Genetic analysis of sucrose concentration in soybean seeds using a historical soybean genomic panel.

KEY MESSAGE: Significant QTL for sucrose concentration have been identified using a historical soybean genomic panel, which could aid in the development of food-grade soybean cultivars. Soybean (Glycine max (L.) Merr) is a crop of global importance for both human and animal consumption, which was domesticated in China more than 6000 years ago. A concern about losing genetic diversity as a result of decades of breeding has been expressed by soybean researchers. In order to develop new cultivars, it is critical for breeders to understand the genetic variability present for traits of interest in their program germplasm. Sucrose concentration is becoming an increasingly important trait for the production of soy-food products. The objective of this study was to use a genome-wide association study (GWAS) to identify putative QTL for sucrose concentration in soybean seed. A GWAS panel consisting of 266 historic and current soybean accessions was genotyped with 76 k genotype-by-sequencing (GBS) SNP data and phenotyped in four field locations in Ontario (Canada) from 2015 to 2017. Seven putative QTL were identified on chromosomes 1, 6, 8, 9, 10, 13 and 14. A key gene related to sucrose synthase (Glyma.06g182700) was found to be associated with the QTL located on chromosome 6. This information will facilitate efforts to increase the available genetic variability for sucrose concentration in soybean breeding programs and develop new and improved high-sucrose soybean cultivars suitable for the soy-food industry.

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study.

Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.

Haplotype diversity underlying quantitative traits in Canadian soybean breeding germplasm.

KEY MESSAGE: Identification of marker-trait associations and trait-associated haplotypes in breeding germplasm identifies regions under selection and highlights changes in haplotype diversity over decades of soybean improvement in Canada. Understanding marker-trait associations using genome-wide association in soybean is typically carried out in diverse germplasm groups where identified loci are often not applicable to soybean breeding efforts. To address this challenge, this study focuses on defining marker-trait associations in breeding germplasm and studying the underlying haplotypes in these regions to assess genetic change through decades of selection. Phenotype data were generated for 175 accessions across multiple environments in Ontario, Canada. A set of 76,549 SNPs were used in the association analysis. A total of 23 genomic regions were identified as significantly associated with yield (5), days to maturity (5), seed oil (3), seed protein (5) and 100-seed weight (5), of which 14 are novel. Each significant region was haplotyped to assess haplotype diversity of the underlying genomic region, identifying ten regions with trait-associated haplotypes in the breeding germplasm. The range of genomic length for these regions (7.2 kb to 6.8 Mb) indicates variation in regional LD for the trait-associated regions. Six of these regions showed changes between eras of breeding, from historical to modern and experimental soybean accessions. Continued selection on these regions may necessitate introgression of novel parental genetic diversity as some haplotypes were fixed within the breeding germplasm. This finding highlights the importance of studying associations and haplotype diversity at a breeding program scale to understand breeders' selections and trends in soybean improvement over time. The haplotypes may also be used as a tool for selection of parental germplasm to inform breeder's decisions on further soybean improvement.

Genome-wide genetic diversity is maintained through decades of soybean breeding in Canada.

KEY MESSAGE: Genetic diversity in Canadian soybean is maintained over decades of selection in two public breeding programs. Breeders have used a portion of the genetic diversity available in germplasm collections. Both public and private breeding efforts have been critical for the development of soybean cultivars grown around the world. Global genetic diversity of soybean has been well characterized; however, this diversity is not well studied at the breeding program scale. The objective of this study was to characterize genetic diversity over decades of breeding in two public soybean breeding programs at the University of Guelph, Canada. To address this objective, a pedigree-related panel combining 296 soybean accessions from the Ridgetown and Guelph Campus breeding programs was studied. The accessions were genotyped using genotyping-by-sequencing, imputed using the GmHapMap reference genotypes resulting in more than 3.8M SNPs, further filtered to 77k SNPs. Population structure analysis did not identify structure between the breeding programs and historical germplasm. The linkage disequilibrium decay ranged from 400 to 600 kb on average in euchromatic regions. Nucleotide diversity over decades of breeding shows that historical accessions had the highest nucleotide diversity, with significant decreases corresponding to the initial breeding activity in Canada; however, genetic diversity has increased in the last 20 years in both breeding programs. Maturity gene E2 was nearly fixed for e2 in Ridgetown accessions, while unfixed in Guelph accessions. Comparison of the breeding programs to the USDA germplasm collection reveals that breeders have only used a portion of the available genetic diversity, allowing future breeders to exploit this untapped resource. The approach used in this study may be of interest to other breeding programs for evaluating changes in genetic diversity resulting from breeding activities.

Assessment of the clinical effects of cholesteryl ester transfer protein inhibition with evacetrapib in patients at high-risk for vascular outcomes: Rationale and design of the ACCELERATE trial.

BACKGROUND: Potent pharmacologic inhibition of cholesteryl ester transferase protein by the investigational agent evacetrapib increases high-density lipoprotein cholesterol by 54% to 129%, reduces low-density lipoprotein cholesterol by 14% to 36%, and enhances cellular cholesterol efflux capacity. The ACCELERATE trial examines whether the addition of evacetrapib to standard medical therapy reduces the risk of cardiovascular (CV) morbidity and mortality in patients with high-risk vascular disease. STUDY DESIGN: ACCELERATE is a phase 3, multicenter, randomized, double-blind, placebo-controlled trial. Patients qualified for enrollment if they have experienced an acute coronary syndrome within the prior 30 to 365 days, cerebrovascular accident, or transient ischemic attack; if they have peripheral vascular disease; or they have diabetes with coronary artery disease. A total of 12,092 patients were randomized to evacetrapib 130 mg or placebo daily in addition to standard medical therapy. The primary efficacy end point is time to first event of CV death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization. Treatment will continue until 1,670 patients reached the primary end point; at least 700 patients reach the key secondary efficacy end point of CV death, myocardial infarction, and stroke, and the last patient randomized has been followed up for at least 1.5 years. CONCLUSIONS: ACCELERATE will establish whether the cholesteryl ester transfer protein inhibition by evacetrapib improves CV outcomes in patients with high-risk vascular disease.

Characterization of the genetic changes in a multi-generational pedigree of an elite Canadian soybean cultivar.

Genotyping through the pedigrees of elite soybean [Glycine max (L.) Merr.] cultivars developed by a breeding program represents an opportunity to explore and characterize various molecular and genetic changes that are a direct result of long-term selection by soybean breeders. For soybeans bred for Ontario Canada, one such elite cultivar was OAC Bayfield, which had exceptional commercial success as well as being a parent of a number of successful cultivars developed by multiple independent breeding programs. A total of 42 genotypes from six different breeding programs, comprising the multi-generational pedigree of OAC Bayfield were genotyped with molecular markers and chromosomal inheritance was tracked throughout the pedigree. Cluster analysis showed high congruence with the known pedigree and identified three distinct ancestral groups. The ancestral genotypes contained the majority of the rare alleles, with the cultivar CNS having the greatest number of unique alleles. The graphical genotype profile for the 20 chromosomes revealed conserved allelic composition which has been assembled in certain chromosomes in the form of specific linkage blocks, which were either a result of recombination involving ancestral linkage blocks or linkage blocks introduced from the cultivar Fiskeby-V. The identification of highly structured, conserved genomic regions are important for future breeding efforts as they are indicators of preferentially selected regions, or conversely, may be a contributing factor to low genetic gains due to mass fixation across a breeding program's germplasm.

Heterosis for carotenoid concentration and profile in maize hybrids.

Production of high-lutein maize grain is of particular interest as a value-added feed source to produce high-lutein eggs. In this paper, it is demonstrated that heterosis for total carotenoid concentration and for the ratio of lutein to zeaxanthin (L:Z ratio), or profile type, exists infrequently in yellow dent crosses. However, yellow dent inbred maize lines A619 and CG102, both possessing high-lutein profiles, produce F1 seed with a classic overdominant expression of lutein levels (i.e., 49 µg/g dry weight (DW) above the high-parent value). Reciprocal crosses of A619 and CG102 with one another and with two high-zeaxanthin (i.e., low lutein), high-carotenoid lines both suggest that the A619 and CG102 high-lutein phenotypes are achieved by different and complementary genotypes. The contribution of CG102 to the heterotic response was examined using a QTL-based approach that involved phenotyping the mapping population in a testcross to A619. Significant QTL were found at loci known to be involved in the carotenoid pathway but also at loci proximate to, but separate from, known carotenoid pathway steps. Exploiting an overdominant heterotic response for lutein and total carotenoids should be given strong consideration as a viable method of producing high-carotenoid hybrid maize lines.

Impact of postharvest handling on carotenoid concentration and composition in high-carotenoid maize (Zea mays L.) kernels.

High carotenoid maize is an ideal source of high value dietary carotenoids, especially lutein and zeaxanthin, in human and animal feed and has been proposed as a feedstock for high carotenoid egg production. A modified analytical method was demonstrated to have reliability, reproducibility, and improved run-time and separation of xanthophylls. This method was used to confirm the localization of carotenoids in endosperm and to determine the effects of drying and storage on carotenoid levels in maize grain. A preliminary trial using room temperature drying indicated that while carotenoid profiles remain stable during storage, carotenoid levels decrease significantly from initial levels between 3 and 6 months of storage, but then remain stable for another year. A more rigorous trial using three drying and storage regimes (freeze-drying and storage at -80 degrees C; room temperature drying and storage; 90 degrees C drying and room temperature storage) indicated that extreme caution is needed to maintain carotenoid levels in maize during handling and storage, but in situations where freeze-drying is not possible, high heat drying is no more detrimental than low heat drying.

Chitosan nanoparticles are compatible with respiratory epithelial cells in vitro.

The aim of this work was to evaluate the biocompatibility of novel respirable powder formulations of nanoparticles (NP) entrapped in mannitol microspheres using human respiratory epithelial cell lines. Microspheres formulated at NP:mannitol ratios of 10:90, 20:80 and 40:60 were evaluated using the Calu-3 and A549 cell lines. The MTT cell viability assay revealed an absence of overt toxicity to Calu-3 or A549 cells following exposure to the formulations containing <1.3mg NP/ml (equivalent to 0.87 mg NP/cm(2)) for up to 48 h. Transepithelial electrical resistance (TER) and solute permeability in Calu-3 cell layers were determined following exposure of the cells to the NP:mannitol 20:80 formulation. After administration of the formulation dissolved in serum-free cell culture medium (1.3mg/ml NP suspension) to the cells, neither TER nor permeability were altered compared to untreated cell layers. Confocal microscopy did not reveal any NP internalisation under the conditions used in this study, although evidence of mucoadhesion was observed. All the data presented are encouraging with respect to the development of chitosan NP-containing microspheres for the pulmonary administration of therapeutic macromolecules. Not only do the formulations possess suitable aerodynamic characteristics and the capacity to encapsulate proteins as shown previously; they have now been shown to exhibit in vitro biocompatibility.

Measles vaccination of macaques by dry powder inhalation.

Measles vaccination via the aerosol route has proven effective under field conditions, using vaccine reconstituted prior to nebulization. Inhalation of a dry powder aerosol vaccine would have additional benefits, including easier logistics of administration, reduced cold chain dependence and the potential of single dose administration. We have evaluated two candidate dry powder measles vaccine formulations in macaques. Specific immune responses were demonstrated, but levels of immunity were lower than in animals vaccinated by injection or by nebulized aerosol. These studies provide proof of principle that dry powder inhalation is a possible route for measles vaccination, but suggest that either the vaccine formulation or the method of delivery need to be improved for a better immune response.

Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier.

PURPOSE: The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function. METHODS: Cell layers were grown on 0.33 cm2 Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue. RESULTS: Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 ohms cm2 at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 ohms cm2 at 11-13 days. Apparent permeability (P(app)) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively. CONCLUSION: Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.