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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.

Summary 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.

Developmental plasticity of root system architecture is crucial for plant performance in nutrient-poor soils. Roots of plants grown under mild nitrogen (N) deficiency show a foraging response characterized by increased root length but mechanisms underlying this developmental plasticity are still elusive. By employing natural variation in Arabidopsis accessions, we show that the brassinosteroid (BR) signaling kinase BSK3 modulates root elongation under mild N deficiency. In particular, a proline to leucine substitution in the predicted kinase domain of BSK3 enhances BR sensitivity and signaling to increase the extent of root elongation. We further show that low N specifically upregulates transcript levels of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation. Altogether, our results uncover a role of BR signaling in root elongation under low N. The BSK3 alleles identified here provide targets for improving root growth of crops growing under limited N conditions., Plant roots elongate under mild nitrogen deficiency as part of a foraging response that facilitates nutrient uptake. Here the authors show that natural variation in this response among Arabidopsis accessions depends on the brassinosteroid (BR) signaling kinase BSK3, which can enhance BR sensitivity and root growth.

Key messageComplementing 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 napusL./canola) employing different omics profiles, individually and in combination. To this end, a population of 950 F1hybrids 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.

We assessed early vegetative growth in a population of 382 accessions ofArabidopsis thalianausing automated non-invasive high-throughput phenotyping. All accessions were imaged daily from seven to 18 days 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 (RGR). In addition, initial seed size was determined. The generated data sets 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-localisation of MTAs occurred at 33 genomic positions. At 21 of these positions, sequential co-localisation of MTAs for two to nine consecutive days was observed. The detected MTAs for PLA and RGR could be grouped according to their temporal expression patterns, emphasising 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.HighlightA genome-wide association study including the factor time highlighted that early plant growth in Arabidopsis is governed by several medium and many small effect loci, most of which act only during short phases of two to nine days.

A major QTL controlling glutamate sensitivity of Arabidopsis root development has been mapped and additional loci identified that epistatically regulate its response to environmental changes., 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 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.

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 (δ13C). However, in the genetic backgrounds tested here, drought tolerance traits, stomatal conductance, δ13C. 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., Accelerated flowering time achieved through nonfunctional and weak alleles of flowering time genes FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) reduce water use in Arabidopsis thaliana without detrimentally impacting reproductive output.

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.

Summary 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) with function in carbon assimilation and nitrogen utilization. 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 kb 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 middle/eastern European accessions, while the C24 type allele is prevalent on the Iberian Peninsula, western of the Rhine, and the British islands. Our findings support FUM2's role in diurnal carbon storage and point to a growth advantage of accessions carrying the FUM2 Col-0 allele. This article is protected by copyright. All rights reserved.

Nitrogen (N) is a macronutrient determining crop yield. The application of N fertilisers can substantially increase the yield, but excess use also causes the nitrate pollution of water resources and increases production costs. Increasing N use efficiency (NUE) in crop plants is an important step to implement low-input agricultural systems. We used Arabidopsis thaliana as model system to investigate the natural genetic diversity in traits related to NUE. Natural variation was used to study adaptive growth patterns and changes in gene expression associated with limited nitrate availability. A genome-wide association study revealed an association of eight SNP markers on Chromosome 1 with shoot growth under limited N. The identified linkage disequilibrium (LD) interval includes the DNA sequences of three cysteine/histidine-rich C1 domain proteins in tandem orientation. These genes differ in promoter structure, methylation pattern and expression level among accessions, correlating with growth performance under N deficiency. Our results suggest the involvement of epigenetic regulation in the expression of NUE-related traits.

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 (TE). 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 but also 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 (QTL) mapping and validation through near-isogenic lines identified two causal QTLs, which showed that a combination of weak and non-functional alleles of the FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) genes substantially reduced plant water-use without penalising reproductive performance. Drought tolerance traits, stomatal conductance, intrinsic water use efficiency (δ13C) and rosette water-use were independent of allelic variation at FRI and FLC, suggesting that flowering is critical in determining life-time plant water use, but not leaf-level traits.

Background Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. Results We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (ΦPSII). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of ΦPSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. Conclusions The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A. thaliana accessions. Individual probes of the GeneChip® that show sequence polymorphisms between different A. thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A. thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A. thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Highlight A comprehensive metabolic study of Arabidopsis seeds revealed mQTL hotspots, candidate genes, epistatic interactions, and a potential master regulatory locus of metabolism on the short arm of chromosome IV., To gain insight into genetic factors controlling seed metabolic composition and its relationship to major seed properties, an Arabidopsis recombinant inbred line (RIL) population, derived from accessions Col-0 and C24, was studied using an MS-based metabolic profiling approach. Relative intensities of 311 polar primary metabolites were used to identify associated genomic loci and to elucidate their interactions by quantitative trait locus (QTL) mapping. A total of 786 metabolic QTLs (mQTLs) were unequally distributed across the genome, forming several hotspots. For the branched-chain amino acid leucine, mQTLs and candidate genes were elucidated in detail. Correlation studies displayed links between metabolite levels, seed protein content, and seed weight. Principal component analysis revealed a clustering of samples, with PC1 mapping to a region on the short arm of chromosome IV. The overlap of this region with mQTL hotspots indicates the presence of a potential master regulatory locus of seed metabolism. As a result of database queries, a series of candidate regulatory genes, including bZIP10, were identified within this region. Depending on the search conditions, metabolic pathway-derived candidate genes for 40–61% of tested mQTLs could be determined, providing an extensive basis for further identification and characterization of hitherto unknown genes causal for natural variation of Arabidopsis seed metabolism.

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.

Summary Like protein-coding genes, loci that produce microRNAs (miRNAs) are generally considered to be under purifying selection [1–3], consistent with miRNA polymorphisms being able to cause disease [4]. Nevertheless, it has been hypothesized that variation in miRNA genes may contribute to phenotypic diversity [1, 3, 5, 6]. Here we demonstrate that a naturally occurring polymorphism in the MIR164A gene affects leaf shape and shoot architecture in Arabidopsis thaliana , with the effects being modified by additional loci in the genome. A single base pair substitution in the miRNA complementary sequence alters the predicted stability of the miRNA:miRNA ∗ duplex. It thereby greatly reduces miRNA accumulation, probably because it interferes with precursor processing. We demonstrate that this is not a rare exception and that natural strains of Arabidopsis thaliana harbor dozens of similar polymorphisms that affect processing of a wide range of miRNA precursors. Our results suggest that natural variation in miRNA biogenesis resulting from cis mutations is a common contributor to phenotypic variation in plants.

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.

The biochemical diversity in the plant kingdom is estimated to well exceed 100,000 distinct compounds (Weckwerth, 2003) and 4000 to 20,000 metabolites per species seem likely (Fernie et al., 2004). In recent years extensive progress has been made towards the identification of enzymes and regulatory genes working in a complex network to generate this large arsenal of metabolites. Genetic loci influencing quantitative traits, e.g. metabolites or biomass, may be mapped to associated molecular markers, a method called quantitative trait locus mapping (QTL mapping), which may facilitate the identification of novel genes in biochemical pathways. Arabidopsis thaliana, as a model organism for seed plants, is a suitable target for metabolic QTL (mQTL) studies due to the availability of highly developed molecular and genetic tools, and the extensive knowledge accumulated on the metabolite profile. While intensely studied, in particular since the availability of its complete sequence, the genome of Arabidopsis still comprises a large proportion of genes with only tentative function based on sequence homology. From a total number of 33,518 genes currently listed (TAIR 9, http://www.arabidopsis.org), only about 25% have direct experimental evidence for their molecular function and biological process, while for more than 30% no biological data are available. Modern metabolomics approaches together with continually extended genomic resources will facilitate the task of assigning functions to those genes. In our previous study we reported on the identification of mQTL (Lisec et al., 2008). In this paper, we summarize the current status of mQTL analyses and causal gene identification in Arabidopsis and present evidence that a candidate gene located within the confidence interval of a fumarate mQTL (AT5G50950) encoding a putative fumarase is likely to be the causal gene of this QTL. The total number of genes molecularly identified based on mQTL studies is still limited, but the advent of multi-parallel analysis techniques for measurement of gene expression, as well as protein and metabolite abundances and for rapid gene identification will assist in the important task of assigning enzymes and regulatory genes to the growing network of known metabolic reactions.

Plants that constitutively express otherwise inducible disease resistance traits often suffer a depressed seed yield in the absence of a challenge by pathogens. This has led to the view that inducible disease resistance is indispensable, ensuring that minimal resources are diverted from growth, reproduction and abiotic stress tolerance. The Arabidopsis genotype C24 has enhanced basal resistance, which was shown to be caused by permanent expression of normally inducible salicylic acid (SA)-regulated defences. However, the seed yield of C24 was greatly enhanced in comparison to disease-resistant mutants that display identical expression of SA defences. Under both water-replete and -limited conditions, C24 showed no difference and increased seed yield, respectively, in comparison with pathogen-susceptible genotypes. C24 was the most drought-tolerant genotype and showed elevated water productivity, defined as seed yield per plant per millilitre water consumed, and achieved this by displaying adjustments to both its development and transpiration efficiency (TE). Therefore, constitutive high levels of disease resistance in C24 do not affect drought tolerance, seed yield and seed viability. This study demonstrates that it will be possible to combine traits that elevate basal disease resistance and improve water productivity in crop species, and such traits need not be mutually exclusive.

Summary 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 F1 hybrids was

The main objective of this study was to identify genomic regions involved in biomass heterosis using QTL, generation means, and mode-of-inheritance classification analyses. In a modified North Carolina Design III we backcrossed 429 recombinant inbred line and 140 introgression line populations to the two parental accessions, C24 and Col-0, whose F 1 hybrid exhibited 44% heterosis for biomass. Mid-parent heterosis in the RILs ranged from −31 to 99% for dry weight and from −58 to 143% for leaf area. We detected ten genomic positions involved in biomass heterosis at an early developmental stage, individually explaining between 2.4 and 15.7% of the phenotypic variation. While overdominant gene action was prevalent in heterotic QTL, our results suggest that a combination of dominance, overdominance and epistasis is involved in biomass heterosis in this Arabidopsis cross., Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1330

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 F1 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.

In our analysis of 5′ and 3′ end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5′ termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F1 hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5′ end generation of mitochondrial transcripts.

Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P 80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24-67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.

The decline of available fossil fuel reserves has triggered world- wide efforts to develop alternative energy sources based on plant biomass. Detailed knowledge of the relations of metabolism and biomass accumulation can be expected to yield powerful novel tools to accelerate and enhance energy plant breeding programs. We used metabolic profiling in the model Arabidopsis to study the relation between biomass and metabolic composition using a recombinant inbred line (RIL) population. A highly significant canonical correlation (0.73) was observed, revealing a close link between biomass and a specific combination of metabolites. Di- viding the entire data set into training and test sets resulted in a median correlation between predicted and true biomass of 0.58. The demonstrated high predictive power of metabolic composition for biomass features this composite measure as an excellent biomarker and opens new opportunities to enhance plant breeding specifically in the context of renewable resources. biomass canonical correlation metabolic profiling recombinant inbred line population biomarker

Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nucleotide long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing, while 24-nucleotide long siRNAs are involved in transcriptional gene silencing in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced post-transcriptional gene silencing. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C.elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in RGRs depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth.

A new large set of reciprocal recombinant inbred lines (RILs) was created between the Arabidopsis accessions Col-0 and C24 for quantitative trait mapping approaches, consisting of 209 Col-0 x C24 and 214 C24 x Col-0 F(7 )RI lines. Genotyping was performed using 110 evenly distributed framework single nucleotide polymorphism markers, yielding a genetic map of 425.70 cM, with an average interval of 3.87 cM. Segregation distortion (SD) was observed in several genomic regions during the construction of the genetic map. Linkage disequilibrium analysis revealed an association between a distorted region at the bottom of chromosome V and a non-distorted region on chromosome IV. A detailed analysis of the RILs for these two regions showed that an SD occurred when homozygous Col-0 alleles on chromosome IV coincided with homozygous C24 alleles at the bottom of chromosome V. Using nearly isogenic lines segregating for the distorted region we confirmed that this genotypic composition leads to reduced fertility and fitness.

The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5' sequences up to nucleotide position -584, the coding regions and the 3' flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position -584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5' end is abundant. This terminus and the other major 5' ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5' terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5' mRNA end.

We examined diversity levels and patterns of 19 nuclear microsatellites and four chloroplast microsatellites in 275 genotypes of wild barley Hordeum spontaneum, in seven stations at the ‘Evolution Canyon’ (EC) microsite, Lower Nahal Oren, Mt. Carmel, Israel. EC is sharply subdivided ecologically into a tropical savannoid, ‘African’, xeric, south-facing slope (SFS) abutting the temperate, dense, liveoak, brushwood, ‘European’, mesic, north-facing slope (NFS). We found the following. (i) 17 of 19 (89.5%) nuDNA simple sequence repeats (SSRs) were polymorphic across all seven subpopulations and three chDNA SSRs were polymorphic. (ii) A total of 216 nuDNA SSR alleles, with a maximum of 23 alleles in a nuclear locus, and ten chDNA SSRs, with a maximum of four alleles in a locus, were registered. (iii) There were striking and significant inter- and intraslope diversities, based on the 19 nuDNA SSRs, climaxing with a remarkable genetic distance between the mid-slope stations on opposite slopes (DA = 0.481), across a distance of 200 m. This genetic distance is as large as that between the H. spontaneum populations of Jerusalem and Sede Boqer, which are separated by 100 km (× 500 larger in transect length). (iv) Slope-unique alleles (103 = 45.6%) were higher on the ‘European’ than on the ‘African’ slope. Slope-specific (predominant) alleles (17) were equal on opposite slopes. (v) nuDNA SSR gene diversity was higher on the ‘European’ slope and the opposite was found for the chDNA SSR. (vi) nuDNA SSR genic differentiation was very high between opposite slopes, with Gst = 0.187; for chDNA SSR this value was 0.127. Our results are inexplicable by stochastic processes and suggest that: (i) microclimatic diversifying selection is the major evolutionary, fast-acting, interslope force, overriding migration and drift, and (ii) ecological stress can generate local, regional and global adaptive patterns, suggesting that natural selection is a major differentiating force of both coding and noncoding SSRs linking micro- and macroevolutionary processes. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84, 205–224.

Barley traits related to salt tolerance are mapped in a population segregating for a dwarfing gene associated with salt tolerance. Twelve quantitative trait loci (QTLs) were detected for seven seedling traits in doubled haploids from the spring barley cross Derkado x B83-12/21/5 when given saline treatment in hydroponics. The location of QTLs for seedling growth stage (leaf appearance rate), stem weight prior to elongation, and tiller number are reported for the first time. In addition, four QTLs were found for the mature plant traits grain nitrogen and plot yield. In total, seven QTLs are co-located with the dwarfing genes sdw1, on chromosome 3H, and ari-e.GP, on chromosome 5H, including seedling leaf response (SGa) to gibberellic acid (GA(3)). QTLs controlling the growth of leaves (GS2) on chromosomes 2H and 3H and emergence of tillers (TN2) and grain yield were independent of the dwarfing genes. Field trials were grown in eastern Scotland and England to estimate yield and grain composition. A genetic map was used to compare the positions of QTLs for seedling traits with the location of QTLs for the mature plant traits. The results are discussed in relation to the study of barley physiology and the location of genes for dwarf habit and responses to GA.

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. We observed little DNA methylation divergence whole-genome wide. Nonetheless, 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 in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.

A number of random inbred lines of barley, grown at two sites, were assessed for high gravity spirit yield. A range of values was obtained and there was good agreement, for nearly all samples, between high gravity spirit yield and spirit yield predicted from extracts and fermentabilities measured by Institute of Brewing recommended methods. One sample, which gave a low predicted spirit yield but good high gravity spirit yield, was shown to have modified poorly and unevenly. Over both sites, high gravity spirit yield appeared to correlate negatively with grain nitrogen content, but this reflected large differences between sites in grain nitrogen. High gravity spirit yield did not correlate with soluble nitrogen, but did with fermentability, so samples with low levels of fermentability had either low or moderate levels of high gravity spirit yield. It was concluded that identification of genetic factors, influencing extract and fermentability, would enhance breeding of barley cultivars suitable for malt whisky distilling.

PCR-based markers were developed from mononucleotide simple-sequence repeats in the chloroplast genome of Nicotiana tabacum and applied to the analysis of genetic diversity. These markers were found to detect high levels of polymorphism at three taxonomic levels in Solanaceous plants. Of 36 chloroplast loci examined, 26 show some degree of polymorphism among potato accessions. Among a set of 30 tetraploid potato cultivars it is apparent that a single chloroplast haplotype is prevalent, presumably a result of the widespread use as a female parent of the imported US cultivar Rough Purple Chili in the latter half of the 19th century. Nonetheless, there is considerable chloroplast diversity in the cultivated potato, and it is clear that a large proportion of this variability has arisen through the use of wild or primitive cultivated species of potato in introgression programmes. This variability should be used in future breeding programmes. An examination of single accessions from 24 potato species, as well as representatives from tobacco and other members of the Solanaceae, reveals high levels of inter-specific chloroplast DNA variation. These data, and the ease of use and potential for multiplexing of these markers, suggest that cpSSRs will be of great utility in population genetics, germplasm management, evolutionary and phylogenetic studies as well as in, the analysis of material from introgression and somatic-fusion experiments. Interestingly, the polymorphism arising from one of the more-polymorphic chloroplast loci examined, does not originate solely from the SSR, and is due to variation in the copy number of two tandemly arrayed sequence elements.

Field resistance to Phytophthora infestans, the causal agent of foliage and tuber blight in cultivated potatoes, earliness (maturity) and vigour, were examined in a diploid segregating potato population grown in replicated trials over three consecutive growing seasons. A genetic linkage map of this population was constructed in parallel using PCR-based SSR, AFLP and CAPS markers. Analysis of the trait scores alongside the marker segregation data allowed the identification of regions of the genome which were significantly correlated with components of the respective characters. The most significant associations for all four traits were with marker alleles on potato linkage group V originating from the male (susceptible) parent. In the case of foliage resistance to late blight, the positions of the majority of the effects, which were located on eleven of the twelve potato linkage groups, have been detected in previous [16] and parallel studies [21]. The absence of Solanum demissum-derived R genes for hypersensitive response to late blight and the co-localisation of QTL for resistance, vigour and earliness suggest that developmental and/or physiological factors play a major role in determining the level of foliage resistance in this population. In contrast with previous findings, a negative correlation was found between foliage and tuber blight resistance.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the β-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.

Seventy eight clones from the cross between SCRI clone 12601ab1 and cv Stirling were used to explore the possibility of genetical linkage analysis in tetraploid potato (Solanum tuberosum subsp. tuberosum). Clone 12601ab1 had quantitative resistance to Globodera pallida Pa2/3 derived from S. tuberosum subsp. andigena. The strategy adopted involved identifying single- (simplex) and double- (duplex) dose AFLP markers in the parents from segregation ratios that could be unambiguously identified in their offspring, detecting linkage between a marker and a putative quantitative trait locus (QTL) for resistance, and placing the QTL on the linkage map of markers. The numbers of scorable segregating markers were 162 simplex ones present only in 12601ab1, 87 present in Stirling, and 32 present in both; and 72 duplex markers present only in 12601ab1 and 45 present in Stirling. The total map length was 990.9 cM in 12601ab1 and 484.6 cM in Stirling. A QTL with a resistance allele present in double dose (QQqq) in 12601ab1 was inferred from the associations between resistance scores (square root of female counts) and two duplex markers linked in coupling, which, in turn, were linked in coupling to four simplex markers also associated with resistance, but to a lesser degree. The largest marker class difference was the one for the duplex marker P61M34=15. It accounted for 27.8% of the phenotypic variance in resistance scores, or approximately 30% of the genotypic variance. Subsequently, this duplex marker was found to be linked in coupling with a duplex SSR allele Stm3016=a, whose locus was shown to be on chromosome IV in a diploid reference mapping population. The other QTLs for resistance segregating in the progeny were not identified for one or more of the following reasons: the markers did not cover the whole of the genome, there were unfavourable repulsion linkages between the QTLs and markers, or the gene effects were not large enough to be detected in an experiment of the size conducted. It is concluded that prospects appear good for detecting QTLs and using marker-assisted selection in a tetraploid potato breeding programme, provided that, in future, the population size is increased to over 250 and more SSR markers are used to complement the AFLPs; the same is likely to be true for other autotetraploid crops.

A simulation study was performed to investigate methods for mapping single-dose (simplex) and double-dose (duplex) markers, and for identification of homologous chromosomes in an autotetraploid species, and to see how the map accuracy depends on the population size. An initial population of 1000 individuals was simulated, with 30 simplex and 10 duplex markers, and recombination fractions and lod scores were calculated between all pairs of markers. These were used to test the feasibility of mapping the simplex and duplex markers simultaneously. Smaller populations, from 500 to 75 individuals, were then simulated, and the estimates of the pairwise recombination fractions and the derived maps were compared with the true map. It was found that the accuracy of the estimates depended strongly on the type of markers involved, with simplex–simplex coupling pairs being most reliable and simplex–simplex repulsion pairs and duplex–duplex pairs in any configuration but coupling being least reliable. Maps can be assembled using recombination fractions and lod scores from pairs of simplex–simplex markers (coupling and repulsion), duplex–simplex (coupling and repulsion) and duplex–duplex (coupling). The agreement between the map order and the true order was good, although the map distance was generally underestimated at small sample sizes.

The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed. Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.

A 4.3 kb genomic clone from potato ( Solanum tuberosum ) cv. Saturna has been isolated which contains the full coding sequence of a protein kinase gene ( Stpk1 ), 1 kb of 3′ sequence, and the first exon of a neighbouring gene encoding a sucrolytic enzyme. An identical organisation could be detected in a genomic clone carrying a gene that encodes a protein kinase homologue in Arabidopsis thaliana (Hayashida et al., Gene, 121 (1992) 325–330). Southern and RT-PCR analyses indicate that Stpk1 is a single copy gene which is expressed throughout the plant with an increase in transcript abundance in leaf after infection with Phytophthora infestans . While comparisons using the deduced amino acid sequence for STPK1 indicate that it is most closely related to cyclic nucleotide-dependent protein kinases, it contains an additional plant-specific insert of 85 amino acids in the catalytic domain.

In the Arabidopsis multiparent recombinant inbred line mapping population, a limited number of plants were detected that lacked axillary buds in most of the axils of the cauline (stem) leaves, but formed such buds in almost all rosette axils. Genetic analysis showed that polymorphisms in at least three loci together constitute this phenotype, which only occurs in late-flowering plants. Early flowering is epistatic to two of these loci, called REDUCED SHOOT BRANCHING1 (RSB1) and RSB2, which themselves do not affect flowering time. Map-based cloning and confirmation by transformation with genes from the region where RSB1 was identified by fine-mapping showed that a specific allele of AGAMOUS-Like6 from accession C24 conferred reduced branching in the cauline leaves. Site-directed mutagenesis in the Columbia allele revealed the causal amino acid substitution, which behaved as dominant negative, as was concluded from a loss-of-function mutation that showed the same phenotype in the late-flowering genetic background. This causal allele occurs at a frequency of 15% in the resequenced Arabidopsis thaliana accessions and correlated with reduced stem branching only in late-flowering accessions. The data show the importance of natural variation and epistatic interactions in revealing gene function.

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.

A recombinant inbred line (RIL) population, derived from two Arabidopsis thaliana accessions, and the corresponding testcrosses with these two original accessions were used for the development and validation of machine learning models to predict the biomass of hybrids. Genetic and metabolic information of the RILs served as predictors. Feature selection reduced the number of variables (genetic and metabolic markers) in the models by more than 80% without impairing the predictive power. Thus, potential biomarkers have been revealed. Metabolites were shown to bear information on inherited macroscopic phenotypes. This proof of concept could be interesting for breeders. The example population exhibits substantial mid-parent biomass heterosis. The results of feature selection could therefore be used to shed light on the origin of heterosis. In this respect, mainly dominance effects were detected., Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1324