Wageningen University
Laboratory of Genetics
An attractive way to improve our understanding of sex determination evolution is to study the underlying mechanisms in closely related species and in a phylogenetic perspective. Hymenopterans are well suited owing to the diverse sex... more
An attractive way to improve our understanding of sex determination evolution is to study the underlying mechanisms in closely related species and in a phylogenetic perspective. Hymenopterans are well suited owing to the diverse sex determination mechanisms, including different types of Complementary Sex Determination (CSD) and maternal control sex determination. We investigated different types of CSD in four species within the braconid wasp genus Asobara that exhibit diverse life-history traits. Nine to thirteen generations of inbreeding were monitored for diploid male production, brood size, offspring sex ratio, and pupal mortality as indicators for CSD. In addition, simulation models were developed to compare these observations to predicted patterns for multilocus CSD with up to ten loci. The inbreeding regime did not result in diploid male production, decreased brood sizes, substantially increased offspring sex ratios nor in increased pupal mortality. The simulations further allowed us to reject CSD with up to ten loci, which is a strong refutation of the multilocus CSD model. We discuss how the absence of CSD can be reconciled with the variation in life-history traits among Asobara species, and the ramifications for the phylogenetic distribution of sex determination mechanisms in the Hymenoptera.
- by Bart Pannebakker
- •
ceptibility to F. solani . Breeding systems need to be developed to aid in the transfer of Fusarium root rot [caused by the soil borne pathogen Fusarium resistance into temperate adapted large-seeded, detersolani (Mart.) Appel & Wr. f.... more
ceptibility to F. solani . Breeding systems need to be developed to aid in the transfer of Fusarium root rot [caused by the soil borne pathogen Fusarium resistance into temperate adapted large-seeded, detersolani (Mart.) Appel & Wr. f. sp. phaseoli (Burk.) Snyd. & Hans.] is a major constraint to common bean (Phaseolus vulgaris L.) produc-minate Andean cultivars that are based on knowledge of tion worldwide. The objectives of this study were to transfer Fusarium the genomic location of rot root resistance in tropically root rot resistance from small-seeded Middle American black bean adapted, small-seeded, indeterminate Middle American into highly susceptible large-seeded Andean kidney and cranberry bean germplasm. bean genotypes and identify quantitative trait loci (QTL) associated Since traits such as root rot resistance are genetically with Fusarium root rot resistance in common bean. Two inbred backcomplex and difficult to evaluate, the efficiency of phecross line (IBL) populations, developed from crosses between smallnotypic selection is low resulting in limited progress in seeded resistant and large-seeded genotypes, were evaluated for reacbreeding for resistance. Breeders are increasingly using tion to root rot during 2 yr in field and greenhouse experiments. Significant genetic variation that displayed continuous transgressive QTL analysis to quantify and locate quantitative resissegregation toward root rot susceptibility confirmed the quantitative tance in plant genomes, since the approach can overnature of resistance. Highly significant treatment-by-environmental come some of the common limitations encountered by effects were observed. Narrow-sense heritability (h 2 N ) estimates for conventional selection for quantitative traits (Asíns, resistance ranged from 0.10 to 0.51 for the kidney and from 0.20 2002; Beavis, 1998; Kelly and Vallejo, 2005). Indirect to 0.82 for the cranberry IBL populations. Nine QTL significantly selection for root rot resistance based on markers linked associated with Fusarium root rot resistance in the field and green-
In Arabidopsis recombinant inbred line (RIL) populations are widely used for quantitative trait locus (QTL) analyses. However, mapping analyses with this type of population can be limited because of the masking effects of major QTL and... more
In Arabidopsis recombinant inbred line (RIL) populations are widely used for quantitative trait locus (QTL) analyses. However, mapping analyses with this type of population can be limited because of the masking effects of major QTL and epistatic interactions of multiple QTL. An alternative type of immortal experimental population commonly used in plant species are sets of introgression lines. Here we introduce the development of a genomewide coverage near-isogenic line (NIL) population of Arabidopsis thaliana, by introgressing genomic regions from the Cape Verde Islands (Cvi) accession into the Landsberg erecta (Ler) genetic background. We have empirically compared the QTL mapping power of this new population with an already existing RIL population derived from the same parents. For that, we analyzed and mapped QTL affecting six developmental traits with different heritability. Overall, in the NIL population smaller-effect QTL than in the RIL population could be detected although the localization resolution was lower. Furthermore, we estimated the effect of population size and of the number of replicates on the detection power of QTL affecting the developmental traits. In general, population size is more important than the number of replicates to increase the mapping power of RILs, whereas for NILs several replicates are absolutely required. These analyses are expected to facilitate experimental design for QTL mapping using these two common types of segregating populations.
Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted... more
Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted metabolomics of plant tissues, based on reversed phase liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF MS) of aqueous methanol extracts. Dedicated software, MetAlign, is used for automated baseline correction and alignment of all extracted mass peaks across all samples, producing detailed information on the relative abundance of thousands of mass signals representing hundreds of metabolites. Subsequent statistics and bioinformatics tools can be used to provide a detailed view on the differences and similarities between (groups of) samples or to link metabolomics data to other systems biology information, genetic markers and/or specific quality parameters. The complete procedure from metabolite extraction to assembly of a data matrix with aligned mass signal intensities takes about 6 days for 50 samples.
Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this... more
Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISRinducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.
Gene expression differences between individuals within a species can be largely explained by differences in genetic background. The effect of genetic variants (alleles) of genes on expression can be studied in a multifactorial way by the... more
Gene expression differences between individuals within a species can be largely explained by differences in genetic background. The effect of genetic variants (alleles) of genes on expression can be studied in a multifactorial way by the application of genetical genomics or expression quantitative trait locus mapping. In this paper, we present a strategy to construct regulatory networks by the application of genetical genomics in combination with transcript profiling of mutants that are disrupted in single genes. We describe the network identification downstream of the receptor-like kinase ERECTA in Arabidopsis (Arabidopsis thaliana). Extending genetical genomics on the Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population with expression profiling of monogenic mutants enabled the identification of regulatory networks in the so far elusive ERECTA signal transduction cascade. We provide evidence that ERECTA is the causal gene for the major hotspot for transcript regulation in the Arabidopsis Ler/Cvi recombinant inbred population. We further propose additional genetic variation between Ler and Cvi in loci of the signaling pathway downstream of ERECTA and suggest candidate genes underlying these loci. Integration of publicly available microarray expression data of other monogenic mutants allowed us to link ERECTA to a downstream mitogen-activated protein kinase signaling cascade. Our study shows that microarray data of monogenic mutants can be effectively used in combination with genetical genomics data to enhance the identification of genetic regulatory networks.
Plants display enormous diversity in their metabolism. Although the biosynthesis and function of the myriads of plant metabolites has been studied for decades, we have little understanding of the interactions between metabolites,... more
Plants display enormous diversity in their metabolism. Although the biosynthesis and function of the myriads of plant metabolites has been studied for decades, we have little understanding of the interactions between metabolites, metabolite signaling, interactions with development and the role of metabolism in genotype-to-phenotype relationships. Technologies for the analysis of metabolites have made tremendous progress in recent years, both in terms of the number of metabolites that are identified and of throughput. Recent developments allow the construction of metabolic networks, and study of the role of these networks in plant growth and development. In this review, we discuss what types of information can be obtained from measurements of metabolites and what requirements they have with respect comprehensiveness of coverage and precision of identification and quantification, and outline procedures that can be implemented to validate the measurements. We then discuss what sorts of perturbations can be used to disturb metabolic networks, including environmental and physiological treatments, chemicals, reverse genetics and the use of natural genetic diversity. www.plant.org on April 27, 2016 -Published by www.plantphysiol.org Downloaded from K (2008) Prime: A web site that assembles tools for metabolomics and transcriptomics. In Silico Biol 8: 339-345 Alonso-Blanco C, Aarts MG, Bentsink L, Keurentjes JJB, Reymond M, Vreugdenhil D, Koornneef M (2009) What has natural variation taught us about plant development, physiology, and adaptation? Plant Cell 21: 1877-1896 Antoniewicz MR, Kelleher JK, Stephanopoulos G (2007) Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. Anal. Chem. 79: 7554-7559 Arrivault S, Guenther M, Ivakov A, Feil R, Vosloh D, van Dongen JT, Sulpice R, Stitt M (2009) Use of reverse-phase liquid chromatography, linked to tandem mass spectrometry, to profile the calvin cycle and other metabolic intermediates in arabidopsis rosettes at different carbon dioxide concentrations. Plant J. 59: 824-839 Balasubramanian S, Schwartz C, Singh A, Warthmann N, Kim MC, Maloof JN, Loudet O, Trainer GT, Dabi T, Borevitz JO, Chory J, Weigel D (2009) Qtl mapping in new arabidopsis thaliana advanced intercross-recombinant inbred lines. PLoS One 4: e4318 Barton DB, Pappl B, Delneri D , Oliver SB, Rattrav M, Bergman CM (2008) Systems biology of energetic and atomic costs in the yeast transcriptome, proteome, and metabolome. Nature Precedings. hdl:10101/npre.2008.1841.2 Barton NH (2000) Genetic hitchhiking. Phil. Trans. R. Soc. B 355: 1553-1562 Bassham JA (2003) Mapping the carbon reduction cycle: A personal retrospective. Photosynthesis Res. 76: 37-52 Bassham JA, Krause GH (1969) Free energy changes and metabolic regulation in steady-state photosynthetic carbon reduction. Biochim. Biophys. Acta 189: 207-221 Bennett BD, Yuan J, Kimball EH, Rabinowitz JD (2002) Absolute quantitation of intracellular metaboliteconcentrations by an isotope ratio-based approach. Nat. Protoc. 3, 1299-1311 Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in escherichia coli. Nat. Chem. Biol. 5: 593-599 Benson AA (2002) Following the path of carbon in photosynthesis: A personal story. Photosynthesis Res. 73: 31-49 Bikard D, Patel D, Le Mette C, Giorgi V, Camilleri C, Bennett MJ, Loudet O (2009) Divergent evolution of duplicate genes leads to genetic incompatibilities within a.
Recent advances in -omics technologies such as transcriptomics, metabolomics, and proteomics along with genotypic profiling have permitted dissection of the genetics of complex traits represented by molecular phenotypes in nonmodel... more
Recent advances in -omics technologies such as transcriptomics, metabolomics, and proteomics along with genotypic profiling have permitted dissection of the genetics of complex traits represented by molecular phenotypes in nonmodel species. To identify the genetic factors underlying variation in primary metabolism in potato (Solanum tuberosum), we have profiled primary metabolite content in a diploid potato mapping population, derived from crosses between S. tuberosum and wild relatives, using gas chromatography-time of flight-mass spectrometry. In total, 139 polar metabolites were detected, of which we identified metabolite quantitative trait loci for approximately 72% of the detected compounds. In order to obtain an insight into the relationships between metabolic traits and classical phenotypic traits, we also analyzed statistical associations between them. The combined analysis of genetic information through quantitative trait locus coincidence and the application of statistical learning methods provide information on putative indicators associated with the alterations in metabolic networks that affect complex phenotypic traits.
Water limitation is one of the most important factors limiting crop productivity world-wide and has likely been an important selective regime influencing the evolution of plant physiology. Understanding the genetic and physiological basis... more
Water limitation is one of the most important factors limiting crop productivity world-wide and has likely been an important selective regime influencing the evolution of plant physiology. Understanding the genetic and physiological basis of drought adaptation is therefore important for improving crops as well as for understanding the evolution of wild species. Here, results are presented from quantitative trait loci (QTL) mapping of flowering time (a drought escape mechanism) and carbon stable isotope ratio (d d d d 13 C) (a drought-avoidance mechanism) in Arabidopsis thaliana. Whole-genome scans were performed using multiple-QTL models for both additive and epistatic QTL effects. We mapped five QTL affecting flowering time and five QTL affecting d d d d 13 C, but two genomic regions contained QTL with effects on both traits, suggesting a potential pleiotropic relationship. In addition, we observed QTL-QTL interaction for both traits. Two d d d d 13 C QTL were captured in nearisogenic lines to further characterize their physiological basis. These experiments revealed allelic effects on d d d d 13 C through the upstream trait of stomatal conductance with subsequent consequences for whole plant transpiration efficiency and water loss. Our findings document considerable natural genetic variation in whole-plant, drought resistance physiology of Arabidopsis and highlight the value of quantitative genetic approaches for exploring functional relationships regulating physiology.
The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis.... more
The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase activities than with sucrose synthase activities. The highest correlations were observed with activities in the elongating zones of roots. The genetic basis of these correlations was studied by using QTL analysis. Several loci, affecting invertase activity, colocated with loci that had an effect on root or hypocotyl length. Further fine mapping of a major locus for root length, but not for hypocotyl length (top chromosome 1), consistently showed colocation with the locus for invertase activity containing a gene coding for a vacuolar invertase. The analysis of a functional knockout line confirmed the role of this invertase in root elongation, whereas other invertase genes might play a role in hypocotyl elongation. Thus, we show the power of QTL analysis, combined for morphological and biochemical traits, followed by fine-mapping and mutant analysis, in unraveling the function of genes and their role in growth and development.
Molecular biologists typically restrict systems biology to cellular levels. By contrast, ecologists define biological systems as communities of interacting individuals at different trophic levels that process energy, nutrient and... more
Molecular biologists typically restrict systems biology to cellular levels. By contrast, ecologists define biological systems as communities of interacting individuals at different trophic levels that process energy, nutrient and information flows. Modern plant breeding needs to increase agricultural productivity while decreasing the ecological footprint. This requires a holistic systems biology approach that couples different aggregation levels while considering the variables that affect these biological systems from cell to community. The challenge is to generate accurate experimental data that can be used together with modelling concepts and techniques that allow experimentally verifying in silico predictions. The coupling of aggregation levels in plant sciences, termed Integral Quantification of Biological Organization (IQ BiO ), might enhance our abilities to generate new desired plant phenotypes.
Rising demand for food and bioenergy makes it imperative to breed for increased crop yield. Vegetative plant growth could be driven by resource acquisition or developmental programs. Metabolite profiling in 94 Arabidopsis accessions... more
Rising demand for food and bioenergy makes it imperative to breed for increased crop yield. Vegetative plant growth could be driven by resource acquisition or developmental programs. Metabolite profiling in 94 Arabidopsis accessions revealed that biomass correlates negatively with many metabolites, especially starch. Starch accumulates in the light and is degraded at night to provide a sustained supply of carbon
Accessions of a plant species can show considerable genetic differences that are analyzed effectively by using recombinant inbred line (RIL) populations. Here we describe the results of genome-wide expression variation analysis in an RIL... more
Accessions of a plant species can show considerable genetic differences that are analyzed effectively by using recombinant inbred line (RIL) populations. Here we describe the results of genome-wide expression variation analysis in an RIL population of Arabidopsis thaliana. For many genes, variation in expression could be explained by expression quantitative trait loci (eQTLs). The nature and consequences of this variation are discussed based on additional genetic parameters, such as heritability and transgression and by examining the genomic position of eQTLs versus gene position, polymorphism frequency, and gene ontology. Furthermore, we developed an approach for genetic regulatory network construction by combining eQTL mapping and regulator candidate gene selection. The power of our method was shown in a case study of genes associated with flowering time, a well studied regulatory network in Arabidopsis. Results that revealed clusters of coregulated genes and their most likely reg...
- by Joost Keurentjes and +1
- •
- Genetics, Polymorphism, Gene expression, Multidisciplinary
de Boer, M., Bom, P., Kindt, F., Keurentjes, J. J. B., van der Sluis, I., van Loon, L. C., and Bakker, P. A. H. M. 2003. Control of Fusarium wilt of radish by combining Pseudomonas putida strains that have different disease-suppressive... more
de Boer, M., Bom, P., Kindt, F., Keurentjes, J. J. B., van der Sluis, I., van Loon, L. C., and Bakker, P. A. H. M. 2003. Control of Fusarium wilt of radish by combining Pseudomonas putida strains that have different disease-suppressive mechanisms. Phytopathology 93:626-632.
Heritability is a central parameter in quantitative genetics, both from an evolutionary and a breeding perspective. For plant traits heritability is traditionally estimated by comparing within and between genotype variability. This... more
Heritability is a central parameter in quantitative genetics, both from an evolutionary and a breeding perspective. For plant traits heritability is traditionally estimated by comparing within and between genotype variability. This approach estimates broad-sense heritability, and does not account for different genetic relatedness. With the availability of high-density markers there is growing interest in marker based estimates of narrow-sense heritability, using mixed models in which genetic relatedness is estimated from genetic markers. Such estimates have received much attention in human genetics but are rarely reported for plant traits. A major obstacle is that current methodology and software assume a single phenotypic value per genotype, hence requiring genotypic means. An alternative that we propose here, is to use mixed models at individual plant or plot level. Using statistical arguments, simulations and real data we investigate the feasibility of both approaches, and how these affect genomic prediction with G-BLUP and genome-wide association studies. Heritability estimates obtained from genotypic means had very large standard errors and were sometimes biologically unrealistic. Mixed models at individual plant or plot level produced more realistic estimates, and for simulated traits standard errors were up to 13 times smaller. Genomic prediction was also improved by using these mixed models, with up to a 49% increase in accuracy. For GWAS on simulated traits, the use of individual plant data gave almost no increase in power. The new methodology is applicable to any complex trait where multiple replicates of individual genotypes can be scored. This includes important agronomic crops, as well as bacteria and fungi.
- by Joost Keurentjes and +1
- •
- Genetics, Algorithms, Genomics, Computational Biology
We here describe the MetaNetwork protocol to reconstruct metabolic networks using metabolite abundance data from segregating populations. MetaNetwork maps metabolite quantitative trait loci (mQTLs) underlying variation in metabolite... more
We here describe the MetaNetwork protocol to reconstruct metabolic networks using metabolite abundance data from segregating populations. MetaNetwork maps metabolite quantitative trait loci (mQTLs) underlying variation in metabolite abundance in individuals of a segregating population using a two-part model to account for the often observed spike in the distribution of metabolite abundance data. MetaNetwork predicts and visualizes potential associations between metabolites using correlations of mQTL profiles, rather than of abundance profiles. Simulation and permutation procedures are used to assess statistical significance. Analysis of about 20 metabolite mass peaks from a mass spectrometer takes a few minutes on a desktop computer. Analysis of 2,000 mass peaks will take up to 4 days. In addition, MetaNetwork is able to integrate high-throughput data from subsequent metabolomics, transcriptomics and proteomics experiments in conjunction with traditional phenotypic data. This way MetaNetwork will contribute to a better integration of such data into systems biology. 7 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 685 PROTOCOL