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Purported benefits of human-grade pet foods include reduced inflammation, enhanced coat quality, and improved gut health, but research is scarce. Therefore, we compared gene expression, skin and coat health measures, and the fecal microbiome of dogs consuming a mildly cooked human-grade or extruded kibble diet. Twenty beagles (BW = 10.25 ± 0.82 kg; age = 3.85 ± 1.84 yr) were used in a completely randomized design. Test diets included: 1) chicken and brown rice recipe [feed-grade; extruded; blue buffalo (BB)]; and 2) chicken and white rice [human-grade; mildly cooked; Just Food for Dogs (JFFD)]. The study consisted of a 4-week baseline when all dogs ate BB, and a 12-week treatment phase when dogs were randomized to either diet (n = 10/group). After the baseline and treatment phases, fresh fecal samples were scored and collected for pH, dry matter (DM), and microbiome analysis; blood samples were collected for gene expression analysis; hair samples were microscopically imaged; and skin was analyzed for delayed-type hypersensitivity (DTH), sebum concentration, hydration status, and transepidermal water loss (TEWL). Data were analyzed as a change from baseline (CFB) using the Mixed Models procedure of SAS (version 9.4). At baseline, fecal pH was higher (P0.05) and hair surface score, superoxide dismutase (SOD) expression, and tumor necrosis factor-α (TNF-α) expression was lower (P0.05) in dogs allotted to JFFD. The decrease in CFB fecal pH and DM was greater (P0.05) in dogs fed JFFD, but fecal scores were not different. The increase in CFB hair surface score was higher (P0.05) in dogs fed JFFD. The decrease in CFB TEWL (back region) was greater (P0.05) in dogs fed JFFD, but TEWL (inguinal and ear regions), hydration status, and sebum concentrations in all regions were not different. Hair cortex scores and DTH responses were not affected by diet. The increase in CFB gene expression of SOD, COX-2, and TNF-α was greater (P0.05) in dogs fed JFFD. PCoA plots based on Bray-Curtis distances of bacterial genera and species showed small shifts over time in dogs fed BB, but dramatic shifts in those fed JFFD. JFFD increased (adj. P0.05) relative abundances of 4 bacterial genera, 11 bacterial species, 68 KEGG pathways, and 167 MetaCyc pathways, and decreased (adj. P0.05) 16 genera, 25 species, 98 KEGG pathways, and 87 MetaCyc pathways. In conclusion, the JFFD diet dramatically shifted the fecal microbiome but had minor effects on skin and coat measures and gene expression.This study tested the effects of a mildly cooked human-grade diet and a feed-grade extruded kibble diet on the fecal microbiome, skin and coat health measures, and expression of genes related to inflammation and oxidative stress in healthy adult dogs. During a 4-week baseline, 20 beagles consumed the kibble diet. After baseline, 10 dogs continued to consume that diet, while 10 dogs consumed the mildly cooked diet for 12 weeks. After baseline and treatment phases, fresh fecal, blood, and hair samples were collected and skin was analyzed. The mildly cooked diet led to lower fecal pH and dry matter percentage, but fecal scores were not affected. The mildly cooked diet dramatically altered the fecal microbiome, shifting the relative abundances of over 30 bacterial species and 165 bacterial metabolic pathways. Measures of skin sebum content and hydration status were not different between groups, but skin water loss was lower in dogs consuming the mildly cooked diet. Baseline and post-treatment gene expression and hair surface scores were noted, but hair cortex and delayed-type hypersensitivity testing were not altered by diet. Our results demonstrate that mildly cooked diets dramatically change the fecal microbiome, but may not impact skin and coat in healthy adult dogs over a short time period.

CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-κB led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntington’s disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.

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., The perennial grass Miscanthus is a promising biomass crop. Here, via genomics and transcriptomics, the authors reveal its allotetraploid origin, characterize gene expression associated with rhizome development and nutrient recycling, and describe the hybrid origin of the triploid M. x giganteus.

Dietary fiber has become increasingly recognized as a key factor in maintaining gastrointestinal health. Dietary fiber sources are often comprised of several different fiber fractions, each with unique physicochemical properties. These properties can have varying physiological effects on the gastrointestinal tract that include modulation of microbiota, production of fermentation-derived metabolites, and laxation. The objectives of this study were 1) to determine the effects of a novel dietary fiber source, miscanthus grass fiber (MF), and prebiotic and fiber blends on gastrointestinal tolerance, apparent total tract digestibility, fecal metabolites, and fecal microbiota and 2) to evaluate the palatability of extruded diets containing MF in comparison to traditional dietary fiber sources. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. Six dietary treatments were formulated to meet or exceed the AAFCO nutrient profile of 2018 and included either cellulose (CO), beet pulp (BP), MF, or a blend of MF and tomato pomace, MF and resistant starch, or MF and fructooligosaccharide. A total of 12 adult neutered female beagles (mean age 5.8 ± 1.1 yr; mean body weight 10.9 ± 1.0 kg; mean body condition score 5.7 ± 0.7) were randomly assigned to one of the six treatment diets in a replicated 6 × 6 Latin square design. Each dog was fed their assigned diet for a treatment period of 21 d with 17 d of diet adaptation followed by 4 d of total and fresh fecal collection. All diets were well accepted and digested by the dogs. Dogs fed BP had greater fecal total short-chain fatty acid concentration than the CO treatment (P0.05), while the dogs fed diets containing MF were intermediate. In a two-bowl palatability trial, no significant preference was observed between the extruded diets containing MF and CO (P0.05). However, a significant preference for the extruded diet containing BP over the diet containing only MF was observed (P0.05). The α-diversity of fecal microbial communities was not impacted by treatment (P0.05), but β-diversity indicated that dogs fed the BP diet differed from the other treatment groups (P0.05). The data from this study suggest that miscanthus grass can be successfully utilized in fiber blends in extruded diets for adult dogs, with modulatory effects similar to the traditional dietary fiber source, cellulose.There are many ingredients utilized in dog foods that provide a source of dietary fiber. However, new ingredient sources can help to add variety to diet formulas and may provide benefits to pet food processing and animal health. Miscanthus grass is a novel ingredient for dog food that provides an excellent source of dietary fiber. In a dog feeding trial and palatability test, diets containing miscanthus grass had similar results to diets containing cellulose, a traditionally used dietary fiber ingredient. The inclusion of this novel ingredient did not produce any observed negative effects on digestion, stool quality, diet palatability, or overall animal health. This indicates that miscanthus grass is a viable ingredient for use in commercial dog foods as a source of dietary fiber.

A recent association between the inclusion of pulses in canine diets and taurine deficiency has become a prevalent issue in the pet food industry. Although dogs do not currently have a nutritional requirement for taurine, taurine deficiencies that do occur can result in serious health issues, such as dilated cardiomyopathy. The objective of this study was to determine the circulating concentrations of plasma and whole blood taurine, indispensable and dispensable amino acid concentrations in the plasma, and taurine and creatinine concentrations in the urine of adult dogs fed a green lentil diet. Twelve adult, intact, female beagles were randomly assigned to a diet containing 45% green lentils (GLD) or a poultry byproduct meal diet (CON) for 90 d. Fresh urine samples were collected every 30 d and analyzed for taurine and creatinine concentrations. A blood sample was also collected every 30 d and analyzed for amino acids including taurine. Animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. All diets were formulated to meet or exceed the nutrient requirements for adult dogs at maintenance. The concentrations of taurine in the plasma and whole blood showed no differences (P > 0.05) between dietary treatments or across time points. Similarly, no differences (P > 0.05) in plasma methionine concentrations were observed between treatments or across time points. A treatment effect (P < 0.05) showed dogs fed GLD had higher total primary fecal bile acid excretion compared with dogs fed CON. The differential abundance of fecal microbial communities showed Firmicutes as the predominant phyla in dogs fed both GLD and CON, with Bacteroidaceae, Erysipelotrichaceae, and Lactobacillaceae as predominant families in dogs fed GLD. The α-diversity of dogs fed GLD (P < 0.05) was lower than in dogs fed CON. These data suggest that the inclusion of 45% green lentil in extruded diets does not lower whole blood and plasma taurine concentrations during a 90-d period and is appropriate for use in a complete and balanced formulation for dogs.

Background Given the economic and environmental importance of allopolyploids and other species with highly duplicated genomes, there is a need for methods to distinguish paralogs, i.e. duplicate sequences within a genome, from Mendelian loci, i.e. single copy sequences that pair at meiosis. The ratio of observed to expected heterozygosity is an effective tool for filtering loci but requires genotyping to be performed first at a high computational cost, whereas counting the number of sequence tags detected per genotype is computationally quick but very ineffective in inbred or polyploid populations. Therefore, new methods are needed for filtering paralogs. Results We introduce a novel statistic, Hind/HE, that uses the probability that two reads sampled from a genotype will belong to different alleles, instead of observed heterozygosity. The expected value of Hind/HE is the same across all loci in a dataset, regardless of read depth or allele frequency. In contrast to methods based on observed heterozygosity, it can be estimated and used for filtering loci prior to genotype calling. In addition to filtering paralogs, it can be used to filter loci with null alleles or high overdispersion, and identify individuals with unexpected ploidy and hybrid status. We demonstrate that the statistic is useful at read depths as low as five to 10, well below the depth needed for accurate genotype calling in polyploid and outcrossing species. Conclusions Our methodology for estimating Hind/HE across loci and individuals, as well as determining reasonable thresholds for filtering loci, is implemented in polyRAD v1.6, available at https://github.com/lvclark/polyRAD. In large sequencing datasets, we anticipate that the ability to filter markers and identify problematic individuals prior to genotype calling will save researchers considerable computational time.

To improve the efficiency of breeding of Miscanthus for biomass yield, there is a need to develop genomics-assisted selection for this long-lived perennial crop by relating genotype to phenotype and breeding value across a broad range of environments. We present the first genome-wide association (GWA) and genomic prediction study of Miscanthus that utilizes multilocation phenotypic data. A panel of 568 Miscanthus sinensis accessions was genotyped with 46,177 single nucleotide polymorphisms (SNPs) and evaluated at one subtropical and five temperate locations over 3 years for biomass yield and 14 yield-component traits. GWA and genomic prediction were performed separately for different years of data in order to assess reproducibility. The analyses were also performed for individual field trial locations, as well as combined phenotypic data across groups of locations. GWA analyses identified 27 significant SNPs for yield, and a total of 504 associations across 298 unique SNPs across all traits, sites, and years. For yield, the greatest number of significant SNPs was identified by combining phenotypic data across all six locations. For some of the other yield-component traits, greater numbers of significant SNPs were obtained from single site data, although the number of significant SNPs varied greatly from site to site. Candidate genes were identified. Accounting for population structure, genomic prediction accuracies for biomass yield ranged from 0.31 to 0.35 across five northern sites and from 0.13 to 0.18 for the subtropical location, depending on the estimation method. Genomic prediction accuracies of all traits were similar for single-location and multilocation data, suggesting that genomic selection will be useful for breeding broadly adapted M. sinensis as well as M. sinensis optimized for specific climates. All of our data, including DNA sequences flanking each SNP, are publicly available. By facilitating genomic selection in M. sinensis and Miscanthus × giganteus, our results will accelerate the breeding of these species for biomass in diverse environments. © 2019 The Authors. GCB Bioenergy Published by John Wiley & Sons Ltd

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.

Dietary fiber plays an important role in gastrointestinal health as it modulates the gut environment and promotes microbiome homeostasis. However, dietary fiber sources vary in composition, physico-chemical properties, and physiological effects. The objective of this study was to evaluate the effects of miscanthus grass fiber and prebiotic fiber blends on fecal microbiota, fecal metabolites, and apparent total tract digestibility in comparison to traditional dietary fiber sources. Animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. Six dietary treatments were formulated to meet or exceed AAFCO nutrient profile (2018) and included either cellulose, beet pulp, miscanthus fiber, or a blend of miscanthus fiber and tomato pomace, miscanthus fiber and resistant starch, or miscanthus fiber and fructooligosaccharide. Twelve female adult beagles were randomly assigned one of the six treatments in a replicated 6x6 Latin square design and fed for 21 d including 17 d of diet adaptation followed by 4 d of total and fresh fecal collection. All diets were well digested by the animals. Dogs fed beet pulp had greater fecal total short-chain fatty acid concentration than the cellulose treatment (P < 0.05), while the dogs fed diets containing miscanthus fiber were intermediate. No difference in the α-diversity of fecal microbial communities was observed among treatments (P >0.05), while β-diversity of dogs fed the beet pulp treatment differed from the other treatment groups. Miscanthus grass can be utilized successfully in diets for adult dogs with tomato pomace and resistant starch blends resulting in similar physiological effects to cellulose.

Schizophrenia is a severe neuropsychiatric disorder associated with a wide array of transcriptomic and neurobiochemical changes. Genome-wide transcriptomic profiling conducted in postmortem brain have provided novel insights into the pathophysiology of this disorder, and identified biological processes including immune/inflammatory-related responses, metabolic, endocrine, and synaptic function. However, few studies have investigated whether similar changes are present in peripheral tissue. Here, we used RNA-sequencing to characterize transcriptomic profiles of lymphocytes in 18 nonpsychotic controls and 19 individuals with schizophrenia. We identified 2819 differentially expressed transcripts (Pnominal < .05) in the schizophrenia group when compared to controls. Bioinformatic analyses conducted on a subset of 293 genes (Pnominal < .01 and |log2 FC| > 0.5) highlighted immune/inflammatory responses as key biological processes in our dataset. Differentially expressed genes in lymphocytes were highly enriched in gene expression profiles associated with cortex layer 5a and immune cells. Thus, we investigated whether the changes in transcripts levels observed in lymphocytes could also be detected in the prefrontal cortex (PFC, BA10) in a second replication cohort of schizophrenia subjects. Remarkably, mRNA levels detected in the PFC and lymphocytes were in strong agreement, and measurements obtained using RNA-sequencing positively correlated with data obtained by reverse transcriptase-quantitative polymerase chain reaction analysis. Collectively, our work supports a role for immune dysfunction in the pathogenesis of schizophrenia and suggests that peripheral markers can be used as accessible surrogates to investigate putative central nervous system disruptions.

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

Honey bees (Apis melliferaL) suffer from many brood pathogens, including viruses. Despite considerable research, the molecular responses and dynamics of honey bee pupae to viral pathogens remain poorly understood. Israeli Acute Paralysis Virus (IAPV) is emerging as a model virus since its association with severe colony losses. Using worker pupae, we studied the transcriptomic and methylomic consequences of IAPV infection over three distinct time points after inoculation. Contrasts of gene expression and 5mC DNA methylation profiles between IAPV-infected and control individuals at these time points—corresponding to the pre-replicative (5 hr), replicative (20 hr), and terminal (48 hr) phase of infection—indicate that profound immune responses and distinct manipulation of host molecular processes accompany the lethal progression of this virus. We identify the temporal dynamics of the transcriptomic response to with more genes differentially expressed in the replicative and terminal phases than in the pre-replicative phase. However, the number of differentially methylated regions decreased dramatically from the pre-replicative to the replicative and terminal phase. Several cellular pathways experienced hyper- and hypo-methylation in the pre-replicative phase and later dramatically increased in gene expression at the terminal phase, including the MAPK, Jak-STAT, Hippo, mTOR, TGF-beta signaling pathways, ubiquitin mediated proteolysis, and spliceosome. These affected biological functions suggest that adaptive host responses to combat the virus are mixed with viral manipulations of the host to increase its own reproduction, all of which are involved in anti-viral immune response, cell growth, and proliferation. Comparative genomic analyses with other studies of viral infections of honey bees and fruit flies indicated that similar immune pathways are shared. Our results further suggest that dynamic DNA methylation responds to viral infections quickly, regulating subsequent gene activities. Our study provides new insights of molecular mechanisms involved in epigenetic that can serve as foundation for the long-term goal to develop anti-viral strategies for honey bees, the most important commercial pollinator.Author SummaryHoney bees, the most important managed pollinators, are experiencing unsustainable mortality. Israeli Acute Paralysis Virus (IAPV) causes economically important disease in honey bees, and it is emerging as a model system to study viral pathogen-host interactions in pollinators. The pupation stage is important for bee development but individuals are particularly vulnerable for parasitic mite infestations and viral infections. Currently, it is unclear how honey bee pupae respond to this virus. However, these responses, including gene expression and DNA methylomic changes, are critical to understand so that anti-viral genes can be identified and new anti-viral strategies be developed. Here, we use next-generation sequencing tools to reveal the dynamic changes of gene expression and DNA methylation as pupae succumb to IAPV infections after 5, 20, and 48 hours. We found that IAPV causes changes in regions of DNA methylation more at the beginning of infection than later. The activity of several common insect immune pathways are affected by the IAPV infections, as are some other fundamental biological processes. Expression of critical enzymes in DNA methylation are also induced by IAPV in a temporal manner. By comparing our results to other virus studies of honey bees and fruit flies, we identified common anti-viral immune responses. Thus, our study provides new insight on the genome responses of honey bees over the course of a fatal virus infection with theoretical and practical implications.

Background and AimsMiscanthus, 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.MethodsCollections 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 ResultsSix 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.ConclusionsGenetic 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.

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.

Improving biomass yield is a major goal of Miscanthus breeding. We conducted a study on one interspecific Miscanthus sinensis × Miscanthus sacchariflorus F1 population and two intraspecific M. sinensis F1 populations, each of which shared a common parent. A field trial was established at Urbana, IL during spring 2011, and phenotypic data were collected in 2012 and 2013 for fourteen yield traits. Six high-density parental genetic maps, as well as a consensus genetic map integrating M. sinensis and M. sacchariflorus, were developed via the pseudotestcross strategy for noninbred parents with ≥1214 single-nucleotide polymorphism markers generated from restriction site-associated DNA sequencing. We confirmed for the first time a whole-genome duplication in M. sacchariflorus relative to Sorghum bicolor, similar to that observed previously for M. sinensis. Four quantitative trait locus (QTL) analysis methods for detecting marker-trait associations were compared: (1) individual parental map composite interval mapping analysis, (2) individual parental map stepwise analysis, (3) consensus map single-population stepwise analysis and (4) consensus map joint-population stepwise analysis. These four methods detected 288, 264, 133 and 109 total QTLs, which resolved into 157, 136, 106 and 86 meta-QTLs based on QTL congruency, respectively, including a set of 59 meta-QTLs common to all four analysis methods. Composite interval mapping and stepwise analysis co-identified 118 meta-QTLs across six parental maps, suggesting high reliability of stepwise regression in QTL detection. Joint-population stepwise analysis yielded the highest resolution of QTLs compared to the other three methods across all meta-QTLs. Strong, frequently advantageous transgressive segregation in the three populations indicated a promising future for breeding new higher-yielding cultivars of Miscanthus.

Although energycane (Saccharum spp.hybrids) is widely used as a source of lignocellulosic biomass for bioethanol, breeding this crop for disease resistance is challenging due to its narrow genetic base. Therefore, efforts are underway to introgress novel sources of genetic resistance fromMiscanthusinto energycane. Given that disease resistance in energycane could be either qualitative or quantitative in nature, careful examination of a wide variety of genomic-enabled breeding approaches will be crucial to the success of such an undertaking. Here we examined the efficiency of both genomic selection (GS) and marker-assisted selection (MAS) for traits simulated under different genetic architectures in F1and BC1populations ofMiscanthus×Miscanthusand sugarcane × sugarcane crosses. We observed that the performance of MAS was comparable and sometimes superior to GS for traits simulated with four quantitative trait nucleotides (QTNs). In contrast, as the number of simulated QTN increased, all four GS models that were evaluated tended to outperform MAS, select more phenotypically optimal F1individuals, and accurately predict simulated trait values in subsequent BC1generations. We therefore conclude that GS is preferable to MAS for introgressing genetic sources of horizontal disease resistance fromMiscanthusto energycane, while MAS remains a suitable option for introgressing vertical disease resistance.

Background and aims Miscanthus is a genus of perennial C4 grasses native to East Asia. It includes the emerging ligno-cellulosic biomass crop M. ×giganteus, a hybrid between M. sinensis and M. sacchariflorus. Biomass yield and cold tolerance are of particular interest in Miscanthus, given that this crop is more temperate adapted than its C4 relatives maize, sorghum and sugarcane. Methods A plant exploration was conducted in eastern Russia, at the northern extreme of the native range for Miscanthus, with collections including 174 clonal germplasm accessions (160 M. sacchariflorus and 14 M. sinensis) from 47 sites. Accessions were genotyped by restriction site-associated DNA sequencing (RAD-seq) and plastid microsatellites. Key Results Miscanthus sinensis was found in maritime climates near Vladivostok (43·6°N) and on southern Sakhalin Island (46·6°N). Miscanthus sacchariflorus was found inland at latitudes as high as 49·3°N, where M. sinensis was absent. Most M. sacchariflorus accessions were diploid, but approx. 2 % were tetraploids. Molecular markers revealed little population structure (Jost’s D < 0·007 among diploid groups) but high genetic diversity (expected heterozygosity = 0·14) within the collection of Russian M. sacchariflorus. Genome-wide association (GWA) analysis for traits measured at the collection sites revealed three M. sacchariflorus single nucleotide polymorphisms (SNPs) significantly associated with the number of stems per unit area, one with height and one with basal stem diameter; three were near or within previously described sorghum quantitative trait loci for related traits. Conclusions This new Miscanthus germplasm collection from eastern Russia will be useful for breeding Miscanthus and sugarcane cultivars with improved adaptation to cold. Moreover, a strategy is proposed to facilitate the rapid utilization of new germplasm collections: by implementing low-cost SNP genotyping to conduct GWA studies of phenotypic data obtained at collection sites, plant breeders can be provided with actionable information on which accessions have desirable traits and alleles.

This is the accepted manuscript of the paper "Breeding progress and preparedness for mass‐scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar", published as final paper in "Global Change Biology Bioenergy Volume 11, Issue 1, 08 February 2019, Pages 118–151 https://doi.org/10.1111/gcbb.12566”. Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output–input energy ratios: namelyPanicum virgatum(switchgrass), species of the generaMiscanthus(miscanthus),Salix(willow) andPopulus(poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed‐based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass‐scale deployment of PBCs.

Highlight Homologues of CONSTANS/Heading date 1 were cloned from Miscanthus sinensis and named MsiHd1. MsiHd1a in most Japanese accessions contained non-functional alleles, whereas Asian mainland accessions harboured only functional alleles., The genus Miscanthus is a perennial C4 grass native to eastern Asia and is a promising candidate bioenergy crop for cool temperate areas. Flowering time is a crucial factor governing regional and seasonal adaptation; in addition, it is also a key target trait for extending the vegetative phase to improve biomass potential. Homologues of CONSTANS (CO)/Heading date 1(Hd1) were cloned from Miscanthus sinensis and named MsiHd1. Sequences of MsiHd1 homologues were compared among 24 wild M. sinensis accessions from Japan, 14 from China, and three from South Korea. Two to five MsiHd1 alleles in each accession were identified, suggesting that MsiHd1 consists of at least three loci in the Miscanthus genome. Verifying the open reading frame in MsiHd1, they were classified as putative functional alleles without mutations or non-functional alleles caused by indels. The Neighbor–Joining tree indicated that one of the multiple MsiHd1 loci is a pseudogene locus without any functional alleles. The pseudogene locus was named MsiHd1b, and the other loci were considered to be part of the MsiHd1a multi-locus family. Interestingly, in most Japanese accessions 50% or more of the MsiHd1a alleles were non-functional, whereas accessions from the East Asian mainland harboured only functional alleles. Five novel miniature inverted transposable elements (MITEs) (MsiMITE1–MsiMITE5) were observed in MsiHd1a/b. MsiMITE1, detected in exon 1 of MsiHd1a, was only observed in Japanese accessions and its revertant alleles derived from retransposition were predominantly in Chinese accessions. These differences in MsiHd1a show that the dependency on functional MsiHd1a alleles is different between accessions from the East Asian mainland and Japan.

Miscanthus × giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type-specimen. A subset of accessions was also evaluated by restriction-site associated DNA sequencing (RAD-seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD-seq, the former is currently more cost-effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type-specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much-needed variation to growers.

In facultatively asexual invasive species, an understanding of the origins and diversity of clones can reveal introduction and invasion pathways and inform management efforts. In this study, we use microsatellite and chloroplast DNA markers to infer clonal diversity of the Rubus fruticosus agg. invasion in the Western United States, determine the relationship of these clones to clones found in other exotic ranges, and determine the geographic and genetic origins of the invasive clones. We found two invasive clones in the Western United States, where the invasion had previously been thought to consist of a single asexual lineage. The most common clone was genetically identical to the microspecies R. armeniacus from the native range of Germany, while the second clone was identical to the microspecies R. anglocandicans in the invaded range of Australia and closely related to samples from the native ranges of England and Serbia. A third distinct clone was identified in a collection from the exotic range of Chile. Our results demonstrate that cryptic genetic diversity may be present in asexual invasions that are thought to be homogeneous. However, the asexual relationships between R. fruticosus agg. clones in the native and multiple exotic ranges indicate that preadaptation has played an important role in invasion success in this species aggregate.

We present an R package to help remedy the lack of software for manipulating and analysing autopolyploid and allopolyploid microsatellite data. POLYSAT can handle genotype data of any ploidy, including populations of mixed ploidy, and assumes that allele copy number is always ambiguous in partial heterozygotes. It can import and export genotype data in eight different formats, calculate pairwise distances between individuals using a stepwise mutation and infinite alleles model, estimate ploidy based on allele counts and estimate allele frequencies and pairwise F(ST) values. This software is freely available through the Comprehensive R Archive Network (http://cran.r-project.org/) and includes a thorough tutorial.

In grapevines (Vitis spp.), the timing of growth cessation in the fall is an important aspect of adaptation and a key objective in breeding new grape cultivars suitable for continental climates. Growth cessation is a complex biological process that is initiated by environmental cues such as daylength and temperature, as well as water and nutrient availability. The genetic control of growth cessation in grapevines was studied by mapping quantitative trait loci (QTL) in a hybrid grape population. An F2 mapping population was developed by selfing a single F1 plant derived from a cross between an accession of the North American species Vitis riparia and the Vitis hybrid wine cultivar Seyval (Seyve-Villard 5–276). A linkage map was constructed using 115 simple sequence repeat (SSR) markers and six candidate genes in a population of 119 F2 progeny. The markers provided coverage of the 19 Vitis linkage groups with an average distance between markers of 8.4 cM. The critical photoperiod for growth cessation in lateral buds for the parents and F2 progeny was determined in a replicated field trial in 2001 and 2002 and under controlled photoperiod treatments in a greenhouse in 2002, 2003, and 2004. QTL analysis using composite interval mapping identified a single major QTL in the field and greenhouse trials. However, the field and greenhouse QTL mapped to different linkage groups in the two different environments, suggesting the presence of additional, nonphotoperiodic cues for induction of growth cessation in the field. In the greenhouse, where noninducing temperatures were maintained, a QTL on linkage group (LG) 13 explained 80.0% to 96.6% of the phenotypic variance of critical photoperiod for growth cessation. In the field, where vines experienced natural fluctuations in temperature and rainfall in addition to the naturally decreasing photoperiod, a QTL on LG 11 explained 85.4% to 94.3% of phenotypic variance.

A major limitation in the analysis of genetic marker data from polyploid organisms is non-Mendelian segregation, particularly when a single marker yields allelic signals from multiple, independently segregating loci (isoloci). However, with markers such as microsatellites that detect more than two alleles, it is sometimes possible to deduce which alleles belong to which isoloci. Here we describe a novel mathematical property of codominant marker data when it is recoded as binary (presence/absence) allelic variables: under random mating in an infinite population, two allelic variables will be negatively correlated if they belong to the same locus, but uncorrelated if they belong to different loci. We present an algorithm to take advantage of this mathematical property, sorting alleles into isoloci based on correlations, then refining the allele assignments after checking for consistency with individual genotypes. We demonstrate the utility of our method on simulated data, as well as a real microsatellite dataset from a natural population of octoploid white sturgeon (Acipenser transmontanus). Our methodology is implemented in the R packagepolysatversion 1.5.