A framework genetic map for Miscanthus sinensis from RNAseq-based markers shows recent tetraploidy

Abstract Background Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting large-scale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. Results We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genome-wide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. Conclusions The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion

A framework genetic map for \u3ci\u3eMiscanthus sinensis\u3c/i\u3e from RNAseq-based markers shows recent tetraploidy

Background: Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting largescale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. Results: We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genomewide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. Conclusions: The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion

Achieving tolerant CO₂ electro-reduction catalyst in real water matrix

In order to achieve practical application of electrochemical CO₂ conversion technologies, the development of durable catalyst in real water matrix is essential because the use of catalysts only showing high performance within a well-refined environment cannot guarantee their feasibility in realistic conditions. Here, we report a design strategy for a catalyst, which shows excellent tolerance to deactivation factors, using a carbon-based material under more practical condition implemented by real tap water. Screening analyses on various components in tap water elucidated that the impurity group, which can be deposited on the catalyst surface and impede the active sites, such as copper, zinc, and especially iron are the main factors responsible for deactivation. Based on these findings, the structural modified nitrogen-doped carbon nanotube (denoted as ball mill N-CNT) was adopted as a catalyst design to secure durability. Consequently, the ball mill N-CNT revealed tolerance to the disclosed deactivation factors and showed stable performance during unprecedented long-time of 120 h in tap water media

Molecular characterization and genogrouping of VP1 of aquatic birnavirus GC1 isolated from rockfish Sebastes schlegeli in Korea

The cDNA nucleotide sequence of genome segment B encoding the VP1 protein was determined for the aquatic birnavirus GC1 isolated from the rockfish Sebastes schlegeli in Korea. The VP1 protein of GC1 contains a 2,538 bp open reading frame, which encodes a protein comprising 846 amino acid residues that has a predicted MW of 94 kDa. The sequence contains 6 potential Asn-X-Ser/Thr motifs. Eight potential Ser phosphorylation sites and 1 potential Tyr phophorylation site were also identified. GC1 contains the Leu-Lys-Asn (LKN) motif instead of the typical Gly-Asp-Asp (GDD) motif found in other aquatic birnaviruses. We also identified the GLPYIGKT motif, the putative GTP-binding site at amino acid position 248. In total, the VP1 regions of 22 birnavirus strains were compared for analyzing the genetic relationship among the family Birnaviridae. Based on the deduced amino acid sequences, GC1 was observed to be more closely related to the infectious pancreatic necrosis virus (IPNV) from the USA, Japan, and Korea than the IPNV from Europe. Further, aquatic birnaviruses containing GC1 and IPNV have genogroups that are distinct from those in the genus Avibirnaviruses and Entomo-birnaviruses. The birnavirusstrains were clustered into 5 genogroups based on their amino acid sequences. The marine aquatic birnaviruses (MABVs) containing GC1 were included in the MABV genogroup; the IPNV strains isolated from Korea, Japan, and the USA were included in genogroup 1 and the IPNV strains isolated primarily from Europe were included in genogroup 2. Avibirnaviruses and entomobirnaviruses were included in genogroup 3 and 4, respectively

Induction of Neuronal Death by Microglial AGE-Albumin: Implications for Alzheimer’s Disease

Advanced glycation end products (AGEs) have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders such as Alzheimer’s disease (AD). In this study, we demonstrate that AGE-albumin, the most abundant AGE product in human AD brains, is synthesized in activated microglial cells and secreted into the extracellular space. The rate of AGE-albumin synthesis in human microglial cells is markedly increased by amyloid-β exposure and oxidative stress. Exogenous AGE-albumin upregulates the receptor protein for AGE (RAGE) and augments calcium influx, leading to apoptosis of human primary neurons. In animal experiments, soluble RAGE (sRAGE), pyridoxamine or ALT-711 prevented Aβ-induced neuronal death in rat brains. Collectively, these results provide evidence for a new mechanism by which microglial cells promote death of neuronal cells through synthesis and secretion of AGE-albumin, thereby likely contributing to neurodegenerative diseases such as AD

Genome biology of the paleotetraploid perennial biomass crop Miscanthus

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. x giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Cytogenetics and genome structure in genus Miscanthus, a potential source of bioenergy feedstocks

Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses. Accessions in the genus Miscanthus are potential crop candidates of lignocellulosic biomass for energy production, whose high biomass productivity is attractive as a biofuel feedstock. Miscanthus is not native to the United States and has been introduced as ornamental accessions by private nurseries and research institutes. Comprehensive taxonomic studes have not been conducted on these U.S. accessions. Previous taxonomic studies have been conducted on the genus Miscanthus using morphology and DNA sequence variation. This study (chaper 2) combines information on genome size and ploidy levels and DNA sequence variation to classify Miscanthus accessions to aid potential biomass crop improvement programs and to investigate the evolution of the genus. We observed that Miscanthus accessions fell into 4 groups, including section Miscanthus, section Triarrhena and two groups intermediate between two sections, based on morphology and genome size. Sixteen simple sequence repeat (SSR) primer pairs were selected based on amplification and polymorphism across three genera, Miscanthus, Saccharum and Erianthus. Morphology, genome size and SSR genotyping of 42 accessions including diploid and triploid interspecific hybrid progeny suggested that there are three Miscanthus species (M. sinensis, M. sacchariflorus and M. x giganteus), one M. sacchariflorus variety and one putative hybrid among Miscanthus accessions, which were clearly separated from the other two genera. The species status of M. floridulus remains in question. The evolution of Miscanthus and related genera is discussed based on genome size, ploidy level, cluster analysis and geographical distribution. Based on genome size and chromosome number comparision between Miscanthus and other related genera, we hypothesize large-scale duplications have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. As described in chapter 3, a cross between two M. sinensis accessions, ‘Grosse fontaine’ and ‘Undine’ was made to produce 221 segrating progeny as a mapping population. Simple sequence repeat (SSR) markers from sugarcane expressed sequence tags (EST) and genomic sequences were screened in the two parental M. sinensis accessions. Single nucleotide polymorphism (SNP) markers from deep transcriptome sequencing (RNAseq) were also used for map construction. A total of 210 SSR markers and 658 single nucleotide polymorphism (SNP) markers were validated via segregation in the full sib F1 mapping population. A genetic map for M. sinensis was constructed that was resolved into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis revealed genome-wide duplication in Miscanthus relative to S. bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in sorghum. The M. sinensis map and comparative mapping with sorghum suggests that the genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The genetic map for Miscanthus is useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion. In chapter 4, artificial genome doubling of various Miscanthus accessions with antimototic agents was used to understand the phenotypic responses to whole genome duplication in Miscanthus. Interspecific manipulation of ploidy levels is also a potential strategy for Miscanthus crop improvement to generate superior germplasm and to circumvent reproductive barriers for the introduction of new genetic variation into core germplasm. Therefore, synthetic autotetraploid lines of M. sacchariflorus and M. sinensis, and autoallohexaploid M. x giganteus were produced in tissue culture from oryzalin treatments to seed- and immature inflorescence-derived callus lines. Genome doubling of diploid M. sinensis, M. sacchariflorus, and triploid M. x giganteus to generate tetraploid and hexaploid lines was confirmed by stomata size, nuclear DNA content, and chromosome counts. A putative pentaploid line was also identified among the M. x giganteus synthetic polyploid lines by nuclear DNA content and chromosome counts. Comparisons of phenotypic performance of synthetic polyploid lines with their diploid and triploid progenitors in the greenhouse found species-specific differences in plant tiller number, height, and flowering time among the doubled lines. Stem diameter tended to increase after polyploidization but there were no significant improvement in biomass traits. Under field conditions, M. x giganteus synthetic polyploid lines showed greater phenotypic variation, in terms of plant height, stem diameter and tiller number, than their progenitor lines. Production of synthetic autopolyploid lines displaying significant phenotypic variation suggests that ploidy manipulation can introduce genetic diversity in the limited Miscanthus germplasm currently available in the United States. The role of polyploidization in the evolution and breeding of the genus Miscanthus is discussed

The First Identification of <i>Cryptosporidium parvum</i> Virus-1 (CSpV1) in Hanwoo (<i>Bos taurus coreanae</i>) Calves in Korea

Cryptosporidium is an obligate coccidian parasite that causes enteric diseases in bovine species. A double-stranded RNA virus associated with C. parvum oocysts, Cryptosporidium parvum virus-1 (CSpV1), has been characterized. However, the relationship between the abovementioned coccidian parasite and the virus has not been studied in the context of the known clinical outcomes. This study aimed to characterize the prevalence and molecular traits of CSpV1 in diarrheal feces of Hanwoo (Korean indigenous cattle) calves. Of the 140 fecal samples previously tested for C. parvum, which were obtained from Hanwoo calves aged 60 days, 70 tested positive and 70 tested negative. These samples were included in this study. By using the polymerase chain reaction (PCR) analysis targeting the RdRp gene of CSpV1, we detected CSpV1 in 28 samples (20.0%), with infection rates of 31.4% (22/70) in C. parvum-positive and 8.6% (6/70) in C. parvum-negative samples. CSpV1 samples detected in the same farm were clustered together. To the best of our knowledge, this is the first study to report the prevalence and molecular characteristics of CSpV1 in Hanwoo calves in the Republic of Korea, providing important insights into the relationship between C. parvum and CSpV1 in bovine hosts