Integrated multi-omics analyses and genome-wide association studies reveal prime candidate genes of metabolic and vegetative growth variation in canola.

Genome-wide association studies (GWAS) identified thousands of genetic loci associated with complex plant traits, including many traits of agronomical importance. However, functional interpretation of GWAS results remains challenging because of large candidate regions due to linkage disequilibrium. High-throughput omics technologies, such as genomics, transcriptomics, proteomics and metabolomics open new avenues for integrative systems biological analyses and help to nominate systems information supported (prime) candidate genes. In the present study, we capitalise on a diverse canola population with 477 spring-type lines which was previously analysed by high-throughput phenotyping of growth-related traits and by RNA sequencing and metabolite profiling for multi-omics-based hybrid performance prediction. We deepened the phenotypic data analysis, now providing 123 time-resolved image-based traits, to gain insight into the complex relations during early vegetative growth and reanalysed the transcriptome data based on the latest Darmor-bzh v10 genome assembly. Genome-wide association testing revealed 61 298 robust quantitative trait loci (QTL) including 187 metabolite QTL, 56814 expression QTL and 4297 phenotypic QTL, many clustered in pronounced hotspots. Combining information about QTL colocalisation across omics layers and correlations between omics features allowed us to discover prime candidate genes for metabolic and vegetative growth variation. Prioritised candidate genes for early biomass accumulation include A06p05760.1_BnaDAR (PIAL1), A10p16280.1_BnaDAR, C07p48260.1_BnaDAR (PRL1) and C07p48510.1_BnaDAR (CLPR4). Moreover, we observed unequal effects of the Brassica A and C subgenomes on early biomass production.

Strong prevalence of light regime-specific QTL in Arabidopsis detected using automated high-throughput phenotyping in fluctuating or constant light.

Plants have evolved and adapted under dynamic environmental conditions, particularly to fluctuating light, but plant research has often focused on constant growth conditions. To quantitatively asses the adaptation to fluctuating light, a panel of 384 natural Arabidopsis thaliana accessions was analyzed in two parallel independent experiments under fluctuating and constant light conditions in an automated high-throughput phenotyping system upgraded with supplemental LEDs. While the integrated daily photosynthetically active radiation was the same under both light regimes, plants in fluctuating light conditions accumulated significantly less biomass and had lower leaf area during their measured vegetative growth than plants in constant light. A total of 282 image-derived architectural and/or color-related traits at six common time points, and 77 photosynthesis-related traits from one common time point were used to assess their associations with genome-wide natural variation for both light regimes. Out of the 3000 significant marker-trait associations (MTAs) detected, only 183 (6.1%) were common for fluctuating and constant light conditions. The prevalence of light regime-specific QTL indicates a complex adaptation. Genes in linkage disequilibrium with fluctuating light-specific MTAs with an adjusted repeatability value >0.5 were filtered for gene ontology terms containing "photo" or "light", yielding 15 selected candidates. The candidate genes are involved in photoprotection, PSII maintenance and repair, maintenance of linear electron flow, photorespiration, phytochrome signaling, and cell wall expansion, providing a promising starting point for further investigations into the response of Arabidopsis thaliana to fluctuating light conditions.

Dynamic growth QTL action in diverse light environments: characterization of light regime-specific and stable QTL in Arabidopsis.

Plant growth is a complex process affected by a multitude of genetic and environmental factors and their interactions. To identify genetic factors influencing plant performance under different environmental conditions, vegetative growth was assessed in Arabidopsis thaliana cultivated under constant or fluctuating light intensities, using high-throughput phenotyping and genome-wide association studies. Daily automated non-invasive phenotyping of a collection of 382 Arabidopsis accessions provided growth data during developmental progression under different light regimes at high temporal resolution. Quantitative trait loci (QTL) for projected leaf area, relative growth rate, and PSII operating efficiency detected under the two light regimes were predominantly condition-specific and displayed distinct temporal activity patterns, with active phases ranging from 2 d to 9 d. Eighteen protein-coding genes and one miRNA gene were identified as potential candidate genes at 10 QTL regions consistently found under both light regimes. Expression patterns of three candidate genes affecting projected leaf area were analysed in time-series experiments in accessions with contrasting vegetative leaf growth. These observations highlight the importance of considering both environmental and temporal patterns of QTL/allele actions and emphasize the need for detailed time-resolved analyses under diverse well-defined environmental conditions to effectively unravel the complex and stage-specific contributions of genes affecting plant growth processes.

Opportunities and limits of controlled-environment plant phenotyping for climate response traits.

Rising temperatures and changing precipitation patterns will affect agricultural production substantially, exposing crops to extended and more intense periods of stress. Therefore, breeding of varieties adapted to the constantly changing conditions is pivotal to enable a quantitatively and qualitatively adequate crop production despite the negative effects of climate change. As it is not yet possible to select for adaptation to future climate scenarios in the field, simulations of future conditions in controlled-environment (CE) phenotyping facilities contribute to the understanding of the plant response to special stress conditions and help breeders to select ideal genotypes which cope with future conditions. CE phenotyping facilities enable the collection of traits that are not easy to measure under field conditions and the assessment of a plant's phenotype under repeatable, clearly defined environmental conditions using automated, non-invasive, high-throughput methods. However, extrapolation and translation of results obtained under controlled environments to field environments is ambiguous. This review outlines the opportunities and challenges of phenotyping approaches under controlled environments complementary to conventional field trials. It gives an overview on general principles and introduces existing phenotyping facilities that take up the challenge of obtaining reliable and robust phenotypic data on climate response traits to support breeding of climate-adapted crops.

Combining next-generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals.

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M2 ) family derived from a heterozygous (M1 ) parent were identified using whole-genome shotgun (WGS) sequencing of a small number of (M2 ) individuals with mutant and wild-type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self-pollinated (M3 ) offspring. Finally, we filtered for segregating EMS-induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M2 ) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non-synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.

A naturally occurring promoter polymorphism of the Arabidopsis FUM2 gene causes expression variation, and is associated with metabolic and growth traits.

Fumarate and malate are known intermediates of the TCA cycle, a mitochondrial metabolic pathway generating NADH for respiration. Arabidopsis thaliana and other Brassicaceae contain an additional cytosolic fumarase (FUM2) that functions in carbon assimilation and nitrogen use. Here, we report the identification of a hitherto unknown FUM2 promoter insertion/deletion (InDel) polymorphism found between the Col-0 and C24 accessions, which also divides a large number of Arabidopsis accessions carrying either the Col-0 or the C24 allele. The polymorphism consists of two stretches of 2.1 and 3.8 kb, which are both absent from the promotor region of Col-0 FUM2. By analysing mutants as well as mapping and natural populations with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate/malate ratio in leaves. In a large population of 174 natural accessions, the polymorphism was also found to be associated with the fumarate/malate ratio, malate and fumarate levels, and with dry weight at 15 days after sowing (DAS). The association with biomass production was confirmed in an even larger (251) accession population for dry weight at 22 DAS. The dominant Col-0 allele that results in increased fumarate/malate ratios and enhanced biomass production is predominantly found in central/eastern European accessions, whereas the C24 type allele is prevalent on the Iberian Peninsula, west of the Rhine and in the British Isles. Our findings support the role of FUM2 in diurnal carbon storage, and point to a growth advantage of accessions carrying the FUM2 Col-0 allele.

Natural plant growth and development achieved in the IPK PhenoSphere by dynamic environment simulation.

In plant science, the suboptimal match of growing conditions hampers the transfer of knowledge from controlled environments in glasshouses or climate chambers to field environments. Here we present the PhenoSphere, a plant cultivation infrastructure designed to simulate field-like environments in a reproducible manner. To benchmark the PhenoSphere, the effects on plant growth of weather conditions of a single maize growing season and of an averaged season over three years are compared to those of a standard glasshouse and of four years of field trials. The single season simulation proves superior to the glasshouse and the averaged season in the PhenoSphere: The simulated weather regime of the single season triggers plant growth and development progression very similar to that observed in the field. Hence, the PhenoSphere enables detailed analyses of performance-related trait expression and causal biological mechanisms in plant populations exposed to weather conditions of current and anticipated future climate scenarios.