Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum (Lib) de Bary (S. sclerotiorum), is one of the most important diseases that causes significant soybean [Glycine max (L.) Merr.] seed yield and quality losses in Canada and globally. Initiation of plant defense mechanisms is crucial for establishing partial resistance to the pathogenic fungus. To understand plant response to S. sclerotiorum, we conducted a temporal (1, 3, and 5 days post-inoculation [DPI]) assessment of gene expression changes in the stem of soybean genotypes with contrasting phenotypic response. We focused on four genes that have been previously reported as associated with SSR partial resistance and are known to be involved in defense-related functions such as cell wall modification, signaling, response to wounding, and response to fungus. The results showed a higher and earlier expression of the genes in partially resistant cultivars compared to the susceptible. Expression of some genes increased up to 11- (Glyma.02G059700) to 16-fold (Glyma.09G232100) by 3 DPI in the partially resistant cultivar, OAC Drayton, while the genes were generally downregulated in the susceptible cultivar, OAC Shire, at the same DPI. This study improves our understanding of expression patterns of genes involved in plant defense against fungal pathogens in soybean. More importantly, the knowledge of genes that are essential in defense against S. sclerotiorum can be used to fine-map the quantitative trait loci for SSR resistance and facilitate accelerated breeding of SSR-resistant cultivars through gene-based marker-assisted selection.

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.

Palmer amaranth is one of the most economically important and widespread weeds of arable land in the United States. Although no populations are currently known to exist in Canada, its distribution has expanded northward such that it is present in many of the states bordering Canada and multiple pathways exist for its introduction. In this short communication, we report on the transport of viable Palmer amaranth seed on imported sweet potato slips. A reproductive pair of Palmer amaranth seedlings were identified from soil accompanying imported sweet potato slips in 2018. Identification was confirmed using species-specific single nucleotide polymorphisms.

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.

Abstract 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 76k 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 found 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.

Soybean [Glycine max (L.) Merr.] is the world's leading legume crop and the largest oilseed crop. It forms a symbiotic relationship with rhizobia bacteria residing in root nodules that provide fixed nitrogen to host plants through symbiotic nitrogen fixation (SNF). In soybean, it has been widely reported that the highest SNF occurs at the pod-filling stage, associated with the peak demand for nitrogen. However, the majority of seed nitrogen is derived from remobilizing root/shoot nitrogen, representing cumulative SNF from the seedling stage to the pre-pod-fill stage. Therefore, the question arises as to whether there has also been selection for improved SNF at these earlier stages, or whether pre-pod-fill SNF traits have drifted. To test this hypothesis, in this study, pre-pod SNF-related traits were evaluated in soybean cultivars that span 100 years of breeding selection in the Canadian Province of Ontario. Specifically, we evaluated SNF traits in 19 pedigree-related historical cultivars and 25 modern cultivars derived from the University of Guelph soybean breeding program. Field trials were conducted at Woodstock, Ontario, Canada in 2016 and 2017, and various SNF-related traits were measured at pre-pod-fill stages (R1-R3), including nitrogen fixation capacity. Considerable variation was observed among Canadian soybean cultivars released over the past 100 years for pre-pod-fill nitrogen fixation. The modern soybean cultivars had similar or moderately higher pre-pod-fill SNF compared to the historical lines in terms of the percentage of nitrogen derived from the atmosphere (%Ndfa) and total shoot fixed nitrogen. These findings suggest that, despite no direct selection by breeders, pre-pod-fill nitrogen fixation, and associated SNF traits have been maintained and possibly improved in modern soybean breeding. However, the low level of pre-pod-fill SNF in some modern cultivars, and generally wide variation observed in SNF between them, suggest some level of genetic drift for this trait in some pedigrees. Specific historical and modern soybean cultivars were identified as potential parents to enable targeted breeding for improved pre-pod-fill SNF. This retrospective study sheds light on our understanding of the impact of decades of recent selective breeding on pre-pod-fill nitrogen fixation traits in soybean in a temperate environment.

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.

1 Abstract Cannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several reports have been published demonstrating a response. Most reports show low levels of regeneration from somatic tissues, but a landmark publication by Lata et al . in 2010 reported regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant biotechnology this protocol has not been used in subsequent papers in the decade since it was published, raising the concern that it is not reproducible. Many researchers are looking to build research programmes in this growing field, and it is important that the reproducibility and robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to ensure they are reproducible across the species. Replication studies in this burgeoning field will help research groups avoid lost time and resources which arise from pursuing protocols that are not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive genotype. However, the most successful shoot induction medium developed in the 2010 paper failed to induce regeneration in any of the cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it is evident that the existing regeneration protocol is not robust and could not be replicated in any of the 10 genotypes tested.

Improvement of agronomic and seed quality traits in soybean [Glycine max (L.) Merr.] occurs in breeding programs as a result of crossing elite soybean genotypes and selection of superior offspring in each cycle. The objective of this study was to evaluate changes in traits over 100 yr of selection in Ontario, Canada, in two soybean breeding programs at the University of Guelph: Guelph Campus (Maturity Group [MG] 0 and I) and Ridgetown Campus (MG II) programs. Fifty‐six Guelph cultivars (released between 1983 and 2016), 46 Ridgetown cultivars (released between 1987 and 2015), and 37 Guelph‐pedigree related historical cultivars (from 1913 to 1985) were grown in field trials over 3 yr (Guelph Campus) and 2 yr (Ridgetown Campus) at two locations per breeding program to measure 13 traits. Yield increased in the Guelph Campus cultivars by 17.1 kg ha−1 yr−1 and by 15.7 kg ha−1 yr−1 in the Ridgetown Campus cultivars, while yield per day of maturity per year of release significantly increased in elite cultivars. Protein in the Ridgetown Campus cultivars increased at 0.76 g kg−1 yr−1, whereas the Guelph Campus cultivars showed no trend. In Ridgetown Campus cultivars 100‐seed weight significantly increased at 0.1 g yr−1. Within the historical cultivars, 100‐seed weight was significantly correlated with both protein (r = 0.50) and oil (r = −0.42). Genotype × trait biplot analyses revealed differential patterns of trait relationships between the elite and historical cultivars. Oil and yield were associated within the Guelph Campus cultivars. The association of 100‐seed weight and protein was only found in the historical cultivars. Yield has increased in both breeding programs while seed traits have been altered over time.

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.

The apparent efficacy of linuron to control pigweeds (Amaranthus spp.) has declined in Ontario, Canada, in past decades, possibly due to resistance. Samples were collected in multiple fields across Ontario with reported linuron failure. These were characterized at the whole-plant and molecular levels. Screening with linuron revealed resistance in six out of nine green pigweed (Amaranthus powellii Wats.) populations and 36 out of 38 populations of redroot pigweed (Amaranthus retroflexus L.). Sequencing of the psbA gene showed resistant plants had mutations conferring resistance to photosystem II (PSII) inhibitors. The most commonly seen mutation was coding for a Val(219)Ile substitution, while other populations had Ala(251)Val or Phe(274)Val. Two populations were documented with a double mutation at Val(219)Ile and Phe(274)Val. All substitutions endowed plants with low to moderate resistance to linuron, with various levels of cross resistance to other PSII inhibitors. The double mutants were characterized by higher levels of resistance to linuron and diuron compared with each single substitution. The widespread failure of linuron to control pigweed species in many carrot fields in Ontario is due to the selection of PSII mutants. This is the first report of double mutation in psbA in any weed species and the first report of Ala(251)Val and Phe(273)Val in pigweed species. The presence of a double mutation is probably the result of continuous selection of plants already resistant due to a single mutation. Our results illustrate the need for diversified weed management strategies in crops where herbicide options are limited.

Genetic hitchhiking methods used to uncover selection signatures related to traits of agronomic importance in crops have primarily been used at the level of domestication by comparing groups of wild germplasm to landraces or elite breeding lines. In this study, two groups of cultivars defined by an elite Canadian soybean cultivar, 'OAC Bayfield', were compared to identify selection signatures related to long-term breeding within a specific region. Cultivars were assigned to either a pre- or post-OAC Bayfield group. Of the 162 simple sequence repeat markers used to genotype members of the pedigree, 14 were fixed and 19 exhibited a selective signature. An in silico analysis compared the results in this study to quantitative trait loci (QTL) reported in SoyBase and showed that 18 out of the 19 markers with a selective signature were associated with at least one QTL. From the 80 QTL associated with the 18 markers, half were related to plant architecture, yield, or maturity. In addition, the number and type of QTL associated with the fixed versus selected loci differed, particularly for yield. Genomic regions exhibiting a selection signature may contain important loci that either need to be conserved for agronomic performance or be targeted for introgressive breeding and germplasm enrichment.

Glyphosate-resistant populations ofConyza canadensishave been spreading at a rapid rate in Ontario, Canada, since first being documented in 2010. Determining the genetic relationship among existing Ontario populations is necessary to understand the spread and selection of the resistant biotypes. The objectives of this study were to: (1) characterize the genetic variation ofC. canadensisaccessions from the province of Ontario using simple sequence repeat (SSR) markers and (2) investigate the molecular mechanism (s) conferring resistance in these accessions. Ninety-eightC. canadensisaccessions were genotyped using 8 SSR markers. Germinable accessions were challenged with glyphosate to determine their dose response, and the sequences of 5-enolpyruvylshikimate-3-phosphate synthase genes 1 and 2 were obtained. Results indicate that a majority of glyphosate-resistant accessions from Ontario possessed a proline to serine substitution at position 106, which has previously been reported to confer glyphosate resistance in other crop and weed species. Accessions possessing this substitution demonstrated notably higher levels of resistance than non–target site resistant (NTSR) accessions from within or outside the growing region and were observed to form a subpopulation genetically distinct from geographically proximate glyphosate-susceptible and NTSR accessions. Although it is unclear whether other non–target site resistance mechanisms are contributing to the levels of resistance observed in target-site resistant accessions, these results indicate that, at a minimum, selection for Pro-106-Ser has occurred in addition to selection for non–target site resistance and has significantly enhanced the levels of resistance to glyphosate inC. canadensisaccessions from Ontario.

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.

ABSTRACTSoybean [Glycine max (L.) Merr.] oil from conventional cultivars typically contains ~30 g kg−1 stearic acid of the total seed oil. Increased stearic acid concentration in the seed oil of soybeans is desirable for certain food and industrial uses. To date a small number of mutants have been developed with increased stearic acid levels three to six times that of normal. At least two such lines were found to possess separate mutations in the delta‐9‐stearoyl‐acyl carrier protein desaturase C gene (SACPD‐C) that dramatically increased seed stearic acid concentration. We now report additional independent mutations in this gene that increase seed stearic acid levels of the soybean germplasm line RG7 to ~116 g kg−1. An F5 recombinant inbred line (RIL) population was developed to determine the relationship between the RG7 SACPD‐C mutation and stearic acid concentration. Transgressive segregation in the progeny of the cross between mutant lines RG7 and RG2 (low palmitic acid) further increases seed stearic acid to almost 180 g kg−1, suggesting the presence of yet another gene having a significant effect on stearic acid accumulation. We also discerned an independent, presumably allelic, mutation within the SACPD‐C gene in line RG8, which has about 106 g kg−1 stearic acid. Molecular markers diagnostic for the RG7 and RG8 SACPD‐C mutations were developed, enabling rapid selection for these mutations in the development of cultivars with increased seed stearic acid content in the seed oil.

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.

ABSTRACTLutein and zeaxanthin are commonly referred to as the macular carotenoids, as they are localized to ocular tissues and their loss is associated with age‐related macular degeneration. Thirty‐four high‐carotenoid (HiC) lines exhibiting uniquely high concentrations of carotenoids resulted from allele mining of the Orange Flint race using traditional breeding techniques and visual selection for deep orange endosperm color. Total carotenoid concentrations of the HiC lines ranged from 50 to 101 μg g−1 dry weight (DW) with lutein and zeaxanthin concentrations as high as 66 and 65 μg g−1 DW, respectively, levels higher than reported in previous germplasm surveys. The HiC lines fall into three classes based on the accumulation of the major carotenoid: “high‐lutein,” “high‐zeaxanthin,” and “balanced.” Significant year effects were observed for carotenoid concentrations but not for profiles. During kernel development the pattern of carotenoid accumulation in the HiC lines did not appear to be different than in yellow corn belt dent lines. Collectively, the HiC lines represented only one y1 haplotype, 10 unique haplotypes of lcyE, and two unique haplotypes at crtRB1 Interestingly, previously identified diagnostic polymorphisms within lcyE did not appear to be useful in distinguishing between high‐lutein and high‐zeaxanthin HiC lines, and high β‐carotene levels were achieved despite the presence of a suboptimal crtRB1 allele. The HiC lines illustrate the utility of mining allelic variation from exotic sources coupled with the power of simple visual selection and the potential limitations of diagnostic polymorphisms.

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.