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.

Temporal dynamics of QTL effects on vegetative growth in Arabidopsis thaliana.

We assessed early vegetative growth in a population of 382 accessions of Arabidopsis thaliana using automated non-invasive high-throughput phenotyping. All accessions were imaged daily from 7 d to 18 d after sowing in three independent experiments and genotyped using the Affymetrix 250k SNP array. Projected leaf area (PLA) was derived from image analysis and used to calculate relative growth rates (RGRs). In addition, initial seed size was determined. The generated datasets were used jointly for a genome-wide association study that identified 238 marker-trait associations (MTAs) individually explaining up to 8% of the total phenotypic variation. Co-localization of MTAs occurred at 33 genomic positions. At 21 of these positions, sequential co-localization of MTAs for 2-9 consecutive days was observed. The detected MTAs for PLA and RGR could be grouped according to their temporal expression patterns, emphasizing that temporal variation of MTA action can be observed even during the vegetative growth phase, a period of continuous formation and enlargement of seemingly similar rosette leaves. This indicates that causal genes may be differentially expressed in successive periods. Analyses of the temporal dynamics of biological processes are needed to gain important insight into the molecular mechanisms of growth-controlling processes in plants.

Multi-omics-based prediction of hybrid performance in canola.

KEY MESSAGE: Complementing or replacing genetic markers with transcriptomic data and use of reproducing kernel Hilbert space regression based on Gaussian kernels increases hybrid prediction accuracies for complex agronomic traits in canola. In plant breeding, hybrids gained particular importance due to heterosis, the superior performance of offspring compared to their inbred parents. Since the development of new top performing hybrids requires labour-intensive and costly breeding programmes, including testing of large numbers of experimental hybrids, the prediction of hybrid performance is of utmost interest to plant breeders. In this study, we tested the effectiveness of hybrid prediction models in spring-type oilseed rape (Brassica napus L./canola) employing different omics profiles, individually and in combination. To this end, a population of 950 F1 hybrids was evaluated for seed yield and six other agronomically relevant traits in commercial field trials at several locations throughout Europe. A subset of these hybrids was also evaluated in a climatized glasshouse regarding early biomass production. For each of the 477 parental rapeseed lines, 13,201 single nucleotide polymorphisms (SNPs), 154 primary metabolites, and 19,479 transcripts were determined and used as predictive variables. Both, SNP markers and transcripts, effectively predict hybrid performance using (genomic) best linear unbiased prediction models (gBLUP). Compared to models using pure genetic markers, models incorporating transcriptome data resulted in significantly higher prediction accuracies for five out of seven agronomic traits, indicating that transcripts carry important information beyond genomic data. Notably, reproducing kernel Hilbert space regression based on Gaussian kernels significantly exceeded the predictive abilities of gBLUP models for six of the seven agronomic traits, demonstrating its potential for implementation in future canola breeding programmes.

Strong temporal dynamics of QTL action on plant growth progression revealed through high-throughput phenotyping in canola.

A major challenge of plant biology is to unravel the genetic basis of complex traits. We took advantage of recent technical advances in high-throughput phenotyping in conjunction with genome-wide association studies to elucidate genotype-phenotype relationships at high temporal resolution. A diverse Brassica napus population from a commercial breeding programme was analysed by automated non-invasive phenotyping. Time-resolved data for early growth-related traits, including estimated biovolume, projected leaf area, early plant height and colour uniformity, were established and complemented by fresh and dry weight biomass. Genome-wide SNP array data provided the framework for genome-wide association analyses. Using time point data and relative growth rates, multiple robust main effect marker-trait associations for biomass and related traits were detected. Candidate genes involved in meristem development, cell wall modification and transcriptional regulation were detected. Our results demonstrate that early plant growth is a highly complex trait governed by several medium and many small effect loci, most of which act only during short phases. These observations highlight the importance of taking the temporal patterns of QTL/allele actions into account and emphasize the need for detailed time-resolved analyses to effectively unravel the complex and stage-specific contributions of genes affecting growth processes that operate at different developmental phases.

SIEVE ELEMENT-LINING CHAPERONE1 Restricts Aphid Feeding on Arabidopsis during Heat Stress.

The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural Arabidopsis thaliana accessions, we identified the small heat shock-like SIEVE ELEMENT-LINING CHAPERONE1 (SLI1). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On sli1 mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when SLI1 expression is upregulated. At this moderately high temperature, sli1 mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress.

Genetic controls of short- and long-term stomatal CO2 responses in Arabidopsis thaliana.

BACKGROUND AND AIMS: The stomatal conductance (gs) of most plant species decreases in response to elevated atmospheric CO2 concentration. This response could have a significant impact on plant water use in a future climate. However, the regulation of the CO2-induced stomatal closure response is not fully understood. Moreover, the potential genetic links between short-term (within minutes to hours) and long-term (within weeks to months) responses of gs to increased atmospheric CO2 have not been explored. METHODS: We used Arabidopsis thaliana recombinant inbred lines originating from accessions Col-0 (strong CO2 response) and C24 (weak CO2 response) to study short- and long-term controls of gs. Quantitative trait locus (QTL) mapping was used to identify loci controlling short- and long-term gs responses to elevated CO2, as well as other stomata-related traits. KEY RESULTS: Short- and long-term stomatal responses to elevated CO2 were significantly correlated. Both short- and long-term responses were associated with a QTL at the end of chromosome 2. The location of this QTL was confirmed using near-isogenic lines and it was fine-mapped to a 410-kb region. The QTL did not correspond to any known gene involved in stomatal closure and had no effect on the responsiveness to abscisic acid. Additionally, we identified numerous other loci associated with stomatal regulation. CONCLUSIONS: We identified and confirmed the effect of a strong QTL corresponding to a yet unknown regulator of stomatal closure in response to elevated CO2 concentration. The correlation between short- and long-term stomatal CO2 responses and the genetic link between these traits highlight the importance of understanding guard cell CO2 signalling to predict and manipulate plant water use in a world with increasing atmospheric CO2 concentration. This study demonstrates the power of using natural variation to unravel the genetic regulation of complex traits.

Genetic diversity for nitrogen use efficiency in Arabidopsis thaliana.

MAIN CONCLUSION: The plasticity of plant growth response to differing nitrate availability renders the identification of biomarkers difficult, but allows access to genetic factors as tools to modulate root systems to a wide range of soil conditions. Nitrogen availability is a major determinant of crop yield. While the application of fertiliser substantially increases the yield on poor soils, it also causes nitrate pollution of water resources and high costs for farmers. Increasing nitrogen use efficiency in crop plants is a necessary step to implement low-input agricultural systems. We exploited the genetic diversity present in the worldwide Arabidopsis thaliana population to study adaptive growth patterns and changes in gene expression associated with chronic low nitrate stress, to identify biomarkers associated with good plant performance under low nitrate availability. Arabidopsis accessions were grown on agar plates with limited and sufficient supply of nitrate to measure root system architecture as well as shoot and root fresh weight. Differential gene expression was determined using Affymetrix ATH1 arrays. We show that the response to differing nitrate availability is highly variable in Arabidopsis accessions. Analyses of vegetative shoot growth and root system architecture identified accession-specific reaction modes to cope with limited nitrate availability. Transcription and epigenetic factors were identified as important players in the adaption to limited nitrogen in a global gene expression analysis. Five nitrate-responsive genes emerged as possible biomarkers for NUE in Arabidopsis. The plasticity of plant growth in response to differing nitrate availability in the substrate renders the identification of morphological and molecular features as biomarkers difficult, but at the same time allows access to a multitude of genetic factors which can be used as tools to modulate and adjust root systems to a wide range of soil conditions.

Accelerated flowering time reduces lifetime water use without penalizing reproductive performance in Arabidopsis.

Natural selection driven by water availability has resulted in considerable variation for traits associated with drought tolerance and leaf-level water-use efficiency (WUE). In Arabidopsis, little is known about the variation of whole-plant water use (PWU) and whole-plant WUE (transpiration efficiency). To investigate the genetic basis of PWU, we developed a novel proxy trait by combining flowering time and rosette water use to estimate lifetime PWU. We validated its usefulness for large-scale screening of mapping populations in a subset of ecotypes. This parameter subsequently facilitated the screening of water use and drought tolerance traits in a recombinant inbred line population derived from two Arabidopsis accessions with distinct water-use strategies, namely, C24 (low PWU) and Col-0 (high PWU). Subsequent quantitative trait loci mapping and validation through near-isogenic lines identified two causal quantitative trait loci, which showed that a combination of weak and nonfunctional alleles of the FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) genes substantially reduced plant water use due to their control of flowering time. Crucially, we observed that reducing flowering time and consequently water use did not penalize reproductive performance, as such water productivity (seed produced per unit of water transpired) improved. Natural polymorphisms of FRI and FLC have previously been elucidated as key determinants of natural variation in intrinsic WUE (δ13 C). However, in the genetic backgrounds tested here, drought tolerance traits, stomatal conductance, δ13 C. and rosette water use were independent of allelic variation at FRI and FLC, suggesting that flowering is critical in determining lifetime PWU but not always leaf-level traits.

Century-scale methylome stability in a recently diverged Arabidopsis thaliana lineage.

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.

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.

QTL analysis of the developmental response to L-glutamate in Arabidopsis roots and its genotype-by-environment interactions.

Primary root growth in Arabidopsis and a number of other species has previously been shown to be remarkably sensitive to the presence of external glutamate, with glutamate signalling eliciting major changes in root architecture. Using two recombinant inbred lines from reciprocal crosses between Arabidopsis accessions C24 and Col-0, we have identified one large-effect quantitative trait locus (QTL), GluS1, and two minor QTLs, GluS2 and GluS3, which together accounted for 41% of the phenotypic variance in glutamate sensitivity. The presence of the GluS1 locus on chromosome 3 was confirmed using a set of C24/Col-0 isogenic lines. GluS1 was mapped to an interval between genes At3g44830-At3g46880. When QTL mapping was repeated under a range of environmental conditions, including temperature, shading and nitrate supply, a strong genotype-by-environment interaction in the controls for the glutamate response was identified. Major differences in the loci controlling this trait were found under different environmental conditions. Here we present evidence for the existence of loci on chromosomes 1 and 5 epistatically controlling the response of the GluS1 locus to variations in ambient temperature, between 20°C and 26°C. In addition, a locus on the long arm of chromosome 1 was found to play a major role in controlling the ability of external nitrate signals to antagonize the glutamate effect. We conclude that there are multiple loci controlling natural variation in glutamate sensitivity in Arabidopsis roots and that epistatic interactions play an important role in modulating glutamate sensitivity in response to changes in environmental conditions.

The ABI4-induced Arabidopsis ANAC060 transcription factor attenuates ABA signaling and renders seedlings sugar insensitive when present in the nucleus.

Seedling establishment is inhibited on media containing high levels (∼ 6%) of glucose or fructose. Genetic loci that overcome the inhibition of seedling growth on high sugar have been identified using natural variation analysis and mutant selection, providing insight into sugar signaling pathways. In this study, a quantitative trait locus (QTL) analysis was performed for seedling sensitivity to high sugar in a Col/C24 F2 population of Arabidopsis thaliana. A glucose and fructose-sensing QTL, GSQ11, was mapped through selective genotyping and confirmed in near-isogenic lines in both Col and C24 backgrounds. Allelism tests and transgenic complementation showed that GSQ11 lies within the ANAC060 gene. The Col ANAC060 allele confers sugar insensitivity and was dominant over the sugar-sensitive C24 allele. Genomic and mRNA analyses showed that a single-nucleotide polymorphism (SNP) in Col ANAC060 affects the splicing patterns of ANAC060 such that 20 additional nucleotides are present in the mRNA. The insertion created a stop codon, resulting in a truncated ANAC60 protein lacking the transmembrane domain (TMD) that is present in the C24 ANAC060 protein. The absence of the TMD results in the nuclear localization of ANAC060. The short version of the ANAC060 protein is found in ∼ 12% of natural Arabidopsis accessions. Glucose induces GSQ11/ANAC060 expression in a process that requires abscisic acid (ABA) signaling. Chromatin immunoprecipitation-qPCR and transient expression analysis showed that ABI4 directly binds to the GSQ11/ANAC060 promoter to activate transcription. Interestingly, Col ANAC060 reduced ABA sensitivity and Glc-induced ABA accumulation, and ABI4 expression was also reduced in Col ANAC060 lines. Thus, the sugar-ABA signaling cascade induces ANAC060 expression, but the truncated Col ANAC060 protein attenuates ABA induction and ABA signaling. This negative feedback from nuclear ANAC060 on ABA signaling results in sugar insensitivity.

Mapping parental DMRs predictive of local and distal methylome remodeling in epigenetic F1 hybrids.

F1 hybrids derived from a cross between two inbred parental lines often display widespread changes in DNA methylation and gene expression patterns relative to their parents. An emerging challenge is to understand how parental epigenomic differences contribute to these events. Here, we generated a large mapping panel of F1 epigenetic hybrids, whose parents are isogenic but variable in their DNA methylation patterns. Using a combination of multi-omic profiling and epigenetic mapping strategies we show that differentially methylated regions in parental pericentromeres act as major reorganizers of hybrid methylomes and transcriptomes, even in the absence of genetic variation. These parental differentially methylated regions are associated with hybrid methylation remodeling events at thousands of target regions throughout the genome, both locally (in cis) and distally (in trans). Many of these distally-induced methylation changes lead to nonadditive expression of nearby genes and associate with phenotypic heterosis. Our study highlights the pleiotropic potential of parental pericentromeres in the functional remodeling of hybrid genomes and phenotypes.

Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids.

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids.

Broad-spectrum resistance of Arabidopsis C24 to downy mildew is mediated by different combinations of isolate-specific loci.

Most natural Arabidopsis thaliana accessions are susceptible to one or more isolates of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). However, Arabidopsis C24 has proved resistant to all Hpa isolates tested so far. Here we describe the complex genetic basis of broad-spectrum resistance in C24. The genetics of C24 resistance to three Hpa isolates was analyzed by segregation analysis and quantitative trait locus (QTL) mapping on recombinant inbred and introgression lines. Resistance of C24 to downy mildew was found to be a multigenic trait with complex inheritance. Many identified resistance loci were isolate-specific and located on different chromosomes. Among the C24 resistance QTLs, we found dominant, codominant and recessive loci. Interestingly, none of the identified loci significantly contributed to resistance against all three tested Hpa isolates. Our study demonstrates that broad-spectrum resistance of Arabidopsis C24 to Hpa is based on different combinations of multiple isolate-specific loci. The identified quantitative resistance loci are particularly promising as they provide an important basis for the cloning of susceptibility- and immunity-related genes.

Probing the reproducibility of leaf growth and molecular phenotypes: a comparison of three Arabidopsis accessions cultivated in ten laboratories.

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

Identification of heterotic metabolite QTL in Arabidopsis thaliana RIL and IL populations.

Two mapping populations of a cross between the Arabidopsis thaliana accessions Col-0 and C24 were cultivated and analyzed with respect to the levels of 181 metabolites to elucidate the biological phenomenon of heterosis at the metabolic level. The relative mid-parent heterosis in the F(1) hybrids was <20% for most metabolic traits. The first mapping population consisting of 369 recombinant inbred lines (RILs) and their test cross progeny with both parents allowed us to determine the position and effect of 147 quantitative trait loci (QTL) for metabolite absolute mid-parent heterosis (aMPH). Furthermore, we identified 153 and 83 QTL for augmented additive (Z(1)) and dominance effects (Z(2)), respectively. We identified putative candidate genes for these QTL using the aracyc database (http://www.arabidopsis.org/biocyc), and calculated the average degree of dominance, which was within the dominance and over-dominance range for most metabolites. Analyzing a second population of 41 introgression lines (ILs) and their test crosses with the recurrent parent, we identified 634 significant differences in metabolite levels. Nine per cent of these effects were classified as over-dominant, according to the mode of inheritance. A comparison of both approaches suggested epistasis as a major contributor to metabolite heterosis in Arabidopsis. A linear combination of metabolite levels was shown to significantly correlate with biomass heterosis (r = 0.62).

Unraveling epistasis with triple testcross progenies of near-isogenic lines.

Libraries of near-isogenic lines (NILs) are a powerful plant genetic resource to map quantitative trait loci (QTL). Nevertheless, QTL mapping with NILs is mostly restricted to genetic main effects. Here we propose a two-step procedure to map additive-by-additive digenic epistasis with NILs. In the first step, a generation means analysis of parents, their F(1) hybrid, and one-segment NILs and their triple testcross (TTC) progenies is used to identify in a one-dimensional scan loci exhibiting QTL-by-background interactions. In a second step, one-segment NILs with significant additive-by-additive background interactions are used to produce particular two-segment NILs to test for digenic epistatic interactions between these segments. We evaluated our approach by analyzing a random subset of a genomewide Arabidopsis thaliana NIL library for growth-related traits. The results of our experimental study illustrated the potential of the presented two-step procedure to map additive-by-additive digenic epistasis with NILs. Furthermore, our findings suggested that additive main effects as well as additive-by-additive digenic epistasis strongly influence the genetic architecture underlying growth-related traits of A. thaliana.