Impact of genotype-calling methodologies on genome-wide association and genomic prediction in polyploids.

Discovery and analysis of genetic variants underlying agriculturally important traits are key to molecular breeding of crops. Reduced representation approaches have provided cost-efficient genotyping using next-generation sequencing. However, accurate genotype calling from next-generation sequencing data is challenging, particularly in polyploid species due to their genome complexity. Recently developed Bayesian statistical methods implemented in available software packages, polyRAD, EBG, and updog, incorporate error rates and population parameters to accurately estimate allelic dosage across any ploidy. We used empirical and simulated data to evaluate the three Bayesian algorithms and demonstrated their impact on the power of genome-wide association study (GWAS) analysis and the accuracy of genomic prediction. We further incorporated uncertainty in allelic dosage estimation by testing continuous genotype calls and comparing their performance to discrete genotypes in GWAS and genomic prediction. We tested the genotype-calling methods using data from two autotetraploid species, Miscanthus sacchariflorus and Vaccinium corymbosum, and performed GWAS and genomic prediction. In the empirical study, the tested Bayesian genotype-calling algorithms differed in their downstream effects on GWAS and genomic prediction, with some showing advantages over others. Through subsequent simulation studies, we observed that at low read depth, polyRAD was advantageous in its effect on GWAS power and limit of false positives. Additionally, we found that continuous genotypes increased the accuracy of genomic prediction, by reducing genotyping error, particularly at low sequencing depth. Our results indicate that by using the Bayesian algorithm implemented in polyRAD and continuous genotypes, we can accurately and cost-efficiently implement GWAS and genomic prediction in polyploid crops.

Oral morphine induces spinal 5-hydroxytryptamine (5-HT) release using an opioid receptor-independent mechanism.

Morphine induces spinal 5-hydroxytryptamine (5-HT) release, but the role and mechanism of the spinal 5-HT release induced by morphine are not well understood. The purpose of this study was to define the role and mechanism of spinal 5-HT release induced by oral morphine. We also examined whether persistent pain affected the spinal 5-HT release induced by oral morphine. Spinal 5-HT release was measured using microdialysis of lumbar cerebrospinal fluid (CSF). Two opioids, morphine and oxycodone, were orally administered and 5-HT release was measured in awake rats. Naloxone and ß-funaltrexamine (ß-FNA) were used to determine whether the effect of morphine on 5-HT release was mediated by opioid receptor activation. To study persistent pain, a formalin test was used. At 45 min after oral morphine administration, the formalin test was started and spinal 5-HT release was measured. Oral morphine, but not oral oxycodone, increased 5-HT release at the spinal cord to approximately 4000% of the baseline value. This effect of morphine was not antagonized by either naloxone or ß-FNA at a dose that antagonized the antinociceptive effect of morphine. Formalin-induced persistent pain itself had no effect on spinal 5-HT release but enhanced the oral morphine-induced spinal 5-HT release. Oral morphine-induced spinal 5-HT release was not mediated by opioid receptor activation. Spinal 5-HT induced by oral morphine did not play a major role in the antinociceptive effect of morphine in the hot plate test. Persistent pain increased oral morphine-induced spinal 5-HT release.

Perennial biomass cropping and use: Shaping the policy ecosystem in European countries.

Demand for sustainably produced biomass is expected to increase with the need to provide renewable commodities, improve resource security and reduce greenhouse gas emissions in line with COP26 commitments. Studies have demonstrated additional environmental benefits of using perennial biomass crops (PBCs), when produced appropriately, as a feedstock for the growing bioeconomy, including utilisation for bioenergy (with or without carbon capture and storage). PBCs can potentially contribute to Common Agricultural Policy (CAP) (2023-27) objectives provided they are carefully integrated into farming systems and landscapes. Despite significant research and development (R&D) investment over decades in herbaceous and coppiced woody PBCs, deployment has largely stagnated due to social, economic and policy uncertainties. This paper identifies the challenges in creating policies that are acceptable to all actors. Development will need to be informed by measurement, reporting and verification (MRV) of greenhouse gas emissions reductions and other environmental, economic and social metrics. It discusses interlinked issues that must be considered in the expansion of PBC production: (i) available land; (ii) yield potential; (iii) integration into farming systems; (iv) R&D requirements; (v) utilisation options; and (vi) market systems and the socio-economic environment. It makes policy recommendations that would enable greater PBC deployment: (1) incentivise farmers and land managers through specific policy measures, including carbon pricing, to allocate their less productive and less profitable land for uses which deliver demonstrable greenhouse gas reductions; (2) enable greenhouse gas mitigation markets to develop and offer secure contracts for commercial developers of verifiable low-carbon bioenergy and bioproducts; (3) support innovation in biomass utilisation value chains; and (4) continue long-term, strategic R&D and education for positive environmental, economic and social sustainability impacts.

Oral acetaminophen-induced spinal 5-hydroxytriyptamine release produces analgesic effects in the rat formalin test.

The mechanism by which acetaminophen produces its analgesic effects is not fully understood. One possible mechanism is the activation of the spinal 5-hydroxytryptamine (5-HT) receptor, although direct evidence of spinal 5-HT release has not yet been reported. N-arachidonoylphenolamine (AM404), a metabolite of acetaminophen, is believed to be the key substance that contributes to the analgesic effects of acetaminophen. In this study, we examined whether acetaminophen and AM404 induce spinal 5-HT release and the mechanism through which spinal 5-HT receptor activation exerts analgesic effects in a rat formalin test in an inflammatory pain model. Spinal 5-HT release was examined by intrathecal microdialysis in conscious and freely moving rats. Acetaminophen was administered orally, and AM404 was administered intracerebroventricularly. In rat formalin tests, oral acetaminophen and intracerebroventricular AM404 induced significant spinal 5-HT release and produced analgesic effects. The analgesic effect of oral acetaminophen was partially antagonized by intrathecal administration of WAY100135 (a 5-HT1A receptor antagonist) and SB269970 (a 5-HT7 receptor antagonist). In contrast, the analgesic effect of intracerebroventricular AM404 was completely antagonized by WAY100135, while SB269970 had no effect. Our data suggest that while oral acetaminophen and intracerebroventricular AM404 activate the spinal 5-HT system, the role of the spinal 5-HT system activated by oral acetaminophen differs from that activated by intracerebroventricular AM404.

Analgesic characteristics of a newly developed α2δ ligand, mirogabalin, on inflammatory pain.

Mirogabalin is a novel α2δ ligand approved in Japan for the treatment of peripheral neuropathic pain. However, the sites of action of α2δ ligands to produce analgesic effects on inflammatory pain remain unclear. In this study, we investigated the analgesic effect and site of action of mirogabalin using the rat formalin test, an acute inflammatory pain model. Mirogabalin was administered orally, intrathecally, and intracerebroventricularly. Open field tests were performed to evaluate the effect of oral-, intrathecally, and intracerebroventricularly administered mirogabalin on locomotor activity and orientation ability. Oral mirogabalin produced an analgesic effect when the formalin test was performed 4 h, but not 1 or 2 h, after oral administration. Intrathecal, but not intracerebroventricular, administration of mirogabalin produced analgesic effects when mirogabalin was administered 10 min before formalin injection. These analgesic effects were not antagonized by idazoxan, an α2 adrenergic antagonist; WAY100135, a 5-HT1A antagonist; or naloxone, an opioid receptor antagonist. Mirogabalin attenuated moving distances 1 and 2 h after oral administration and 10 min after intracerebroventricular administration, but not 10 min after intrathecal administration. In the oral administration group, the time course of the analgesic effect was different from that of moving distance. In the intracerebroventricular group, mirogabalin attenuated moving distances but did not produce an analgesic effect. In the intrathecal group, mirogabalin produced an analgesic effect but did not affect moving distances. These findings suggest that the analgesic effect of mirogabalin on the rat formalin test is mediated by spinal action and not by the activation of α2, 5-HT1A, or opioid receptors, and that the inhibitory effect of mirogabalin on moving distances is mediated by the supraspinal brain.

Field Assessment of Six Point-Mutations in SDH Subunit Genes Conferring Varying Resistance Levels to SDHIs in Clarireedia spp.

Dollar spot, caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa F.T. Bennett), is the most economically important turfgrass disease causing considerable damage on golf courses. While cultural practices are available for reducing dollar spot infection, chemical fungicide use is often necessary for maintaining optimal turf quality. Since the release of boscalid in 2003, the succinate dehydrogenase inhibitor (SDHI) class has become an invaluable tool for managing dollar spot. However, resistance to this class has recently been reported in Clarireedia spp. and many other plant pathogenic fungi. After SDHI field failure on four golf courses and one university research plot, a total of six unique SDH mutations conferring differential in vitro sensitivities to SDHIs have been identified in Clarireedia spp. In 2018 and 2019, turf research plots were inoculated with sensitive, non-mutated isolates of Clarireedia spp., as well as resistant isolates harboring each unique identified mutation. Fungicide efficacy trials were conducted on inoculated plots to assess differential sensitivity to five SDHI active ingredients (boscalid, fluxapyroxad, isofetamid, fluopyram, and pydiflumetofen) across mutations under field conditions. Results indicate unique mutations are associated with distinct SDHI field efficacy profiles as shown in in-vitro sensitivity assays. Isolate populations with B subunit mutations (H267Y/R) were more sensitive to fluopyram, whereas isolate populations with C subunit mutations (C-G91R, C-G150R) showed resistance to all SDHIs tested. Mutation-associated differential sensitivity observed under field conditions indicates a need for a nation-wide survey and frequent monitoring of SDHI sensitivity of dollar spot populations on golf courses in the USA. Further, the information gained from this study will be useful in providing sustainable management recommendations for controlling site-specific resistant populations of Clarireedia spp.

Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis.

The optimal flowering time for bioenergy crop Miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in Miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8, a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis, we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species' subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A. The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

Neuropeptide W, an endogenous NPBW1 and NPBW2 ligand, produced an analgesic effect via activation of the descending pain modulatory system during a rat formalin test.

Neuropeptide W (NPW) messenger ribonucleic acid (mRNA) and NPBW1 and/or NPBW2 mRNA are expressed in the descending pain inhibitory system. In the present study, we examined whether NPW microinjected into the descending pain inhibitory system, such as the periaqueductal gray (PAG), locus coeruleus (LC), and rostral ventromedial medulla (RVM), produces an analgesic effect using a rat formalin test. Microinjections of NPW into the PAG ipsilateral and contralateral to the formalin-injected side, LC ipsilateral and contralateral to the formalin-injected side, and RVM produced an analgesic effect. In the RVM study, the analgesic effect was antagonized by WAY100135, a 5-HT1A antagonist, and enhanced by prazosin, an α1 antagonist, and SB269970, a 5-HT7 antagonist. Naloxone, an opioid antagonist, also antagonized the effect of NPW in the RVM study. In the ipsilateral LC study, the analgesic effect was antagonized by WAY100135, idazoxan, an α2 antagonist, and naloxone and was enhanced by prazosin and SB269970. In the contralateral LC study, the analgesic effect was antagonized by prazosin, idazoxan, SB269970, and naloxone. The analgesic effect was antagonized by WAY100135, SB269970, idazoxan, and naloxone in the ipsilateral and contralateral PAG studies. These findings strongly suggest that NPBW1/W2 activation by NPW microinjection into the RVM, LC, and PAG affect the descending pain modulatory system and produce anti-nociceptive and pro-nociceptive effects in the rat formalin test.

Managing flowering time in Miscanthus and sugarcane to facilitate intra- and intergeneric crosses.

Miscanthus is a close relative of Saccharum and a potentially valuable genetic resource for improving sugarcane. Differences in flowering time within and between Miscanthus and Saccharum hinders intra- and interspecific hybridizations. A series of greenhouse experiments were conducted over three years to determine how to synchronize flowering time of Saccharum and Miscanthus genotypes. We found that day length was an important factor influencing when Miscanthus and Saccharum flowered. Sugarcane could be induced to flower in a central Illinois greenhouse using supplemental lighting to reduce the rate at which days shortened during the autumn and winter to 1 min d-1, which allowed us to synchronize the flowering of some sugarcane genotypes with Miscanthus genotypes primarily from low latitudes. In a complementary growth chamber experiment, we evaluated 33 Miscanthus genotypes, including 28 M. sinensis, 2 M. floridulus, and 3 M. ×giganteus collected from 20.9° S to 44.9° N for response to three day lengths (10 h, 12.5 h, and 15 h). High latitude-adapted M. sinensis flowered mainly under 15 h days, but unexpectedly, short days resulted in short, stocky plants that did not flower; in some cases, flag leaves developed under short days but heading did not occur. In contrast, for M. sinensis and M. floridulus from low latitudes, shorter day lengths typically resulted in earlier flowering, and for some low latitude genotypes, 15 h days resulted in no flowering. However, the highest ratio of reproductive shoots to total number of culms was typically observed for 12.5 h or 15 h days. Latitude of origin was significantly associated with culm length, and the shorter the days, the stronger the relationship. Nearly all entries achieved maximal culm length under the 15 h treatment, but the nearer to the equator an accession originated, the less of a difference in culm length between the short-day treatments and the 15 h day treatment. Under short days, short culms for high-latitude accessions was achieved by different physiological mechanisms for M. sinensis genetic groups from the mainland in comparison to those from Japan; for mainland accessions, the mechanism was reduced internode length, whereas for Japanese accessions the phyllochron under short days was greater than under long days. Thus, for M. sinensis, short days typically hastened floral induction, consistent with the expectations for a facultative short-day plant. However, for high latitude accessions of M. sinensis, days less than 12.5 h also signaled that plants should prepare for winter by producing many short culms with limited elongation and development; moreover, this response was also epistatic to flowering. Thus, to flower M. sinensis that originates from high latitudes synchronously with sugarcane, the former needs day lengths >12.5 h (perhaps as high as 15 h), whereas that the latter needs day lengths <12.5 h.

A Rapid Molecular Detection System for SdhB and SdhC Point Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Clarireedia Populations.

Dollar spot, caused by the ascomycete fungus Clarireedia (formerly Sclerotinia), is one of the most resource-demanding diseases on amenity turfgrasses in North America. Differential resistance to the succinate dehydrogenase inhibitor (SDHI) fungicide class, conferred by singular point mutations on the SdhB, SdhC, and SdhD subunits of the succinate dehydrogenase enzyme (SDH), has been reported in dollar spot as well as many other plant-pathogenic fungal diseases. Four unique mutations were previously reported from Clarireedia field isolates collected from two different cool-season golf courses in Japan and Rhode Island: an amino acid substitution H267Y and a silent mutation (CTT to CTC) at codon 181 on the SdhB subunit gene, and amino acid substitutions G91R and G150R on the SdhC subunit gene. To properly diagnose and monitor SDHI resistance in the field, a rapid detection system for known mutations is crucial. As part of this study, additional SDHI-resistant Clarireedia isolates were collected from Rutgers University research plots and in vitro sensitivity to four SDHI active ingredients was assessed. SdhB, SdhC, and SdhD subunits of these isolates were sequenced to reveal an additional mutation on the SdhB subunit gene, H267R, not previously observed in Clarireedia. Cleaved amplified polymorphic sequence (CAPS) and derived CAPS molecular markers were developed to detect five mutations conferring SDHI resistance in Clarireedia isolates and validated using samples from two additional golf courses in Connecticut and Wisconsin experiencing SDHI field failure. This PCR-based molecular detection system will be useful for continued monitoring, assessment, and delay of SDHI resistance in the field.

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. × 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.

Training Population Optimization for Genomic Selection in Miscanthus.

Miscanthus is a perennial grass with potential for lignocellulosic ethanol production. To ensure its utility for this purpose, breeding efforts should focus on increasing genetic diversity of the nothospecies Miscanthus × giganteus (M×g) beyond the single clone used in many programs. Germplasm from the corresponding parental species M. sinensis (Msi) and M. sacchariflorus (Msa) could theoretically be used as training sets for genomic prediction of M×g clones with optimal genomic estimated breeding values for biofuel traits. To this end, we first showed that subpopulation structure makes a substantial contribution to the genomic selection (GS) prediction accuracies within a 538-member diversity panel of predominately Msi individuals and a 598-member diversity panels of Msa individuals. We then assessed the ability of these two diversity panels to train GS models that predict breeding values in an interspecific diploid 216-member M×g F2 panel. Low and negative prediction accuracies were observed when various subsets of the two diversity panels were used to train these GS models. To overcome the drawback of having only one interspecific M×g F2 panel available, we also evaluated prediction accuracies for traits simulated in 50 simulated interspecific M×g F2 panels derived from different sets of Msi and diploid Msa parents. The results revealed that genetic architectures with common causal mutations across Msi and Msa yielded the highest prediction accuracies. Ultimately, these results suggest that the ideal training set should contain the same causal mutations segregating within interspecific M×g populations, and thus efforts should be undertaken to ensure that individuals in the training and validation sets are as closely related as possible.

Collecting wild Miscanthus germplasm in Asia for crop improvement and conservation in Europe whilst adhering to the guidelines of the United Nations' Convention on Biological Diversity.

BACKGROUND AND AIMS: Germplasm with diverse, agronomically relevant traits forms the foundation of a successful plant breeding programme. Since 1993, the United Nations has been advocating the implementation of the Convention on Biological Diversity (CBD) and the subsequent 2002 Bonn Guidelines as international best practice on germplasm collection and use. In 2006, a European team made an expedition to Asia to collect wild germplasm of Miscanthus, a C4 perennial rhizomatous grass, for breeding an environmentally adaptable, resilient and high-yielding bioenergy crop. We outline general aspects of germplasm collection, conservation, breeding and biomass production evaluation while following the CBD's guidelines, respecting biodiversity and conservation needs, and the ethical use of genetic resources. METHODS: Effective protocols, quarantine, methods for collecting seed and rhizomes, and a genebank for conservation were established. Versatile informatics and database architecture were used to assist in selection, flowering synchronization, crossing, evaluation, phenotyping and data integration. Approaches were formulated to comply with the CBD guidelines. KEY RESULTS: A total of 303 accessions of M. sinensis, M. sacchariflorus and M. floridulus were collected from 158 geographically and environmentally diverse locations. These species were shown to accumulate different amounts of aerial biomass due to combinations of stem count, height and thickness. Progeny from one interspecies cross accumulated more biomass in early trials and has shown double the yield performance in years 3-4 compared with the existing commercial cultivar M. × giganteus. An example of an F1 hybrid has already demonstrated the long-term potential of exploiting this collection for a breeding programme. CONCLUSIONS: By conforming to the CBD principles, the authors' international collaboration provides a practical example of implementing the CBD. The collection widened the genetic diversity of Miscanthus available to allow for breeding of novel hybrids that exhibit more diverse traits to increase yield and resilience for growth on marginal land and in climate-challenged environments.

Population structure of Miscanthus sacchariflorus reveals two major polyploidization events, tetraploid-mediated unidirectional introgression from diploid M. sinensis, and diversity centred around the Yellow Sea.

BACKGROUND AND AIMS: Miscanthus, a C4 perennial grass native to East Asia, is a promising biomass crop. Miscanthus sacchariflorus has a broad geographic range, is used to produce paper in China and is one of the parents (along with Miscanthus sinensis) of the important biomass species Miscanthus × giganteus. The largest study of M. sacchariflorus population genetics to date is reported here. METHODS: Collections included 764 individuals across East Asia. Samples were genotyped with 34 605 single nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing (RAD-seq) and ten plastid microsatellites, and were subjected to ploidy analysis by flow cytometry. KEY RESULTS: Six major genetic groups within M. sacchariflorus were identified using SNP data: three diploid groups, comprising Yangtze (M. sacchariflorus ssp. lutarioriparius), N China and Korea/NE China/Russia; and three tetraploid groups, comprising N China/Korea/Russia, S Japan and N Japan. Miscanthus sacchariflorus ssp. lutarioriparius was derived from the N China group, with a substantial bottleneck. Japanese and mainland tetraploids originated from independent polyploidization events. Hybrids between diploid M. sacchariflorus and M. sinensis were identified in Korea, but without introgression into either parent species. In contrast, tetraploid M. sacchariflorus in southern Japan and Korea exhibited substantial hybridization and introgression with local diploid M. sinensis. CONCLUSIONS: Genetic data indicated that the land now under the Yellow Sea was a centre of diversity for M. sacchariflorus during the last glacial maximum, followed by a series of migrations as the climate became warmer and wetter. Overall, M. sacchariflorus has greater genetic diversity than M. sinensis, suggesting that breeding and selection within M. sacchariflorus will be important for the development of improved M. × giganteus. Ornamental M. sacchariflorus genotypes in Europe and North America represent a very narrow portion of the species' genetic diversity, and thus do not well represent the species as a whole.

Resistance of Sclerotinia homoeocarpa Field Isolates to Succinate Dehydrogenase Inhibitor Fungicides.

Sclerotinia homoeocarpa isolates were collected from golf courses in Japan and the United States (2016-2017). Japan isolates were collected during a monitoring study and the U.S. isolates were collected due to field failure. Five succinate dehydrogenase inhibitor (SDHI) active ingredients (boscalid, fluopyram, fluxapyroxad, isofetamid, and penthiopyrad) were examined using in vitro sensitivity assays to determine cross-resistance. Sequence analysis revealed a point mutation leading to an amino acid substitution (H267Y) and a silent mutation (CTT to CTC) at codon 181 in the SdhB subunit gene. Isolates with the B-H267Y (n = 10) mutation were resistant to boscalid and penthiopyrad and had increased sensitivity to fluopyram. SdhB silent mutation 181C>T isolates (n = 2) were resistant to boscalid, isofetamid, and penthiopyrad. Sequence analysis revealed 3 mutations leading to an amino acid substitution (G91R, n = 5; G150R, n = 1; G159W, n = 1) in the SdhC subunit gene. Isolates harboring the SdhC (G91R or G150R) mutations were resistant to boscalid, fluxapyroxad, isofetamid, and penthiopyrad. A genetic transformation system was used to generate mutants from a SDHI sensitive isolate to confirm the B-H267Y and C-G91R mutations were direct determinants of SDHI resistance and associated with in vitro sensitivity assay results.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa.

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the ß-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.

Determination of Leaf Water Content by Visible and Near-Infrared Spectrometry and Multivariate Calibration in Miscanthus.

Leaf water content is one of the most common physiological parameters limiting efficiency of photosynthesis and biomass productivity in plants including Miscanthus. Therefore, it is of great significance to determine or predict the water content quickly and non-destructively. In this study, we explored the relationship between leaf water content and diffuse reflectance spectra in Miscanthus. Three multivariate calibrations including partial least squares (PLS), least squares support vector machine regression (LSSVR), and radial basis function (RBF) neural network (NN) were developed for the models of leaf water content determination. The non-linear models including RBF_LSSVR and RBF_NN showed higher accuracy than the PLS and Lin_LSSVR models. Moreover, 75 sensitive wavelengths were identified to be closely associated with the leaf water content in Miscanthus. The RBF_LSSVR and RBF_NN models for predicting leaf water content, based on 75 characteristic wavelengths, obtained the high determination coefficients of 0.9838 and 0.9899, respectively. The results indicated the non-linear models were more accurate than the linear models using both wavelength intervals. These results demonstrated that visible and near-infrared (VIS/NIR) spectroscopy combined with RBF_LSSVR or RBF_NN is a useful, non-destructive tool for determinations of the leaf water content in Miscanthus, and thus very helpful for development of drought-resistant varieties in Miscanthus.

Determination of hemicellulose, cellulose and lignin content using visible and near infrared spectroscopy in Miscanthus sinensis.

Lignocellulosic components including hemicellulose, cellulose and lignin are the three major components of plant cell walls, and their proportions in biomass crops, such as Miscanthus sinensis, greatly impact feed stock conversion to liquid fuels or bio-products. In this study, the feasibility of using visible and near infrared (VIS/NIR) spectroscopy to rapidly quantify hemicellulose, cellulose and lignin in M. sinensis was investigated. Initially, prediction models were established using partial least squares (PLS), least squares support vector machine regression (LSSVR), and radial basis function neural network (RBF_NN) based on whole wavelengths. Subsequently, 23, 25 and 27 characteristic wavelengths for hemicellulose, cellulose and lignin, respectively, were found to show significant contribution to calibration models. Three determination models were eventually built by PLS, LS-SVM and ANN based on the characteristic wavelengths. Calibration models for lignocellulosic components were successfully developed, and can now be applied to assessment of lignocellulose contents in M. sinensis.

Application of visible and near-infrared spectroscopy to classification of Miscanthus species.

The feasibility of visible and near infrared (NIR) spectroscopy as tool to classify Miscanthus samples was explored in this study. Three types of Miscanthus plants, namely, M. sinensis, M. sacchariflorus and M. fIoridulus, were analyzed using a NIR spectrophotometer. Several classification models based on the NIR spectra data were developed using line discriminated analysis (LDA), partial least squares (PLS), least squares support vector machine regression (LSSVR), radial basis function (RBF) and neural network (NN). The principal component analysis (PCA) presented rough classification with overlapping samples, while the models of Line_LSSVR, RBF_LSSVR and RBF_NN presented almost same calibration and validation results. Due to the higher speed of Line_LSSVR than RBF_LSSVR and RBF_NN, we selected the line_LSSVR model as a representative. In our study, the model based on line_LSSVR showed higher accuracy than LDA and PLS models. The total correct classification rates of 87.79 and 96.51% were observed based on LDA and PLS model in the testing set, respectively, while the line_LSSVR showed 99.42% of total correct classification rate. Meanwhile, the lin_LSSVR model in the testing set showed correct classification rate of 100, 100 and 96.77% for M. sinensis, M. sacchariflorus and M. fIoridulus, respectively. The lin_LSSVR model assigned 99.42% of samples to the right groups, except one M. fIoridulus sample. The results demonstrated that NIR spectra combined with a preliminary morphological classification could be an effective and reliable procedure for the classification of Miscanthus species.

A role for the locus coeruleus in the analgesic efficacy of N-acetylaspartylglutamate peptidase (GCPII) inhibitors ZJ43 and 2-PMPA.

N-acetylaspartylglutamate (NAAG) is the third most prevalent and widely distributed neurotransmitter in the mammalian nervous system. NAAG activates a group II metabotropic glutamate receptor (mGluR3) and is inactivated by an extracellular enzyme, glutamate carboxypeptidase II (GCPII) in vivo. Inhibitors of this enzyme are analgesic in animal models of inflammatory, neuropathic and bone cancer pain. NAAG and GCPII are present in the locus coeruleus, a center for the descending noradrenergic inhibitory pain system. In the formalin footpad model, systemic treatment with GCPII inhibitors reduces both phases of the inflammatory pain response and increases release of spinal noradrenaline. This analgesic efficacy is blocked by systemic injection of a group II mGluR antagonist, by intrathecal (spinal) injection of an alpha 2 adrenergic receptor antagonist and by microinjection of an α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist directly into the contralateral locus coeruleus. Footpad inflammation increases release of glutamate in the contralateral locus coeruleus and systemic treatment with a GCPII inhibitor blocks this increase. Direct injection of GCPII inhibitors into the contralateral or ipsilateral locus coeruleus reduces both phases of the inflammatory pain response in a dose-dependent manner and the contralateral effect also is blocked by intrathecal injection of an alpha 2 adrenergic receptor antagonist. These data support the hypothesis that the analgesic efficacy of systemically administered GCPII inhibitors is mediated, at least in part, by the contralateral locus coeruleus via group II mGluR, AMPA and alpha 2 adrenergic receptors.