Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Identification of genes regulated by lipids from seaweed Susabinori (Pyropia yezoensis) involved in the improvement of hepatic steatosis: Insights from RNA-Seq analysis in obese db/db mice.

Hepatic steatosis is an early stage in the progression of non-alcoholic fatty liver disease (NAFLD) and can lead to the development of non-alcoholic steatohepatitis (NASH), a major cause of liver-related morbidity and mortality. Identification of dietary components that can alleviate hepatic steatosis is crucial for developing effective therapeutic strategies for NAFLD. Recently, we demonstrated the impact of lipids extracted from the marine red alga Susabinori (Pyropia yezoensis) in a murine model of type 2-diabete (db/db). We found that Susabinori lipids (SNL), abundant in eicosapentaenoic acid (EPA)-containing polar lipids, protected against obesity-induced hepatic steatosis in db/db mice. To understand the specific genes or biological pathways underlying the effects of SNL, we conducted RNA-Seq analysis of the hepatic transcriptome. By performing comparative analysis of differentially expressed genes between normal mice and db/db mice consuming a control diet, as well as SNL-fed db/db mice, we identified the 15 SNL-dependent up-regulated genes that were down-regulated in db/db mice but up-regulated by SNL feeding. Gene ontology and pathway analysis on these 15 genes demonstrated a significant association with the metabolisms of arachidonic acid (AA) and linoleic acid (LA). Furthermore, we observed alterations in the expression levels of monoacylglycerol lipase (Magl) and fatty acid-binding protein 4 (Fabp4) in the SNL-fed db/db mice, both of which are implicated in AA and LA metabolism. Additionally, the livers of SNL-fed db/db mice exhibited reduced levels of AA and LA, but a high accumulation of EPA. In conclusion, the SNL diet might affect the metabolisms of AA and LA, which contribute to the improvement of hepatic steatosis. Our findings provide insights into the molecular mechanisms underlying the beneficial effects of SNL.

Characterization and organelle genome sequencing of Pyropia species from Myanmar.

Pyropia is a genus comprising red algae of the Bangiaceae family that is commonly found in intertidal zones worldwide. However, understanding of Pyropia species that are prone to tropical regions remains limited despite recent breakthroughs in genomic research. Within the realm of Pyropia species thriving in tropical regions, P. vietnamensis stands out as a widely recognized species. In this study, we aimed to investigate Pyropia species in the southwest coast of Myanmar using physiological and molecular approaches, culture-based analyses, chloroplast rbcL and nuclear SSU gene sequencing, and whole chloroplast and mitochondrial genome sequencing. Physiological analysis showed that the Myanmar samples were more heat-tolerant than their Japanese counterparts, including those of subtropical origin. Additionally, molecular characterization revealed that the Myanmar samples were closely related to P. vietnamensis from India. This study is the first to sequence the chloroplast and mitochondrial genomes of Pyropia species from tropical regions. A unique deletion event was observed within a ribosomal RNA gene cluster in the chloroplast genome of the studied Pyropia species, which is a deviation from the usual characteristics of most Pyropia species. This study improves current understanding of the physiological and molecular characteristics of this comparatively understudied Pyropia species that grows in tropical regions.

RAD-Seq analysis of wild Japanese garlic (Allium macrostemon Bunge) growing in Japan revealed that this neglected crop was previously actively utilized.

Allium macrostemon Bunge, commonly referred to as "no-biru" in Japan, is a widespread wild onion species found across the country. Despite being deeply entwined in ancient Japanese culture, it remains an underutilized crop in Japan. Determining the origins of its domestic populations and understanding their genetic composition is crucial to highlighting the plant's historical significance in Japan. This study aims to bridge this knowledge gap by examining the genetic diversity of 47 A. macrostemon samples from various regions in Japan using RAD-Seq. Our analyses distinguished unique population structures, dividing the samples into three distinct groups: A, B, and C. Notably, groups A and B showed clear evidence of bulb propagation, while group C did not. Group C formed four subgroups: C1, C2, C3, and C4. Hybridization between subgroup C1 and either group A, B, or both, resulted in the emergence of subgroups C2, C3, and C4. Thus, groups A, B, and C1 are posited as the ancestral populations. Additionally, our morphological observations indicated distinct differences among these three groups. Our findings also suggest that human migration may have influenced the plant's distribution, hinting at active usage in the past that later waned, causing its current underutilized status.

The role of oceanic currents in the dispersal and connectivity of the mangrove Rhizophora mangle on the Southwest Atlantic region.

Dispersal is a crucial mechanism to living beings, allowing them to reach new resources such that populations and species can occupy new environments. However, directly observing the dispersal mechanisms of widespread species can be costly or even impractical, which is the case for mangrove trees. The influence of ocean currents on mangrove dispersal is increasingly evident; however, few studies mechanistically relate the patterns of population distribution with the dispersal by oceanic currents under an integrated framework. Here, we evaluate the role of oceanic currents on connectivity of Rhizophora mangle along the Southwest Atlantic. We inferred population genetic structure and migration rates, simulated the displacement of propagules and tested our hypotheses with Mantel tests and redundancy analysis. We observed populations structured in two major groups, north and south, which is corroborated by other studies with Rhizophora and other coastal plants. Inferred recent migration rates do not indicate ongoing gene flow between sites. Conversely, long-term migration rates were low across groups and contrasting dispersal patterns within each one, which is consistent with long-distance dispersal events. Our hypothesis tests suggest that both isolation by distance and isolation by oceanography (derived from the oceanic currents) can explain the neutral genetic variation of R. mangle in the region. Our findings expand current knowledge of mangrove connectivity and highlight how the association of molecular methods with oceanographic simulations improve the interpretation of the dispersal process. This integrative approach is a cost- and time-efficient strategy to include dispersal and connectivity data into marine protected areas planning and management.

Classification of Japanese Pepper (Zanthoxylum piperitum DC.) from Different Growing Regions Based on Analysis of Volatile Compounds and Sensory Evaluation.

The Japanese pepper (Zanthoxylum piperitum DC.) is an attractive plant that is highly palatable and benefits human health. There are several lineages of pepper plants in Japan. However, the classification of each lineage by analyzing its volatile compounds and studies on the effects of differences in volatile compounds on human flavor perception have not been performed in detail. Herein, we conducted gas chromatography (GC) and GC/mass spectrometry (GC/MS) analysis of volatile compounds and sensory evaluation of flavor by an analytical panel using 10 commercially available dry powdered Japanese pepper products from different regions. GC and GC/MS analysis detected limonene, ß-phellandrene, citronellal, and geranyl acetate as the major volatile compounds of Japanese peppers. The composition of volatile compounds showed different characteristics depending on the growing regions, and cluster analysis of composition classified the products into five groups. The sensory evaluation classified the products into four groups, and the results of both classifications were in good agreement. Our results provide an important basis for proposing cooking and utilization methods that take advantage of the unique characteristics of each lineage based on scientific evidence.

Weekly Observations of Estuarine Microbial Assemblages during Summer in the Inner Part of Ariake Bay, Japan; Microbial Water-sediment Coupling in Turbid Shallow Waters.

Estuarine microbial assemblages are altered by a number of environmental factors, and knowledge of these changes is essential for understanding the functions of microbes in estuarine ecosystems. The aims of the present study were to examine the relationship between microbial assemblages in the water column and sediment surface, and to identify the environmental factors that influence the short-term dynamics of microbial assemblages in these two zones in summer in the inner part of Ariake Bay. The microbial assemblage of each sample consisted of a mean of 71.1% operational taxonomic units (OTUs), which commonly occurred in the water column and sediment surface, although their relative composition markedly differed between the two zones. In the water column, spatiotemporal changes in microbial assemblages correlated with several environmental factors, such as the nitrogen content in suspended particles, turbidity, and salinity. On the other hand, temporal changes in the sediment's microbial assemblages were governed by a single environmental factor, namely, the oxygen reduction potential. These results suggest that the composition of microbial assemblages in the water column and sediment surface differed even in highly turbid brackish waters with high sediment resuspension, and the environmental factors contributing to the change in the assemblage composition also differed between the water column and sediment.

Genetic diversity of loquat (Eriobotrya japonica) revealed using RAD-Seq SNP markers.

Loquat (Eriobotrya japonica) have originated in southeastern China and spread as a cultivated plant worldwide. Many of the loquat genetic resources collected internationally are of unknown origin, and their genetic background requires clarification. This study analyzed the genetic diversity of 95 accessions by using Rad-Seq SNP markers. Data analysis broadly classified loquat into three groups: (1) Japanese and Chinese cultivars and some Japanese strains (wild plants that are not used for commercial cultivation), (2) Vietnamese, Israeli, Greek, USA, and Mexican cultivars and strains, and (3) other Japanese strains. Group 2 is cultivated mostly outside of East Asia and was clearly distinct from the other groups, indicating that varieties of unknown origin with genetic backgrounds different from those of Japanese and Chinese cultivars may have been introduced to Mediterranean countries and North America. Because Japanese and Chinese cultivars belong to group 1, the current Japanese cultivars are derived from genetic resources brought from China. Some of group 1 may have been introduced to Japan before excellent varieties were developed in China, while group 3 may have been indigenous to Japan that have not been introduced by human activities, or may have been brought to Japan by human activities from China.

Draft Genome Sequences of Two Antimicrobial Compound-Producing Strains of Bacillus Species, TM-R and SY1-1.

Two antimicrobial compound-producing strains of Bacillus species, namely, TM-R and SY1-1, have been identified previously. In this study, we report the draft genome sequences of these strains and demonstrate the presence of 12 and 14 gene clusters for secondary metabolite biosynthesis in TM-R and SY1-1, respectively.

Development of Monascus purpureus monacolin K-hyperproducing mutant strains by synchrotron light irradiation and their comparative genome analysis.

Monascus purpureus have been used for making koji and other fermented foods and supplements. M. purpureus characteristically produces monacolin K (MK), a secondary metabolite that competitively inhibits cholesterol synthesis. Synchrotron light irradiation was applied to induce mutation in the strain KUPM5 to improve the MK-producing ability of M. purpureus strain KUPM5. Screening by a bioassay utilizing sensitivities to yeast Saccharomyces cerevisiae from 936 colonies allows isolating three mutant strains: SC01, SC02, and SC03. These mutant strains and the parental strain KUPM5 were subjected to make koji using rice, and their metabolites were compared. All strains SC01, SC02, and SC03 in koji showed higher production of MK than the strain KUPM5. Particularly, the SC02 strain produced MK threefold higher than KUPM5 and maintained the production capabilities of other metabolites, including red, yellow, and orange pigments, mycelial contents, and α-amylase activity comparable to those of the strain KUPM5. Comparative genome analysis among strain KUPM5 and the mutants revealed that synchrotron light irradiation introduced mutations in approximately 90% of the total genes, including SNV, MNV, and indel mutations. The frequencies of SNV substitution in the whole genome occupied 68.96% of all mutations, of which 92.38% were transversions and 7.62% were transitions. This study, therefore, proved the synchrotron light irradiation was highly efficient for the strain improvement of a filamentous fungus, M. purpureus, and provided insights into the properties of mutation in the fungus by this mutagen.

Efficient method for generating citrus hybrids with polyembryonic Satsuma mandarin as the female parent.

Many citrus fruits have polyembryonic traits, and their seeds contain many nucellar embryos along with a single zygotic embryo, affecting the crossbreeding process. Generally, nucellar embryos are considered to have more vigorous growth than zygotic embryos. Therefore, the in vitro method using an embryo rescue culture is often chosen to obtain zygotic embryo-derived individuals. Nevertheless, hybrids can be obtained with a certain probability from the seeds sown in the soil. The in-soil method, which sows seeds in the soil, has distinct advantages over the in vitro method, including lower cost and simpler technology. However, the efficiency of obtaining hybrids from these methods has not been compared in detail. The current study evaluates the effectiveness of these methods for obtaining hybrids using polyembryonic Satsuma mandarin as the female parent. The number of mature embryos per seed using the in-soil method was less than one-third of that produced using the in vitro method. Although the in vitro method produced more hybrids than the in-soil method, the ratio of the hybrids to the resulting population was significantly higher in the in-soil method. Thus, the in-soil method was more efficient and practical than the in vitro method for selecting hybrids from polyembryonic Satsuma mandarin seeds. The observations of the individuals obtained using the in-soil method suggest that zygotic embryos were not poorer in growth than nucellar embryos when using our selected parental combinations. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01324-6.

Genomic diversity of 39 samples of Pyropia species grown in Japan.

Some Pyropia species, such as nori (P. yezoensis), are important marine crops. We conducted a phylogenetic analysis of 39 samples of Pyropia species grown in Japan using organellar genome sequences. A comparison of the chloroplast DNA sequences with those from China showed a clear genetic separation between Japanese and Chinese P. yezoensis. Conversely, comparing the mitochondrial DNA sequences did not separate Japanese and Chinese P. yezoensis. Analysis of organellar genomes showed that the genetic diversity of Japanese P. yezoensis used in this study is lower than that of Chinese wild P. yezoensis. To analyze the genetic relationships between samples of Japanese Pyropia, we used whole-genome resequencing to analyze their nuclear genomes. In the offspring resulting from cross-breeding between P. yezoensis and P. tenera, nearly 90% of the genotypes analyzed by mapping were explained by the presence of different chromosomes originating from two different parental species. Although the genetic diversity of Japanese P. yezoensis is low, analysis of nuclear genomes genetically separated each sample. Samples isolated from the sea were often genetically similar to those being farmed. Study of genetic heterogeneity of samples within a single aquaculture strain of P. yezoensis showed that samples were divided into two groups and the samples with frequent abnormal budding formed a single, genetically similar group. The results of this study will be useful for breeding and the conservation of Pyropia species.

Breeding sake yeast and identification of mutation patterns by synchrotron light irradiation.

Sake yeast is one of the important factors that characterize the aroma and taste of sake. To obtain sake yeast strains with different metabolic capabilities from other strains, breeding of a sake yeast is an effective way. In this study, sake yeast strain Y5201 was mutagenized by synchrotron light irradiation to obtain the mutant strains showing different brewing characteristics from parental strain Y5201, and comparative genome analysis between strain Y5201 and mutant strains was performed to identify mutation points and patterns induced by synchrotron light irradiation. Screening with the drug-resistant and fermentation tests selected the nine mutants (C18, C19, C29, C50, C51, C52, C54, T25, and T49) from the mutagenized Y5201 cells. Principal component analysis results based on the analysis of the small-scale brewing test metabolites showed that the mutant strain C19 was different from other strains, which had higher productivity of ethyl caproate and isoamyl acetate than those of the Y5201. Comparative genome analysis revealed that mutants by synchrotron light irradiation had a higher diversity of single nucleotide substitutions and a higher frequency of Indel (insertion/deletion) in these DNA than ethyl methanesulfonate and UV irradiation. These results suggest that synchrotron light irradiation is an effective and unique mutagen for yeast breeding.

Genetic diversity and population structure of razor clam Sinonovacula constricta in Ariake Bay, Japan, revealed using RAD-Seq SNP markers.

The razor clam Sinonovacula constricta is a commercially important bivalve in Japan. The current distribution of this species in Japan is limited to Ariake Bay, where the fishery stock is declining. It is necessary to understand the genetic population structure of this species in order to restore the fishery stock while preserving the genetic diversity of the clam. Here, we report for the first time the genetic population structure of S. constricta in Ariake Bay, Japan. Paired-end restriction site-associated DNA sequencing (RAD-Seq) analyzed samples of S. constricta collected from seven mudflats located along Ariake Bay. Two different genetic populations exist in Ariake Bay, one inhabiting wild habitats and the other inhabiting the transplanted area of artificial seedlings. Our results suggest that genetic differentiation occurred between these two populations (Fst value = 0.052), and a high level of genetic differentiation is maintained between the two groups. In the future, monitoring the interbreeding status of the two genetically distinct populations and the genetic differentiation within each population is important for conserving the genetic diversity of S. constricta in Japan.

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.

Draft Genome Sequences of Three Strains of Pseudomonas syringae pv. eriobotryae, a Pathogen Causing Canker Disease in Loquat, Isolated in Japan.

The phytopathogenic bacterium Pseudomonas syringae pv. eriobotryae causes canker disease in loquat. Isolates from Japan are classified into three groups based on pathogenicity and pigment production. In this study, we report the draft genome sequences of three strains, one belonging to each of the three groups.

Potential application of simple easy-to-use insertion-deletion (InDel) markers in citrus cultivar identification.

We previously developed insertion-deletion (InDel) markers that distinguish three genotypes (two homozygous and one heterozygous) of diverse citrus cultivars. These InDel markers were codominant and could be clearly detected by using simple agarose gel electrophoresis. We sought to establish a method for cultivar identification using these 28 InDel markers to genotype 31 citrus cultivars. The results revealed that a minimum of 6 markers were required to identify individuals using the three-genotype classification method. Furthermore, we found that a simple method for distinguishing between two genotypes (homozygous and heterozygous) could be used to identify individuals using a minimum of 7 markers. Our findings provide a basis for the development of simple and rapid citrus cultivar identification methods.

Evaluation of the population structure and phylogeography of the Japanese Genji firefly, Luciola cruciata, at the nuclear DNA level using RAD-Seq analysis.

The Genji firefly, Luciola cruciata, is widely distributed throughout the major Japanese islands (Honshu, Shikoku, and Kyushu) and distinguished into two ecological types on the basis of the flash interval of the mate-seeking males (4-sec slow-flash or 2-sec fast-flash intervals). The boundary of the ecological types corresponds to the Fossa Magna, a great rupture zone that separates eastern and western Japan. Although the degree of genetic differentiation of the two types has been evaluated using allozyme and mitochondrial DNA sequence data, it has not been evaluated using genome-wide data. Based on the genome-wide data obtained using single-end restriction-site-associated DNA (RAD-Seq), principal component, gene-level phylogenetic tree, admixture, and Wright's fixation index analyses, we identified three phylogenetic groups in L. cruciata: East-Honshu, West-Honshu, and Kyushu. This grouping corresponds to the ecological types: East-Honshu to the slow-flash type and West-Honshu and Kyushu to the fast-flash type. Although introgression was exceptionally observed around adjacent or artificially transplanted areas, gene flow among the groups was almost absent in the natural populations. The phylogenetic tree under the coalescent model also evaluated differentiation among the East-Honshu, West-Honshu and Kyushu groups. Furthermore, because the distribution patterns of the three groups are consistent with the geological history of Japanese islands, a vicariant speciation scenario of L. cruciata is concluded. In addition, we identified genetic markers that can be used to distinguish the three genetic groups for genetic management of firefly transplantation in nature conservation and regeneration.

Genetic diversities and population structures of four popular Myanmar local cattle breeds.

Local cattle breeds are popular in Myanmar because they make excellent draught animals, so it is of fundamental importance that their genetic diversities and population structures are elucidated for breeding and conservation purposes. In this study, we characterized the genetic diversities and population structures of four popular Myanmar local cattle breeds and five native cattle populations from neighboring countries (Bangladesh, Laos, Cambodia, and Vietnam) using the GGP Bovine 50K array. The mean observed and expected heterozygosity estimates using pruned datasets ranged from 0.317 and 0.322, respectively, in Cambodia to 0.448 and 0.421, respectively, in Vietnam South. The four Myanmar local breeds exhibited similar levels of genetic diversity. However, AMOVA revealed significant genetic differentiation among the nine populations (p < 0.00001), and Neighbor-Net analysis showed that Shan Ngwar Pu was distinct from the other Myanmar local breeds. Furthermore, PCA and population structure analyses revealed that the native cattle from neighboring countries genetically influenced the Myanmar local breeds to some extent and that the genetic origins could also be observed in the local breeds. These findings highlight the importance of Myanmar local breeds as genetic resources and provide useful information for the future development of breeding strategies and conservation management plans.