Justin O. Borevitz

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Activation Tagging Identifies a Conserved MYB Regulator of Phenylpropanoid Biosynthesis

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among ∼5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

The extent of linkage disequilibrium in Arabidopsis thaliana

Linkage disequilibrium (LD), the nonrandom occurrence of alleles in haplotypes, has long been of interest to population geneticists. Recently, the rapidly increasing availability of genomic polymorphism data has fueled interest in LD as a tool for fine-scale mapping, in particular for human disease loci. The chromosomal extent of LD is crucial in this context, because it determines how dense a map must be for associations to be detected and, conversely, limits how finely loci may be mapped. Arabidopsis thaliana is expected to harbor unusually extensive LD because of its high degree of selfing. Several polymorphism studies have found very strong LD within individual loci, but also evidence of some recombination. Here we investigate the pattern of LD on a genomic scale and show that in global samples, LD decays within approximately 1 cM, or 250 kb. We also show that LD in local populations may be much stronger than that of global populations, presumably as a result of founder events. The combination of a relatively high level of polymorphism and extensive haplotype structure bodes well for developing a genome-wide LD map in A. thaliana.

Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel

Arabidopsis thaliana is native to Eurasia and is naturalized across the world. Its ability to be easily propagated and its high phenotypic variability make it an ideal model system for functional, ecological and evolutionary genetics. To date, analyses of the natural genetic variation of A. thaliana have involved small numbers of individual plants or genetic markers. Here we genotype 1,307 worldwide accessions, including several regional samples, using a 250K SNP chip. This allowed us to produce a high-resolution description of the global pattern of genetic variation. We applied three complementary selection tests and identified new targets of selection. Further, we characterized the pattern of historical recombination in A. thaliana and observed an enrichment of hotspots in its intergenic regions and repetitive DNA, which is consistent with the pattern that is observed for humans but which is strikingly different from that observed in other plant species. We have made the seeds we used to produce this Regional Mapping (RegMap) panel publicly available. This panel comprises one of the largest genomic mapping resources currently available for global natural isolates of a non-human species.

Natural variation in light sensitivity of Arabidopsis.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process—seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

An extracellular aspartic protease functions in Arabidopsis disease resistance signaling

We have used activation tagging with T‐DNA carrying cauliflower mosaic virus 35S enhancers to investigate the complex signaling networks underlying disease resistance in Arabidopsis. From a screen of ∼5000 lines, we identified constitutive disease resistance (CDR1) encoding an apoplastic aspartic protease, the overexpression of which causes dwarfing and resistance to virulent Pseudomonas syringae. These phenotypes reflect salicylic‐acid‐dependent activation of micro‐oxidative bursts and various defense‐related genes. Antisense CDR1 plants were compromised for resistance to avirulent P. syringae and more susceptible to virulent strains than wild type. CDR1 accumulates in intercellular fluid in response to pathogen attacks. Induction of CDR1 generates a small mobile signal, and CDR1 action is blocked by the protease inhibitor pepstatin and by mutations in the protease active sites. We propose that CDR1 mediates a peptide signal system involved in the activation of inducible resistance mechanisms.

The Scale of Population Structure in Arabidopsis thaliana

The population structure of an organism reflects its evolutionary history and influences its evolutionary trajectory. It constrains the combination of genetic diversity and reveals patterns of past gene flow. Understanding it is a prerequisite for detecting genomic regions under selection, predicting the effect of population disturbances, or modeling gene flow. This paper examines the detailed global population structure of Arabidopsis thaliana. Using a set of 5,707 plants collected from around the globe and genotyped at 149 SNPs, we show that while A. thaliana as a species self-fertilizes 97% of the time, there is considerable variation among local groups. This level of outcrossing greatly limits observed heterozygosity but is sufficient to generate considerable local haplotypic diversity. We also find that in its native Eurasian range A. thaliana exhibits continuous isolation by distance at every geographic scale without natural breaks corresponding to classical notions of populations. By contrast, in North America, where it exists as an exotic species, A. thaliana exhibits little or no population structure at a continental scale but local isolation by distance that extends hundreds of km. This suggests a pattern for the development of isolation by distance that can establish itself shortly after an organism fills a new habitat range. It also raises questions about the general applicability of many standard population genetics models. Any model based on discrete clusters of interchangeable individuals will be an uneasy fit to organisms like A. thaliana which exhibit continuous isolation by distance on many scales.

Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary ‘tug of war’, in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

Increased food and ecosystem security via perennial grains

Perennial grains hold promise, especially for marginal landscapes or with limited resources where annual versions struggle. Despite doubling of yields of major grain crops since the 1950s, more than one in seven people suffer from malnutrition (1). Global population is growing; demand for food, especially meat, is increasing; much land most suitable for annual crops is already in use; and production of nonfood goods (e.g., biofuels) increasingly competes with food production for land (2). The best lands have soils at low or moderate risk of degradation under annual grain production but make up only 12.6% of global land area (16.5 million km2) (3). Supporting more than 50% of world population is another 43.7 million km2 of marginal lands (33.5% of global land area), at high risk of degradation under annual grain production but otherwise capable of producing crops (3). Global food security depends on annual grains—cereals, oilseeds, and legumes—planted on almost 70% of croplands, which combined supply a similar portion of human calories (4, 5). Annual grain production, though, often compromises essential ecosystem services, pushing some beyond sustainable boundaries (5). To ensure food and ecosystem security, farmers need more options to produce grains under different, generally less favorable circumstances than those under which increases in food security were achieved this past century. Development of perennial versions of important grain crops could expand options.

Natural Selection Shapes Genome-Wide Patterns of Copy-Number Polymorphism in Drosophila melanogaster

The role that natural selection plays in governing the locations and early evolution of copy-number mutations remains largely unexplored. We used high-density full-genome tiling arrays to create a fine-scale genomic map of copy-number polymorphisms (CNPs) in Drosophila melanogaster. We inferred a total of 2658 independent CNPs, 56% of which overlap genes. These include CNPs that are likely to be under positive selection, most notably high-frequency duplications encompassing toxin-response genes. The locations and frequencies of CNPs are strongly shaped by purifying selection, with deletions under stronger purifying selection than duplications. Among duplications, those overlapping exons or introns, as well as those falling on the X chromosome, seem to be subject to stronger purifying selection.