Revisiting a GWAS peak in Arabidopsis thaliana reveals possible confounding by genetic heterogeneity.

Genome-wide association studies (GWAS) have become a standard approach for exploring the genetic basis of phenotypic variation. However, correlation is not causation, and only a tiny fraction of all associations have been experimentally confirmed. One practical problem is that a peak of association does not always pinpoint a causal gene, but may instead be tagging multiple causal variants. In this study, we reanalyze a previously reported peak associated with flowering time traits in Swedish Arabidopsis thaliana population. The peak appeared to pinpoint the AOP2/AOP3 cluster of glucosinolate biosynthesis genes, which is known to be responsible for natural variation in herbivore resistance. Here we propose an alternative hypothesis, by demonstrating that the AOP2/AOP3 flowering association can be wholly accounted for by allelic variation in two flanking genes with clear roles in regulating flowering: NDX1, a regulator of the main flowering time controller FLC, and GA1, which plays a central role in gibberellin synthesis and is required for flowering under some conditions. In other words, we propose that the AOP2/AOP3 flowering-time association may be yet another example of a spurious, "synthetic" association, arising from trying to fit a single-locus model in the presence of two statistically associated causative loci. We conclude that caution is needed when using GWAS for fine-mapping.

A genome-wide association study identifies variants underlying the Arabidopsis thaliana shade avoidance response.

Shade avoidance is an ecologically and molecularly well-understood set of plant developmental responses that occur when the ratio of red to far-red light (R:FR) is reduced as a result of foliar shade. Here, a genome-wide association study (GWAS) in Arabidopsis thaliana was used to identify variants underlying one of these responses: increased hypocotyl elongation. Four hypocotyl phenotypes were included in the study, including height in high R:FR conditions (simulated sun), height in low R:FR conditions (simulated shade), and two different indices of the response of height to low R:FR. GWAS results showed that variation in these traits is controlled by many loci of small to moderate effect. A known PHYC variant contributing to hypocotyl height variation was identified and lists of significantly associated genes were enriched in a priori candidates, suggesting that this GWAS was capable of generating meaningful results. Using metadata such as expression data, GO terms, and other annotation, we were also able to identify variants in candidate de novo genes. Patterns of significance among our four phenotypes allowed us to categorize associations into three groups: those that affected hypocotyl height without influencing shade avoidance, those that affected shade avoidance in a height-dependent fashion, and those that exerted specific control over shade avoidance. This grouping allowed for the development of explicit hypotheses about the genetics underlying shade avoidance variation. Additionally, the response to shade did not exhibit any marked geographic distribution, suggesting that variation in low R:FR-induced hypocotyl elongation may represent a response to local conditions.

Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping.

Quantitative trait loci (QTL) mapping is a powerful tool for investigating the genetic basis of natural variation. QTL can be mapped using a number of different population designs, but recombinant inbred lines (RILs) are among the most effective. Unfortunately, homozygous RIL populations are time consuming to construct, typically requiring at least six generations of selfing starting from a heterozygous F(1). Haploid plants produced from an F(1) combine the two parental genomes and have only one allele at every locus. Converting these sterile haploids into fertile diploids (termed "doubled haploids," DHs) produces immortal homozygous lines in only two steps. Here we describe a unique technique for rapidly creating recombinant doubled haploid populations in Arabidopsis thaliana: centromere-mediated genome elimination. We generated a population of 238 doubled haploid lines that combine two parental genomes and genotyped them by reduced representation Illumina sequencing. The recombination rate and parental allele frequencies in our population are similar to those found in existing RIL sets. We phenotyped this population for traits related to flowering time and for petiole length and successfully mapped QTL controlling each trait. Our work demonstrates that doubled haploid populations offer a rapid, easy alternative to RILs for Arabidopsis genetic analysis.

Amino acid polymorphisms in Arabidopsis phytochrome B cause differential responses to light.

Plants have a sophisticated system for sensing and responding to their light environment. The light responses of populations and species native to different habitats show adaptive variation; understanding the mechanisms underlying photomorphogenic variation is therefore of significant interest. In Arabidopsis thaliana, phytochrome B (PHYB) is the dominant photoreceptor for red light and plays a major role in white light. Because PHYB has been proposed as a candidate gene for several quantitative trait loci (QTLs) affecting light response, we have investigated sequence and functional variation in Arabidopsis PHYB. We examined PHYB sequences in 33 A. thaliana individuals and in the close relative Arabidopsis lyrata. From 14 nonsynonymous polymorphisms, we chose 5 for further study based on previous QTL studies. In a larger collection of A. thaliana accessions, one of these five polymorphisms, I143L, was associated with variation in red light response. We used transgenic analysis to test this association and confirmed experimentally that natural PHYB polymorphisms cause differential plant responses to light. Furthermore, our results show that allelic variation of PHYB activity is due to amino acid rather than regulatory changes. Together with earlier studies linking variation in light sensitivity to photoreceptor genes, our work suggests that photoreceptors may be a common target of natural selection.

The Aquilegia genome provides insight into adaptive radiation and reveals an extraordinarily polymorphic chromosome with a unique history.

The columbine genus Aquilegia is a classic example of an adaptive radiation, involving a wide variety of pollinators and habitats. Here we present the genome assembly of A. coerulea 'Goldsmith', complemented by high-coverage sequencing data from 10 wild species covering the world-wide distribution. Our analyses reveal extensive allele sharing among species and demonstrate that introgression and selection played a role in the Aquilegia radiation. We also present the remarkable discovery that the evolutionary history of an entire chromosome differs from that of the rest of the genome - a phenomenon that we do not fully understand, but which highlights the need to consider chromosomes in an evolutionary context.

The Generation of Doubled Haploid Lines for QTL Mapping.

Recombinant inbred lines (RILs) are an essential tool for quantitative trait locus (QTL) mapping in Arabidopsis thaliana. Conventionally, the development of these lines is a time-consuming and tedious process requiring six to eight generations of selfing. Here, we describe an alternative approach: the rapid generation of RILs in A. thaliana via the creation of doubled haploids. In this method, F1 plants are crossed to an engineered haploid inducer to produce haploid plants. The chromosomes of these haploids then spontaneously double, generating immortalized homozygous F2 lines called doubled haploid RILs (DH RILs). Finally, DH RILs are genotyped using low-coverage whole-genome sequencing and are ready to be used for QTL mapping.

Verification of Arabidopsis stock collections using SNPmatch, a tool for genotyping high-plexed samples.

Large-scale studies such as the Arabidopsis thaliana '1,001 Genomes' Project require routine genotyping of stocks to avoid sample contamination. To genotype samples efficiently and economically, sequencing must be inexpensive and data processing simple. Here we present SNPmatch, a tool that identifies strains (or inbred lines, or accessions) by matching them to a SNP database. We tested the tool by performing low-coverage resequencing of over 2,000 strains from our lab seed stock collection. SNPmatch correctly genotyped samples from 1-fold coverage sequencing data, and could also identify the parents of F1 or F2 individuals. SNPmatch can be run either on the command line or through AraGeno (https://arageno.gmi.oeaw.ac.at), a web interface that permits sample genotyping from a user-uploaded VCF or BED file.

Multiple alleles at a single locus control seed dormancy in Swedish Arabidopsis.

Seed dormancy is a complex life history trait that determines the timing of germination and is crucial for local adaptation. Genetic studies of dormancy are challenging, because the trait is highly plastic and strongly influenced by the maternal environment. Using a combination of statistical and experimental approaches, we show that multiple alleles at the previously identified dormancy locus DELAY OF GERMINATION1 jointly explain as much as 57% of the variation observed in Swedish Arabidopsis thaliana, but give rise to spurious associations that seriously mislead genome-wide association studies unless modeled correctly. Field experiments confirm that the major alleles affect germination as well as survival under natural conditions, and demonstrate that locally adaptive traits can sometimes be dissected genetically.

Native environment modulates leaf size and response to simulated foliar shade across wild tomato species.

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits.