Stéphanie Durand

Quantitative Trait Loci Mapping in Five New Large Recombinant Inbred Line Populations of Arabidopsis thaliana Genotyped With Consensus Single-Nucleotide Polymorphism Markers

Quantitative approaches conducted in a single mapping population are limited by the extent of genetic variation distinguishing the parental genotypes. To overcome this limitation and allow a more complete dissection of the genetic architecture of complex traits, we built an integrated set of 15 new large Arabidopsis thaliana recombinant inbred line (RIL) populations optimized for quantitative trait loci (QTL) mapping, having Columbia as a common parent crossed to distant accessions. Here we present 5 of these populations that were validated by investigating three traits: flowering time, rosette size, and seed production as an estimate of fitness. The large number of RILs in each population (between 319 and 377 lines) and the high density of evenly spaced genetic markers scored ensure high power and precision in QTL mapping even under a minimal phenotyping framework. Moreover, the use of common markers across the different maps allows a direct comparison of the QTL detected within the different RIL sets. In addition, we show that following a selective phenotyping strategy by performing QTL analyses on genotypically chosen subsets of 164 RILs (core populations) does not impair the power of detection of QTL with phenotypic contributions >7%.

Rapid Establishment of Genetic Incompatibility through Natural Epigenetic Variation

Epigenetic variation is currently being investigated with the aim of deciphering its importance in both adaptation and evolution [1]. In plants, epimutations can underlie heritable phenotypic diversity [2-4], and epigenetic mechanisms might contribute to reproductive barriers between [5] or within species [6]. The extent of epigenetic variation begins to be appreciated in Arabidopsis [7], but the origin of natural epialleles and their impact in the wild remain largely unknown. Here we show that a genetic incompatibility among Arabidopsis thaliana strains is related to the epigenetic control of a pair of duplicate genes involved in fitness: a transposition event results in a rearranged paralogous structure that causes DNA methylation and transcriptional silencing of the other copy. We further show that this natural, strain-specific epiallele is stable over numerous generations even after removal of the duplicated, rearranged gene copy through crosses. Finally, we provide evidence that the rearranged gene copy triggers de novo DNA methylation and silencing of the unlinked native gene by RNA-directed DNA methylation. Our findings suggest an important role of naturally occurring epialleles originating from structural variation in rapidly establishing genetic incompatibilities following gene duplication events.

Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, Amplifluor and TaqMan, in diploid and polyploid plants

The application of high-throughput SNP genotyping is a great challenge for many research projects in the plant genetics domain. The GOOD assay for mass spectrometry, Amplifluor® and TaqMan® are three methods that rely on different principles for allele discrimination and detection, specifically, primer extension, allele-specific PCR and hybridization, respectively. First, with the goal of assessing allele frequencies by means of SNP genotyping, we compared these methods on a set of three SNPs present in the herbicide resistance genes CSR, AXR1 and IXR1 of Arabidopsis thaliana. In this comparison, we obtained the best results with TaqMan® based on PCR specificity, flexibility in primer design and success rate. We also used mass spectrometry for genotyping polyploid species. Finally, a combination of the three methods was used for medium- to high-throughput genotyping in a number of different plant species. Here, we show that all three genotyping technologies are successful in discriminating alleles in various plant species and discuss the factors that must be considered in assessing which method to use for a given application.

Biophysical properties of the clinical-stage antibody landscape

Significance In addition to binding to a desired target molecule, all antibody drugs must also meet a set of criteria regarding the feasibility of their manufacture, stability in storage, and absence of off-target stickiness. This suite of characteristics is often termed “developability.” We present here a comprehensive analysis of these properties for essentially the full set of antibody drugs that have been tested in phase-2 or -3 clinical trials, or are approved by the FDA. Surprisingly, many of the drugs or candidates in this set exhibit properties that indicate significant developability risks; however, the number of such red warning flags decreases with advancement toward approval. This reference dataset should help prioritize future drug candidates for development. Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of “developability.” Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.

Outcrossing as an Explanation of the Apparent Unconventional Genetic Behavior of Arabidopsis thaliana hth Mutants

The reappearance of HTH alleles in the offspring of homozygous Arabidopsis hth mutants is not consistent with classical Mendelian genetics. It has been suggested that stored RNA may be used to restore genetic information. However, Peng et al. reported that hth mutants tend to display outcrossing and suggested that outcrossing might provide an alternative explanation for the apparent genetic instability. We have confirmed and extended these results, corroborating that the apparent non-Mendelian behavior of hth mutants can be explained by their susceptibility to outcrossing.

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Significance As the centers of photosynthesis and respiration, chloroplasts and mitochondria play a crucial role in energy metabolism. Nuclear and cytoplasmic genomes are known to be coadapted at the species level, because organelle metabolism relies on the proper interaction of organelle-encoded and nuclear-encoded proteins. We explored the extent of cytonuclear coadaptation at the intraspecific level in the classic model plant Arabidopsis thaliana: we measured in a field experiment 28 adaptive whole-organism traits on cytolines developed by substituting cytoplasmic genomes among natural strains. Our results indicate that interactions between nuclear and cytoplasmic genomes shape natural variation for most of the traits we studied, suggesting that these interactions can affect the evolutionary dynamics of natural populations of A. thaliana. Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.

Building of an Experimental Cline With Arabidopsis thaliana to Estimate Herbicide Fitness Cost

Various management strategies aim at maintaining pesticide resistance frequency under a threshold value by taking advantage of the benefit of the fitness penalty (the cost) expressed by the resistance allele outside the treated area or during the pesticide selection “off years.” One method to estimate a fitness cost is to analyze the resistance allele frequency along transects across treated and untreated areas. On the basis of the shape of the cline, this method gives the relative contributions of both gene flow and the fitness difference between genotypes in the treated and untreated areas. Taking advantage of the properties of such migration–selection balance, an artificial cline was built up to optimize the conditions where the fitness cost of two herbicide-resistant mutants (acetolactate synthase and auxin-induced target genes) in the model species Arabidopsis thaliana could be more accurately measured. The analysis of the microevolutionary dynamics in these experimental populations indicated mean fitness costs of ∼15 and 92% for the csr1-1 and axr2-1 resistances, respectively. In addition, negative frequency dependence for the fitness cost was also detected for the axr2-1 resistance. The advantages and disadvantages of the cline approach are discussed in regard to other methods of cost estimation. This comparison highlights the powerful ability of an experimental cline to measure low fitness costs and detect sensibility to frequency-dependent variations.

Genomic Conflicts that Cause Pollen Mortality and Raise Reproductive Barriers in Arabidopsis thaliana.

Species differentiation and the underlying genetics of reproductive isolation are central topics in evolutionary biology. Hybrid sterility is one kind of reproductive barrier that can lead to differentiation between species. Here, we analyze the complex genetic basis of the intraspecific hybrid male sterility that occurs in the offspring of two distant natural strains of Arabidopsis thaliana, Shahdara and Mr-0, with Shahdara as the female parent. Using both classical and quantitative genetic approaches as well as cytological observation of pollen viability, we demonstrate that this particular hybrid sterility results from two causes of pollen mortality. First, the Shahdara cytoplasm induces gametophytic cytoplasmic male sterility (CMS) controlled by several nuclear loci. Second, several segregation distorters leading to allele-specific pollen abortion (pollen killers) operate in hybrids with either cytoplasm. The complete sterility of the hybrid with the Shahdara cytoplasm results from the genetic linkage of the two causes of pollen mortality, i.e., CMS nuclear determinants and pollen killers. Furthermore, natural variation at these loci in A. thaliana is associated with different male-sterility phenotypes in intraspecific hybrids. Our results suggest that the genomic conflicts that underlie segregation distorters and CMS can concurrently lead to reproductive barriers between distant strains within a species. This study provides a new framework for identifying molecular mechanisms and the evolutionary history of loci that contribute to reproductive isolation, and possibly to speciation. It also suggests that two types of genomic conflicts, CMS and segregation distorters, may coevolve in natural populations.

An Arabidopsis Natural Epiallele Maintained by a Feed-Forward Silencing Loop between Histone and DNA

The extent of epigenetic variation is currently well documented, but the number of natural epialleles described so far remains very limited. Determining the relevance of epigenetic changes for natural variation is an important question of research that we investigate by isolating natural epialleles segregating in Arabidopsis recombinant populations. We previously described a genetic incompatibility among Arabidopsis strains based on the silencing of a gene involved in fitness. Here, we isolated a new epiallele resulting from the silencing of a transfer-RNA editing gene in an Arabidopsis accession from the Netherlands (Nok-1). Crosses with the reference accession Col-0 show a complete incompatibility between this epiallele and another locus localized on a different chromosome. We demonstrate that conversion of an unmethylated version of this allele occurs in hybrids, associated with modifications of small RNA populations. These epialleles can also spontaneously revert within the population. Furthermore, we bring evidence that neither METHYLTRANSFERASE 1, maintaining methylation at CGs, nor components of RNA-directed DNA methylation, are key factors for the transmission of the epiallele over generations. This depends only on the self-reinforcing loop between CHROMOMETHYLASE 3 and KRYPTONITE, involving DNA methylated in the CHG context and histone H3 lysine 9 methylation. Our findings reveal a predominant role of this loop in maintaining a natural epiallele.

Broad epitope coverage of a human in vitro antibody library

ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.