An Arabidopsis example of association mapping in structured samples

A potentially serious disadvantage of association mapping is the fact that marker-trait associations may arise from confounding population structure as well as from linkage to causative polymorphisms. Using genome-wide marker data, we have previously demonstrated that the problem can be severe in a global sample of 95 Arabidopsis thaliana accessions, and that established methods for controlling for population structure are generally insufficient. Here, we use the same sample together with a number of flowering-related phenotypes and data-perturbation simulations to evaluate a wider range of methods for controlling for population structure. We find that, in terms of reducing the false-positive rate while maintaining statistical power, a recently introduced mixed-model approach that takes genome-wide differences in relatedness into account via estimated pairwise kinship coefficients generally performs best. By combining the association results with results from linkage mapping in F2 crosses, we identify one previously known true positive and several promising new associations, but also demonstrate the existence of both false positives and false negatives. Our results illustrate the potential of genome-wide association scans as a tool for dissecting the genetics of natural variation, while at the same time highlighting the pitfalls. The importance of study design is clear; our study is severely under-powered both in terms of sample size and marker density. Our results also provide a striking demonstration of confounding by population structure. While statistical methods can be used to ameliorate this problem, they cannot always be effective and are certainly not a substitute for independent evidence, such as that obtained via crosses or transgenic experiments. Ultimately, association mapping is a powerful tool for identifying a list of candidates that is short enough to permit further genetic study.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Adding floral nectar resources to improve biological control: Potential pitfalls of the fourth trophic level

The effects of floral nectar resources on ecosystem function were investigated by examining the consequences of increasing habitat complexity in field microcosms on the dynamics of a four-trophic-level community, consisting of lucerne (alfalfa), a herbivore (the pea aphid, Acyrthosiphon pisum), its parasitoid (Aphidius ervi) and a hyperparasitoid (Dendrocerus aphidum). The influence of buckwheat (Fagopyrum esculentum) flowers on the parasitism and hyperparasitism by A. ervi and D. aphidum, respectively, was compared with buckwheat-free treatments. Experimental units for this study were 1.8×1.8×2 m³ steel-framed cages covered with a fine mesh. Parasitism and hyperparasitism rates were significantly higher in the presence of flowering buckwheat. Parasitism rates by A. ervi were lower but not significantly, in the presence of D. aphidum in buckwheat and buckwheat-free treatments. A. pisum density was significantly reduced by A. ervi when buckwheat was present, but the density of the aphid was not affected by the hyperparasitoid. The parasitoid's potential to reduce the host population was, therefore, significantly influenced by the presence of floral nectar. Although hyperparasitism rates were significantly increased by buckwheat, this did not ‘cascade’ to the second trophic level, the pea aphid. However, before floral resources are deployed in agro-ecosystems to enhance biological control of pests, the influence of flowers on the second and fourth trophic levels should always be considered.     

A Nonparametric Test Reveals Selection for Rapid Flowering in the Arabidopsis Genome

The detection of footprints of natural selection in genetic polymorphism data is fundamental to understanding the genetic basis of adaptation, and has important implications for human health. The standard approach has been to reject neutrality in favor of selection if the pattern of variation at a candidate locus was significantly different from the predictions of the standard neutral model. The problem is that the standard neutral model assumes more than just neutrality, and it is almost always possible to explain the data using an alternative neutral model with more complex demography. Today's wealth of genomic polymorphism data, however, makes it possible to dispense with models altogether by simply comparing the pattern observed at a candidate locus to the genomic pattern, and rejecting neutrality if the pattern is extreme. Here, we utilize this approach on a truly genomic scale, comparing a candidate locus to thousands of alleles throughout the Arabidopsis thaliana genome. We demonstrate that selection has acted to increase the frequency of early-flowering alleles at the vernalization requirement locus FRIGIDA. Selection seems to have occurred during the last several thousand years, possibly in response to the spread of agriculture. We introduce a novel test statistic based on haplotype sharing that embraces the problem of population structure, and so should be widely applicable.     

Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines

A new collection of 129 Arabidopsis thaliana RFLP markers has been established based upon DNA fragments cloned in the pUC119 plasmid vector and insert end sequences of P1 clones. Dominant/null alleles affecting low-copy number sequences account for nine of the mapped polymorphisms, suggesting that deletions are not rare in A. thaliana . Recombinant inbred (RI) lines were used for mapping these marker loci. RI line-based mapping allows integration of this set of markers with markers previously reported as well as with any markers mapped in the future using this replenishable mapping resource. These markers are useful for map-based gene isolation and genome physical mapping in A. thaliana as well as studies of chromosome colinearity (synteny) with related species.     

Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.     

The Mitochondrial Protein Import Machinery of Plants (MPIMP) database

The Mitochondrial Protein Import Machinery of Plants database (MPIMP) is an Internet-accessible database containing detailed information on the protein import apparatus of plant mitochondria. The Arabidopsis genome was searched for com-ponents of the mitochondrial protein import apparatus using components from the well-characterized model system of Saccharomyces cerevisiae. Twenty six homologues of 34 components could be found, encompassing the essential components for the general and carrier import pathways. The database is available through the Internet at http://millar3.biochem.uwa.edu.au/~lister/index.html.