Isolation of SARS-CoV-2 from COVID-19 Patients and an Asymptomatic Individual.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019. Despite the recent introduction of vaccines against SARS-CoV-2, more effective vaccines and antiviral drugs must be developed. Here, we isolated five SARS-CoV-2 strains from four patients with coronavirus disease (COVID-19) and an asymptomatic individual using pharyngeal swabs, nasopharyngeal swabs, and sputum samples. Cytopathic effects in inoculated Vero cells were observed between days 3 and 7. SARS-CoV-2 infection was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing. Phylogenetic analyses of the whole genome sequences showed that the virus isolates from the clinical samples belonged to the Wuhan and European lineages. These findings and the isolated viruses may contribute to the development of diagnostic tools, vaccines, and antiviral drugs for COVID-19.

Pretreatment of Lignocellulosic Biomass with Cattle Rumen Fluid for Methane Production: Fate of Added Rumen Microbes and Indigenous Microbes of Methane Seed Sludge.

The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor-1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor-1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.

NET-CAGE characterizes the dynamics and topology of human transcribed cis-regulatory elements.

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.

Phylogeographic analysis suggests two origins for the riparian azalea Rhododendron indicum (L.) Sweet.

Ecological speciation is an important factor in the diversification of plants. The distribution of the woody species Rhododendron indicum, which grows along rivers and is able to withstand water flow when rivers flood (i.e. it is a rheophyte), is disjunct, in contrast to the widespread distribution of its relative, Rhododendron kaempferi. This study aimed to elucidate the phylogenetic relationships between R. indicum and R. kaempferi and the evolutionary processes that gave rise to them. The sequences of three non-coding chloroplast DNA regions (total length 1977 bp) were obtained from 21 populations covering the ranges of the two species. In addition, genome-wide SNPs were genotyped from 20 populations using a genotyping by sequencing method. Leaf morphologies were measured for eight representative populations. Two chloroplast DNA haplotypes, which were detected in R. indicum, were shared between the two species. Genome-wide SNPs identified two lineages in R. indicum and these lineages did not constitute a monophyletic group. Each of these two lineages was related to geographically close populations of R. kaempferi. Leaf morphology, which is a characteristic feature in rheophytes, was not differentiated between the two lineages in R. indicum. The morphological similarity between the two heterogeneous lineages may be a result of parallel evolution from R. kaempferi or of introgressive hybridization between the species due to strong selective pressure imposed by flooding.

Development of microsatellite markers for the endangered orchid Calanthe izu-insularis (Orchidaceae).

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

Background and Aims: Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. Methods: Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. Key Results: Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. Conclusions: The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.

Pretreatment of lignocellulosic biomass by cattle rumen fluid for methane production: Bacterial flora and enzyme activity analysis.

We attempted to develop a pretreatment method for methane fermentation of lignocellulosic biomass using cattle rumen fluid, treated as slaughterhouse waste. When rapeseed (Brassica napus L.) was added to the methane fermentation after being solubilized with rumen fluid, 1.5 times more methane was produced compared with untreated rapeseed. Analysis of the bacterial flora during rumen fluid treatment using the MiSeq next-generation sequencer showed that the predominant phylum shifted from Bacteroidetes, composed of amylolytic Prevotella spp., to Firmicutes, composed of cellulolytic and xylanolytic Ruminococcus spp., in only 6 h. In total, 7 cellulolytic, 25 cello-oligosaccharolytic, and 11 xylanolytic bacteria were detected after investigating the most abundant sequences of detected taxa. The relative abundance of two Ruminococcus species (Ruminococcus albus and R. flavefaciens), known as cellulolytic, cello-oligosaccharolytic, and xylanolytic bacteria, increased with increasing cellulose and hemicellulose degradation rates, and, finally, comprised 48% of all operational taxonomic units. The chronological observation of enzyme activities showed that cellulolytic and xylanolytic activities increased 6 h later, and that oligosaccharolytic activity increased 24 h later. This study detected six bacteria that participate in the degradation of aromatics derived from lignin, which have rarely been reported in rumen fluid. The constitution of the detected bacteria suggests that the aromatics were converted into acetate via benzoate. The list of microbes that cover all lignocellulose-degrading candidates will provide fundamental knowledge for future studies focusing on rumen microbes.

Chloroplast DNA sequencing and detailed microsatellite genotyping of all remnant populations suggests that only a single genet survives of the critically endangered plant Rehmannia japonica.

Rehmannia japonica (Thunb.) Makino ex T. Yamaz. is an endangered perennial herb species in Japan. Although earlier the Japanese considered it a variety of R. glutinosa, recent Japanese taxonomists have consistently regarded it as an independent species. According to the historical literature, Rehmannia japonica seems to have been known in China and Japan in the past. However, Chinese taxonomists do not recognize R. japonica at present. In Japan, only two populations are known, and although these populations flower every year, seed reproduction has not been observed. In this study, we aimed to reveal the phylogenetic relationships and levels of genetic diversity of R. japonica. A haplotype network based on two chloroplast DNA regions (trnL-trnF and rps16) showed that the sequences of R. japonica were distinguishable by three or four sites of indels from the most closely related species, R. chingii, consistent with the separate species status of R. japonica. An analysis of genetic diversity using twelve microsatellite loci showed that all of the ramets of R. japonica collected from two geographically isolated populations had an identical multilocus genotype, including identical heterozygous genotypes at six loci. This result indicated asexual origin of all sampled ramets. This study also suggests that the absence of sexual reproduction of R. japonica is explained by self-incompatibility combined with only a single genet remaining in the R. japonica populations.

Lack of genetic variation prevents adaptation at the geographic range margin in a damselfly.

What limits a species' distribution in the absence of physical barriers? Genetic load due to asymmetric gene flow and the absence of genetic variation due to lack of gene flow are hypothesized to constrain adaptation to novel environments in marginal populations, preventing range expansion. Here, we examined the genetic structure and geographic variation in morphological traits in two damselflies (Ischnura asiatica and I. senegalensis) along a latitudinal gradient in Japan, which is the distribution centre of I. asiatica and the northern limit of I. senegalensis. Genomewide genetic analyses found a loss of genetic diversity at the edge of distribution in I. senegalensis but consistently high diversity in I. asiatica. Gene flow was asymmetric in a south-north direction in both species. Although body size and wing loading showed decreasing latitudinal clines (smaller in north) in I. asiatica in Japan, increasing latitudinal clines (larger in north) in these phenotypic markers were observed in I. senegalensis, particularly near the northern boundary, which coincided well with the location where genetic diversity began a sharp decline. In ectothermic animals, increasing latitudinal cline in these traits was suggested to be established when they failed to adapt to thermal gradient. Therefore, our findings support the possibility that a lack of genetic variation rather than geneflow swamping is responsible for the constraint of adaptation at the margin of geographic distribution.

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform.

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities.

The Kuroshio current influences genetic diversity and population genetic structure of a tropical seagrass, Enhalus acoroides.

Information on genetic diversity and differentiation of seagrass populations is essential for the conservation of coastal ecosystems. However, little is known about the seagrasses in the Indo-West Pacific Ocean, where the world's highest diversity of seagrasses occurs. The influence of sea currents on these populations is also unknown. We estimated the genetic diversity and population genetic structure and identified reproductive features in Enhalus acoroides populations from the Yaeyama Islands, Hainan Island and the Philippines. The Philippines are situated at the centre of the E. acoroides range, Yaeyama and Hainan are peripheral populations, and the Yaeyama population is at the northern limit of the species range. The powerful Kuroshio Current flows from the Philippines to Yaeyama. Genetic analyses using nine microsatellite markers indicated that reproduction of E. acoroides is mostly sexual. Clonal diversity does not decrease in northern populations, although genetic diversity does. However, the genetic diversity of the Yaeyama populations is greater than that of the Hainan populations. Significant genetic differentiation among most populations was evident; however, the Yaeyama and north-east Philippines populations were genetically similar, despite being separated by ~1100 km. An assignment test suggested that recruitment occurs from the north-east Philippines to Yaeyama. The strong current in this region is probably responsible for the extant genetic diversity and recruitment patterns.

Development of 10 novel polymorphic microsatellite markers for the Indo-Pacific horned starfish, Protoreaster nodosus.

Ten novel polymorphic nuclear microsatellite simple sequence repeat (SSR) markers were isolated from an Indo-Pacific horned starfish, Protoreaster nodosus. The isolated SSR markers provided polymorphisms of 2-9 alleles per locus in three populations obtained from Indonesia and the Philippines. The expected and observed heterozygosities ranged from 0.049 to 0.691 and from 0.050 to 0.800, respectively. Pairwise FST values among three populations ranged from 0.018 to 0.050 (global FST=0.031). All P. nodosus individuals collected from three populations exhibited different genotypes for the ten identified SSR markers, indicating that P. nodosus reproduces sexually. Then the developed SSR markers will be useful for studying the population and conservation genetics of P. nodosus.

Pollination efficiencies of flower-visiting insects as determined by direct genetic analysis of pollen origin.

The amount and genetic composition of pollen grains that are transported to flowers influence the reproduction and fitness of plants. Despite the importance of insect-pollination systems, an understanding of those systems is still lacking due to the absence of a genetic analysis of pollen grains that are transported to flowers. We evaluated the pollination efficiencies of bumblebees (Apidae, Bombus spp.), flower beetles (Scarabaeidae, subfamily Cetoniinae, Protaetia and Eucetonia sp.), and small beetles (Lagriidae, Arthromacra sp.) that visited the flowers of Magnolia obovata (Magnoliaceae) using quantitative (flower visitation frequency, amount of adherent pollen per insect) and qualitative (origin and genetic diversity of adherent pollen per insect) criteria. Most of the pollen adhering to bumblebees and small beetles was self-pollen. This result suggests that visitation by these insects may cause geitonogamous pollen flow and negatively affect the reproduction of M. obovata, causing inbreeding depression. In contrast, flower beetles transported large amounts of genetically diverse outcross pollen. Our results suggest that certain beetle species contribute quantitatively and qualitatively to the pollination of M. obovata. Direct genetic analysis of pollen grains will advance our understanding of plant mating systems and may shed light on the mutualism and coevolution of plants and flower visitors.