Genetics, not environment, contributed to winter wheat yield gains in Ontario, Canada.

KEY MESSAGE: Changes in entries' market classes and genetic improvements within classes-not environmental changes-enhanced yields over thirty-one years of wheat trials. Correlations between yields and ancestries drove genomic prediction accuracies. Increasing crop yields is important for enhancing farmers' livelihoods, meeting market demands, and reducing the environmental impact of agriculture. We analyzed the yield trends of Ontario winter wheat variety trials between 1988 and 2018. Over this period, wheat yields steadily increased by 38 kg ha-1 yr-1, or 0.68% yr-1 relative to the mean. While fungicide treatment of trials contributed a one-time 670 kg ha-1 yield increase, yields were otherwise unaffected by long-term changes in agronomic practice, climate, or other non-genetic factors. Genetic improvement entirely accounted for yield improvement. Market class changes over the 31 year span accounted for some yield improvement. More importantly, genetic improvement occurred within each market class. Entry yield estimates calculated from genomic prediction models strongly correlated with field estimated yields with a mean r of 0.68. Genomic prediction accuracies were high because yields differed across genetically distinct subpopulations. Despite environmental changes, genetic improvement will likely increase Ontario winter wheat yields into the future.

Patterns of stability and change in the maize genome: a case study of small RNA transcriptomes in two recombinant inbred lines and their progenitors.

Small RNAs (sRNAs) are epigenetic regulators of eukaryotic genes and transposable elements (TEs). Diverse sRNA expression patterns exist within a species, but how this diversity arises is not well understood. To provide a window into the dynamics of maize sRNA patterning, sRNA and mRNA transcriptomes were examined in two related Zea mays recombinant inbred lines (RILs) and their inbred parents. Analysis of these RILs revealed that most clusters of sRNA expression retained the parental sRNA expression level. However, expression states that differ from the parental allele were also observed, predominantly reflecting decreases in sRNA expression. When RIL sRNA expression differed from the parental allele, the new state was frequently similar between the two RILs, and similar to the expression state found at the allele in the other parent. Novel sRNA expression patterns, distinct from those of either parent, were rare. Additionally, examination of sRNA expression over TEs revealed one TE family, Gyma, which showed consistent enrichment for RIL sRNA expression differences compared to those found in parental alleles. These findings provide insights into how sRNA silencing might evolve over generations and suggest that further investigation into the molecular nature of sRNA trans regulators is warranted.

The Induction of the Isoflavone Biosynthesis Pathway Is Associated with Resistance to Common Bacterial Blight in Phaseolus vulgaris L.

Xanthomonas axonopodis infects common bean (Phaseolus vulgaris L.) causing the disease common bacterial blight (CBB). The aim of this study was to investigate the molecular and metabolic mechanisms underlying CBB resistance in P. vulgaris. Trifoliate leaves of plants of a CBB-resistant P. vulgaris recombinant inbred line (RIL) and a CBB-susceptible RIL were inoculated with X. axonopodis or water (mock treatment). Leaves sampled at defined intervals over a 48-h post-inoculation (PI) period were monitored for alterations in global transcript profiles. A total of 800 genes were differentially expressed between pathogen and mock treatments across both RILs; approximately half were differentially expressed in the CBB-resistant RIL at 48 h PI. Notably, there was a 4- to 32-fold increased transcript abundance for isoflavone biosynthesis genes, including several isoflavone synthases, isoflavone 2'-hydroxylases and isoflavone reductases. Ultra-high performance liquid chromatography-tandem mass spectrometry assessed leaf metabolite levels as a function of the PI period. The concentrations of the isoflavones daidzein and genistein and related metabolites coumestrol and phaseollinisoflavan were increased in CBB-resistant RIL plant leaves after exposure to the pathogen. Isoflavone pathway transcripts and metabolite profiles were unaffected in the CBB-susceptible RIL. Thus, induction of the isoflavone pathway is associated with CBB-resistance in P. vulgaris.

Introgressed DNA within a Zea mays near-isogenic line displays lower levels of 24nt sRNA expression than the homologous region from the recurrent parent.

Near-isogenic lines (NILs) are classical genetic tools used to dissect the actions of an allele when placed in a uniform genetic background. Although the goal of NIL creation is to examine the effects of a single allele in isolation, DNA linked to the allele is invariably retained and can confound any allele-specific effects. In addition to genetic variation, highly polymorphic species such as Zea mays will contain introgressed polymorphisms encompassing transposable elements (TEs) and the cis-acting small RNA (sRNA) that represses them. Through transcriptomics, we described the differences in sRNA and TE transcriptional expression between a W22-derived introgression and its homologous B73 region. As anticipated, many differences in sRNA expression were observed. Unexpectedly, however, 24nt sRNA expression over the introgressed region was low overall compared to both the homologous B73 region and the rest of the genome. Across the introgression, low sRNA expression was accompanied by increased TE transcription. Possible explanations for the observed trends in sRNA and TE expression across the introgression region are discussed. These findings support the notion that any introgressed allele is in an epigenetic environment distinct from that found at the allele from the recurrent parent. Additionally, these results suggest that further study of sRNA expression levels during the introgression process is warranted.

The effects of crop attributes, selection, and recombination on Canadian bread wheat molecular variation.

Cultivated germplasm provides an opportunity to investigate how crop agronomic traits, selection for major genes, and differences in crossing-over rates drive patterns of allelic variation. To identify how these factors correlated with allelic variation within a collection of cultivated bread wheat (Triticum aestivum L.), we generated genotypes for 388 accessions grown in Canada over the past 170 yr using filtered single nucleotide polymorphism (SNP) calls from an Illumina Wheat iSelect 90K SNP-array. Entries' breeding program, era of release, grain texture, kernel color, and growth habit contributed to allelic differentiation. Allelic diversity and linkage disequilibrium (LD) of markers flanking some major loci known to affect traits such as gluten strength, growth habit, and grain color were consistent with selective sweeps. Nonetheless, some flanking markers of major loci had low LD and high allelic diversity. Positive selection may have acted upon homoeologous genes that had significant enrichment for the gene ontology terms 'response-to-auxin' and 'response-to-wounding.' Long regions of LD, spanning approximately one-third the length of entire chromosomes, were associated with many pericentromeric regions. These regions were also characterized by low diversity. Enhancing recombination across these regions could generate novel allele combinations to accelerate Canadian wheat improvement.

Zea mays RNA-seq estimated transcript abundances are strongly affected by read mapping bias.

BACKGROUND: Genetic variation for gene expression is a source of phenotypic variation for natural and agricultural species. The common approach to map and to quantify gene expression from genetically distinct individuals is to assign their RNA-seq reads to a single reference genome. However, RNA-seq reads from alleles dissimilar to this reference genome may fail to map correctly, causing transcript levels to be underestimated. Presently, the extent of this mapping problem is not clear, particularly in highly diverse species. We investigated if mapping bias occurred and if chromosomal features associated with mapping bias. Zea mays presents a model species to assess these questions, given it has genotypically distinct and well-studied genetic lines. RESULTS: In Zea mays, the inbred B73 genome is the standard reference genome and template for RNA-seq read assignments. In the absence of mapping bias, B73 and a second inbred line, Mo17, would each have an approximately equal number of regulatory alleles that increase gene expression. Remarkably, Mo17 had 2-4 times fewer such positively acting alleles than did B73 when RNA-seq reads were aligned to the B73 reference genome. Reciprocally, over one-half of the B73 alleles that increased gene expression were not detected when reads were aligned to the Mo17 genome template. Genes at dissimilar chromosomal ends were strongly affected by mapping bias, and genes at more similar pericentromeric regions were less affected. Biased transcript estimates were higher in untranslated regions and lower in splice junctions. Bias occurred across software and alignment parameters. CONCLUSIONS: Mapping bias very strongly affects gene transcript abundance estimates in maize, and bias varies across chromosomal features. Individual genome or transcriptome templates are likely necessary for accurate transcript estimation across genetically variable individuals in maize and other species.

The neonicotinoid insecticide thiamethoxam enhances expression of stress-response genes in Zea mays in an environmentally specific pattern.

Recent studies indicate that thiamethoxam (TMX), a neonicotinoid insecticide, can affect plant responses to environmental stressors, such as neighboring weeds. The molecular mechanisms behind both stable and environmentally specific responses to TMX likely involve genes related to defense and stress responses. We investigated the effect of a TMX seed treatment on global gene expression in maize coleoptiles both under normal conditions and under low ratio red to far-red (R:FR) light stress induced by the presence of neighboring plants. The neighboring plant treatment upregulated genes involved in biotic and abiotic stress responses and affected specific photosynthesis and cell-growth related genes. Low R:FR light may enhance maize resistance to herbivores and pathogens. TMX appears to compromise resistance. The TMX treatment stably repressed many genes that encode proteins involved in biotic stress responses, as well as cell-growth genes. Notably, TMX effects on many genes' expression were conditional on the environment. In response to low R:FR, plants treated with TMX engage genes in the JA pathway, as well as other stress-related response pathways. Neighboring weeds may condition TMX-treated plants to become more stress tolerant.

Genomic regions associated with important seed quality traits in food-grade soybeans.

BACKGROUND: The production of soy-based food products requires specific physical and chemical characteristics of the soybean seed. Identification of quantitative trait loci (QTL) associated with value-added traits, such as seed weight, seed protein and sucrose concentration, could accelerate the development of competitive high-protein soybean cultivars for the food-grade market through marker-assisted selection (MAS). The objectives of this study were to identify and validate QTL associated with these value-added traits in two high-protein recombinant inbred line (RIL) populations. RESULTS: The RIL populations were derived from the high-protein cultivar 'AC X790P' (49% protein, dry weight basis), and two high-yielding commercial cultivars, 'S18-R6' (41% protein) and 'S23-T5' (42% protein). Fourteen large-effect QTL (R2 > 10%) were identified associated with seed protein concentration. Of these QTL, seven QTL were detected in both populations, and eight of them were co-localized with QTL associated with either seed sucrose concentration or seed weight. None of the protein-related QTL was found to be associated with seed yield in either population. Sixteen candidate genes with putative roles in protein metabolism were identified within seven of these protein-related regions: qPro_Gm02-3, qPro_Gm04-4, qPro_Gm06-1, qPro_Gm06-3, qPro_Gm06-6, qPro_Gm13-4 and qPro-Gm15-3. CONCLUSION: The use of RIL populations derived from high-protein parents created an opportunity to identify four novel QTL that may have been masked by large-effect QTL segregating in populations developed from diverse parental cultivars. In total, we have identified nine protein QTL that were detected either in both populations in the current study or reported in other studies. These QTL may be useful in the curated selection of new soybean cultivars for optimized soy-based food products.

Evidence for the Accumulation of Nonsynonymous Mutations and Favorable Pleiotropic Alleles During Wheat Breeding.

Plant breeding leads to the genetic improvement of target traits by selecting a small number of genotypes from among typically large numbers of candidate genotypes after careful evaluation. In this study, we first investigated how mutations at conserved nucleotide sites normally viewed as deleterious, such as nonsynonymous sites, accumulated in a wheat, Triticum aestivum, breeding lineage. By comparing a 150 year old ancestral and modern cultivar, we found recent nucleotide polymorphisms altered amino acids and occurred within conserved genes at frequencies expected in the absence of purifying selection. Mutations that are deleterious in other contexts likely had very small or no effects on target traits within the breeding lineage. Second, we investigated if breeders selected alleles with favorable effects on some traits and unfavorable effects on others and used different alleles to compensate for the latter. An analysis of a segregating population derived from the ancestral and modern parents provided one example of this phenomenon. The recent cultivar contains the Rht-B1b green revolution semi-dwarfing allele and compensatory alleles that reduce its negative effects. However, improvements in traits other than plant height were due to pleiotropic loci with favorable effects on traits and to favorable loci with no detectable pleiotropic effects. Wheat breeding appears to tolerate mutations at conserved nucleotide sites and to only select for alleles with both favorable and unfavorable effects on traits in exceptional situations.

Shared and genetically distinct Zea mays transcriptome responses to ongoing and past low temperature exposure.

BACKGROUND: Cold temperatures and their alleviation affect many plant traits including the abundance of protein coding gene transcripts. Transcript level changes that occur in response to cold temperatures and their alleviation are shared or vary across genotypes. In this study we identify individual transcripts and groups of functionally related transcripts that consistently respond to cold and its alleviation. Genes that respond differently to temperature changes across genotypes may have limited functional importance. We investigate if these genes share functions, and if their genotype-specific gene expression levels change in magnitude or rank across temperatures. RESULTS: We estimate transcript abundances from over 22,000 genes in two unrelated Zea mays inbred lines during and after cold temperature exposure. Genotype and temperature contribute to many genes' abundances. Past cold exposure affects many fewer genes. Genes up-regulated in cold encode many cytokinin glucoside biosynthesis enzymes, transcription factors, signalling molecules, and proteins involved in diverse environmental responses. After cold exposure, protease inhibitors and cuticular wax genes are newly up-regulated, and environmentally responsive genes continue to be up-regulated. Genes down-regulated in response to cold include many photosynthesis, translation, and DNA replication associated genes. After cold exposure, DNA replication and translation genes are still preferentially downregulated. Lignin and suberin biosynthesis are newly down-regulated. DNA replication, reactive oxygen species response, and anthocyanin biosynthesis genes have strong, genotype-specific temperature responses. The ranks of genotypes' transcript abundances often change across temperatures. CONCLUSIONS: We report a large, core transcriptome response to cold and the alleviation of cold. In cold, many of the core suite of genes are up or downregulated to control plant growth and photosynthesis and limit cellular damage. In recovery, core responses are in part to prepare for future stress. Functionally related genes are consistently and greatly up-regulated in a single genotype in response to cold or its alleviation, suggesting positive selection has driven genotype-specific temperature responses in maize.

Distinct gene networks modulate floral induction of autonomous maize and photoperiod-dependent teosinte.

Temperate maize was domesticated from its tropical ancestor, teosinte. Whereas temperate maize is an autonomous day-neutral plant, teosinte is an obligate short-day plant that requires uninterrupted long nights to induce flowering. Leaf-derived florigenic signals trigger reproductive growth in both teosinte and temperate maize. To study the genetic mechanisms underlying floral inductive pathways in maize and teosinte, mRNA and small RNA genome-wide expression analyses were conducted on leaf tissue from plants that were induced or not induced to flower. Transcriptome profiles reveal common differentially expressed genes during floral induction, but a comparison of candidate flowering time genes indicates that photoperiod and autonomous pathways act independently. Expression differences in teosinte are consistent with the current paradigm for photoperiod-induced flowering, where changes in circadian clock output trigger florigen production. Conversely, differentially expressed genes in temperate maize link carbon partitioning and flowering, but also show altered expression of circadian clock genes that are distinct from those altered upon photoperiodic induction in teosinte. Altered miRNA399 levels in both teosinte and maize suggest a novel common connection between flowering and phosphorus perception. These findings provide insights into the molecular mechanisms underlying a strengthened autonomous pathway that enabled maize growth throughout temperate regions.

Proanthocyanidin accumulation and transcriptional responses in the seed coat of cranberry beans (Phaseolus vulgaris L.) with different susceptibility to postharvest darkening.

BACKGROUND: Edible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown. RESULTS: Seed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products. CONCLUSION: Proanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.

Changes in light quality alter physiological responses of soybean to thiamethoxam.

MAIN CONCLUSION: The interaction between neighboring weed-induced far-red enriched light and thiamethoxam can significantly alter soybean seedling morphology, nodulation, isoflavone levels, UV-absorbing phenolics, and carbon and nitrogen content. Neonicotinoid insecticides that are widely used on major crop plants can enhance plant growth and yield. Although the underlying mechanism of this enhanced growth and yield is not clear, recent studies suggest that neonicotinoids such as thiamethoxam (TMX) may exert their effects at least in part via signals that involve salicylic acid (SA) and jasmonic acid (JA). In the current research, effects of TMX on morphological and physiological responses of soybean have been compared under far-red-depleted (FR-D) and far-red-enriched (FR-E) light reflected by neighboring weeds. TMX significantly enhanced shoot and root growth but did not prevent stem elongation under FR-E light. Also, TMX did not prevent reductions in shoot carbon content and shoot carbon to nitrogen ratio under FR-E light. Despite similarities between these TMX effects in soybean and those known for SA and JA in other plant species, TMX significantly enhanced root-nodule numbers per plant and levels of root isoflavones malonyl-daidzin and malonyl-genistin under FR-E light only. These results suggest that the combined effect of FR-E light and TMX triggers a mechanism that operates concomitantly to enhance root isoflavones and nodulation in soybean.

Brevis plant1, a putative inositol polyphosphate 5-phosphatase, is required for internode elongation in maize.

In maize (Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields.

Maize (Zea mays) seeds can detect above-ground weeds; thiamethoxam alters the view.

BACKGROUND: Far red light is known to penetrate soil and delay seed germination. Thiamethoxam as a seed treatment has been observed to enhance seed germination. No previous work has explored the effect of thiamethoxam on the physiological response of buried maize seed when germinating in the presence of above-ground weeds. We hypothesised that the changes in red:far red reflected from above-ground weeds would be detected by maize seed phytochrome and delay seed germination by decreasing the level of GA and increasing ABA. We further hypothesised that thiamethoxam would overcome this delay in germination. RESULTS: Thiamethoxam enhanced seed germination in the presence of above-ground weeds by increasing GA signalling and downregulating DELLA protein and ABA signalling genes. An increase in amylase activity and a degradation of starch were also observed. CONCLUSIONS: Far red reflected from the above-ground weeds was capable of penetrating below the soil surface and was detected by maize seed phytochrome. Thiamethoxam altered the effect of far red on seed germination by stimulating GA and inhibiting ABA synthesis. This is the first study to suggest that the mode of action of thiamethoxam involves both GA synthesis and ABA inhibition.

Thiamethoxam as a seed treatment alters the physiological response of maize (Zea mays) seedlings to neighbouring weeds.

BACKGROUND: Thiamethoxam is a broad-spectrum neonicotinoid insecticide that, when applied to seed, has been observed to enhance seedling vigour under environmental stress conditions. Stress created by the presence of neighbouring weeds is known to trigger the accumulation of hydrogen peroxide (H2 O2 ) in maize seedling tissue. No previous work has explored the effect of thiamethoxam as a seed treatment on the physiological response of maize seedlings emerging in the presence of neighbouring weeds. RESULTS: Thiamethoxam was found to enhance seedling vigour and to overcome the expression of typical shade avoidance characteristics in the presence of neighbouring weeds. These results were attributed to maintenance of the total phenolics content, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity and anthocyanin and lignin contents. These findings were also associated with the activation of scavenging genes, which reduced the accumulation of H2 O2 and the subsequent damage caused by lipid peroxidation in maize seedlings originating from treated seeds even when exposed to neighbouring weeds. CONCLUSIONS: These results suggest the possibility of exploring new chemistries and modes of action as novel seed treatments to upregulate free radical scavenging genes and to maintain the antioxidant system within plants. Such an approach may provide an opportunity to enhance crop competitiveness with weeds.

Regulatory motifs identified from a maize developmental coexpression network.

Transcriptional control is an important determinant of plant development, and distinct modules of coordinated genes characterize the maize developmental transcriptome. Upstream regulatory sequences are often the primary factors that control gene expression pattern and abundance. Here, we identify 244 regulatory motifs that are significantly enriched within 24 gene expression modules previously constructed from transcript abundances of 34,876 Zea mays (maize) gene models from embryogenesis to senescence. Within modules, we identify motifs that have not been characterized. In addition, we identify motifs similar to experimentally verified motifs, and the functions of these motifs overlap with predicted module functions. This work demonstrates the power of transcript-level coexpression modules to identify both variants of known regulatory motifs and novel motifs that control a species' developmental transcriptome.

Beyond the single gene: How epistasis and gene-by-environment effects influence crop domestication.

Domestication is a multifaceted evolutionary process, involving changes in individual genes, genetic interactions, and emergent phenotypes. There has been extensive discussion of the phenotypic characteristics of plant domestication, and recent research has started to identify the specific genes and mutational mechanisms that control domestication traits. However, there is an apparent disconnect between the simple genetic architecture described for many crop domestication traits, which should facilitate rapid phenotypic change under selection, and the slow rate of change reported from the archeobotanical record. A possible explanation involves the middle ground between individual genetic changes and their expression during development, where gene-by-gene (epistatic) and gene-by-environment interactions can modify the expression of phenotypes and opportunities for selection. These aspects of genetic architecture have the potential to significantly slow the speed of phenotypic evolution during crop domestication and improvement. Here we examine whether epistatic and gene-by-environment interactions have shaped how domestication traits have evolved. We review available evidence from the literature, and we analyze two domestication-related traits, shattering and flowering time, in a mapping population derived from a cross between domesticated foxtail millet and its wild progenitor. We find that compared with wild progenitor alleles, those favored during domestication often have large phenotypic effects and are relatively insensitive to genetic background and environmental effects. Consistent selection should thus be able to rapidly change traits during domestication. We conclude that if phenotypic evolution was slow during crop domestication, this is more likely due to cultural or historical factors than epistatic or environmental constraints.