have been identified from test crossing results. For efficient breeding of male-sterile C. japonica trees, more male-sterile individuals and individuals heterozygous for male-sterile genes are required. Therefore, we aimed to develop DNA markers for marker-assisted selection of four types of male-sterile genes from populations without a family structure. First, for four families exhibiting segregation of each male-sterile locus (MS1, MS2, MS3, and MS4), genome-wide single-nucleotide polymorphism and insertion/deletion (indel) genotyping was performed using the Axiom myDesign Targeted Genotyping Array method. Four high-density linkage maps for mapping the MS1, MS2, MS3, and MS4 families were constructed, which included 4923, 1722, 1896, and 2247 markers, respectively. In these maps, 15, 4, 2, and 2 markers were located 0.0, 3.3, 1.1, and 0.0 cM from the MS1, MS2, MS3, and MS4 loci, respectively. Second, for the markers located 0.0 cM from a male-sterile locus (i.e., MS1 and MS4), to clarify the most tightly linked markers, we calculated the prediction rate of male-sterile gene genotypes from marker genotypes for 78 trees. The markers with the highest prediction rates were AX-174127446 (0.95) for MS1 and AX-174121522 (1.00) for MS4. The AX-174121522 marker was considered to be suitable for selecting trees homozygous or heterozygous for the MS4 gene from plus-trees without a pollination test, which requires a large amount of time and effort. The nearest markers to the male-sterile loci found in this study may facilitate the isolation of male-sterile genes in C. japonica via combination with the draft genomic sequence that is currently being collated.

The genus Bebryce (Cnidaria, Octocorallia, Plexauridae) at Japan, with descriptions of three new species.

Three new deep-water species of Bebryce from Japan are described and depicted using Scanning Electron Microscopy: Bebryce otsuchiensis sp. n., Bebryce rotunda sp. n., and Bebryce satsumaensis sp. n. Bebryce studeri Whitelegge, 1897, was reported from Japanese waters for the first time, bringing the total of Japanese Bebryce species to six. Five of these six species seem to be endemic to Japanese waters and all occur in deep water up to 213 m. A key to the Bebryce species is presented.

Species of Elasmogorgia and Euplexaura (Cnidaria, Octocorallia) from Japan with a discussion about the genus Filigella.

Octocorals with thread-like colony shape have been re-examined, mainly from Japanese waters. The holotypes of Elasmogorgia filiformis and Filigella boninensis and a syntype of Filigella mitsukurii have been studied. Euplexaura arbuscula is identified and Euplexaura yayoii sp. n. described.

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags.

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.

Melithaeidae of Japan (Octocorallia, Alcyonacea) re-examined with descriptions of 11 new species.

Japanese melithaeid type material is re-examined and re-described. The sclerites of the different species are depicted using Scanning Electron Microscopy. All Japanese species of the family Melithaeidae treated here belong to the genus Melithaea and are endemic to Japanese waters. Old museum material and newly collected specimens from Japanese waters are identified after comparison with this type material. Acabaria modesta var. abyssicola is regarded a separate species, here named Melithaea abyssicola (Kükenthal, 1909). In addition, 11 new species are described: Melithaea boninensis sp. n., Melithaea doederleini sp. n., Melithaea isonoi sp. n., Melithaea keramaensis sp. n., Melithaea oyeni sp. n., Melithaea ryukyukensis sp. n., Melithaea sagamiensis sp. n., Melithaea satsumaensis sp. n., Melithaea suensoni sp. n., Melithaea tanseii sp. n., and Melithaea tokaraensis sp. n.. Pleurocorallium confusum Moroff, 1902, Pleurocoralloides formosum Moroff, 1902, Melitodes flabellifera Kükenthal, 1908, and Melitodes densa Kükenthal, 1908 are synonymized with Melithaea japonica (Verrill, 1865). We have designated a neotype for Melithaea mutsu Minobe, 1929. A key to the Japanese melithaeids is presented.

Induction of insulin-like growth factor 2 expression in a mesenchymal cell line co-cultured with an ameloblast cell line.

Various growth factors have been implicated in the regulation of cell proliferation and differentiation during tooth development. It has been unclear if insulin-like growth factors (IGFs) participate in the epithelium-mesenchyme interactions of tooth development. We previously produced three-dimensional sandwich co-culture systems (SW) containing a collagen membrane that induce the differentiation of epithelial cells. In the present study, we used the SW system to analyze the expression of IGFs and IGFRs. We demonstrate that IGF2 expression in mesenchymal cells was increased by SW. IGF1R transduces a signal; however, IGF2R does not transduce a signal. Recombinant IGF2 induces IGF1R and IGF2R expression in epithelial cells. IGF1R expression is increased by SW; however, IGF2R expression did not increase by SW. Thus, IGF2 signaling works effectively in SW. These results suggest that IGF signaling acts through the collagen membrane on the interaction between the epithelium and mesenchyme. In SW, other cytokines may be suppressed to induce IGF2R induction. Our results suggest that IGF2 may play a role in tooth differentiation.

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro.

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.