Showing papers by "Detlef Weigel published in 2010"

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

Abstract: 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.

The Rate and Molecular Spectrum of Spontaneous Mutations in Arabidopsis thaliana

Abstract: To take complete advantage of information on within-species polymorphism and divergence from close relatives, one needs to know the rate and the molecular spectrum of spontaneous mutations. To this end, we have searched for de novo spontaneous mutations in the complete nuclear genomes of five Arabidopsis thaliana mutation accumulation lines that had been maintained by single-seed descent for 30 generations. We identified and validated 99 base substitutions and 17 small and large insertions and deletions. Our results imply a spontaneous mutation rate of 7 × 10−9 base substitutions per site per generation, the majority of which are G:C→A:T transitions. We explain this very biased spectrum of base substitution mutations as a result of two main processes: deamination of methylated cytosines and ultraviolet light–induced mutagenesis.

Control of cell proliferation in Arabidopsis thaliana by microRNA miR396

Abstract: Cell proliferation is an important determinant of plant form, but little is known about how developmental programs control cell division. Here, we describe the role of microRNA miR396 in the coordination of cell proliferation in Arabidopsis leaves. In leaf primordia, miR396 is expressed at low levels that steadily increase during organ development. We found that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 accumulates preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, coincides with the activity of the cell proliferation marker CYCLINB1;1. We show that miR396 attenuates cell proliferation in developing leaves, through the repression of GRF activity and a decrease in the expression of cell cycle genes. We observed that the balance between miR396 and the GRFs controls the final number of cells in leaves. Furthermore, overexpression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 severely compromises the shoot meristem. We found that miR396 is expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, we show that miR396 can regulate cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, have higher miR396 and lower GRF levels. These results implicate miR396 as a significant module in the regulation of cell proliferation in plants.

Control of cell proliferation in Arabidopsis thaliana by microRNA miR396

Abstract: Cell proliferation is an important determinant of plant form, but little is known about how developmental programs control cell division. Here, we describe the role of microRNA miR396 in the coordination of cell proliferation in Arabidopsis leaves. In leaf primordia, miR396 is expressed at low levels that steadily increase during organ development. We found that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 accumulates preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, coincides with the activity of the cell proliferation marker CYCLINB1;1. We show that miR396 attenuates cell proliferation in developing leaves, through the repression of GRF activity and a decrease in the expression of cell cycle genes. We observed that the balance between miR396 and the GRFs controls the final number of cells in leaves. Furthermore, overexpression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 severely compromises the shoot meristem. We found that miR396 is expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, we show that miR396 can regulate cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, have higher miR396 and lower GRF levels. These results implicate miR396 as a significant module in the regulation of cell proliferation in plants.

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

Abstract: 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.

The scale of population structure in Arabidopsis thaliana.

Abstract: 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.

A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana.

Abstract: Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

MicroRNA Gene Evolution in Arabidopsis Lyrata and Arabidopsis Thaliana

Abstract: MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (∼10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.

Increased leaf size: different means to an end

Abstract: The final size of plant organs, such as leaves, is tightly controlled by environmental and genetic factors that must spatially and temporally coordinate cell expansion and cell cycle activity However, this regulation of organ growth is still poorly understood The aim of this study is to gain more insight into the genetic control of leaf size in Arabidopsis (Arabidopsis thaliana) by performing a comparative analysis of transgenic lines that produce enlarged leaves under standardized environmental conditions To this end, we selected five genes belonging to different functional classes that all positively affect leaf size when overexpressed: AVP1, GRF5, JAW, BRI1, and GA20OX1 We show that the increase in leaf area in these lines depended on leaf position and growth conditions and that all five lines affected leaf size differently; however, in all cases, an increase in cell number was, entirely or predominantly, responsible for the leaf size enlargement By analyzing hormone levels, transcriptome, and metabolome, we provide deeper insight into the molecular basis of the growth phenotype for the individual lines A comparative analysis between these data sets indicates that enhanced organ growth is governed by different, seemingly independent pathways The analysis of transgenic lines simultaneously overexpressing two growth-enhancing genes further supports the concept that multiple pathways independently converge on organ size control in Arabidopsis

On reconciling the interactions between APETALA2 , miR172 and AGAMOUS with the ABC model of flower development

Abstract: The ABC model of flower development explains how three classes of homeotic genes confer identity to the four types of floral organs. In Arabidopsis thaliana, APETALA2 (AP2) and AGAMOUS (AG) represent A- and C-class genes that act in an antagonistic fashion to specify perianth and reproductive organs, respectively. An apparent paradox was the finding that AP2 mRNA is supposedly uniformly distributed throughout young floral primordia. Although miR172 has a role in preventing AP2 protein accumulation, miR172 was reported to disappear from the periphery only several days after AG activation in the center of the flower. Here, we resolve the enigmatic behavior of AP2 and its negative regulator miR172 through careful expression analyses. We find that AP2 mRNA accumulates predominantly in the outer floral whorls, as expected for an A-class homeotic gene. Its pattern overlaps only transiently with that of miR172, which we find to be restricted to the center of young floral primordia from early stages on. MiR172 also accumulates in the shoot meristem upon floral induction, compatible with its known role in regulating AP2-related genes with a role in flowering. Furthermore, we show that AP2 can cause striking organ proliferation defects that are not limited to the center of the floral meristem, where its antagonist AG is required for terminating stem cell proliferation. Moreover, AP2 never expands uniformly into the center of ag mutant flowers, while miR172 is largely unaffected by loss of AG activity. We present a model in which the decision whether stamens or petals develop is based on the balance between AP2 and AG activities, rather than the two being mutually exclusive.

Local-scale patterns of genetic variability, outcrossing, and spatial structure in natural stands of Arabidopsis thaliana

Abstract: As Arabidopsis thaliana is increasingly employed in evolutionary and ecological studies, it is essential to understand patterns of natural genetic variation and the forces that shape them. Previous work focusing mostly on global and regional scales has demonstrated the importance of historical events such as long-distance migration and colonization. Far less is known about the role of contemporary factors or environmental heterogeneity in generating diversity patterns at local scales. We sampled 1,005 individuals from 77 closely spaced stands in diverse settings around Tubingen, Germany. A set of 436 SNP markers was used to characterize genome-wide patterns of relatedness and recombination. Neighboring genotypes often shared mosaic blocks of alternating marker identity and divergence. We detected recent outcrossing as well as stretches of residual heterozygosity in largely homozygous recombinants. As has been observed for several other selfing species, there was considerable heterogeneity among sites in diversity and outcrossing, with rural stands exhibiting greater diversity and heterozygosity than urban stands. Fine-scale spatial structure was evident as well. Within stands, spatial structure correlated negatively with observed heterozygosity, suggesting that the high homozygosity of natural A. thaliana may be partially attributable to nearest-neighbor mating of related individuals. The large number of markers and extensive local sampling employed here afforded unusual power to characterize local genetic patterns. Contemporary processes such as ongoing outcrossing play an important role in determining distribution of genetic diversity at this scale. Local “outcrossing hotspots” appear to reshuffle genetic information at surprising rates, while other stands contribute comparatively little. Our findings have important implications for sampling and interpreting diversity among A. thaliana accessions.

The Role of the Arabidopsis Morning Loop Components CCA1, LHY, PRR7, and PRR9 in Temperature Compensation

Abstract: A defining, yet poorly understood characteristic of the circadian clock is that it is buffered against changes in temperature such that the period length is relatively constant across a range of physiologically relevant temperatures. We describe here the role of PSEUDO RESPONSE REGULATOR7 (PRR7) and PRR9 in temperature compensation. The Arabidopsis thaliana circadian oscillator comprises a series of interlocking feedback loops, and PRR7 and PRR9 function in the morning loop. The prr7 prr9 double mutant displays a unique phenotype that has not been observed before in other Arabidopsis clock mutants. In the prr7 prr9 mutant, the effects of temperature are overcompensated, apparently due to hyperactivation of the transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Inactivation of CCA1 and LHY fully suppresses the overcompensation defects of prr7 prr9 mutants and rescues their long period phenotype. Overcompensation in prr7 prr9 mutants does not rely on FLOWERING LOCUS C, a previously identified gene required for temperature compensation. Together, our results reveal a role of PRR7 and PRR9 in regulating CCA1 and LHY activities in response to ambient temperature.

Probing the Reproducibility of Leaf Growth and Molecular Phenotypes: A Comparison of Three Arabidopsis Accessions Cultivated in Ten Laboratories

Abstract: A major goal of the life sciences is to understand how molecular processes control phenotypes Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking Metabolite profiles derived from the same leaf displayed a strong genotype × environment (laboratory) component Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories

Genome-wide single nucleotide polymorphisms reveal population history and adaptive divergence in wild guppies.

Abstract: Adaptation of guppies (Poecilia reticulata) to contrasting upland and lowland habitats has been extensively studied with respect to behaviour, morphology and life history traits. Yet population history has not been studied at the whole-genome level. Although single nucleotide polymorphisms (SNPs) are the most abundant form of variation in many genomes and consequently very informative for a genome-wide picture of standing natural variation in populations, genome-wide SNP data are rarely available for wild vertebrates. Here we use genetically mapped SNP markers to comprehensively survey genetic variation within and among naturally occurring guppy populations from a wide geographic range in Trinidad and Venezuela. Results from three different clustering methods, Neighbor-net, principal component analysis (PCA) and Bayesian analysis show that the population substructure agrees with geographic separation and largely with previously hypothesized patterns of historical colonization. Within major drainages (Caroni, Oropouche and Northern), populations are genetically similar, but those in different geographic regions are highly divergent from one another, with some indications of ancient shared polymorphisms. Clear genomic signatures of a previous introduction experiment were seen, and we detected additional potential admixture events. Headwater populations were significantly less heterozygous than downstream populations. Pairwise F(ST) values revealed marked differences in allele frequencies among populations from different regions, and also among populations within the same region. F(ST) outlier methods indicated some regions of the genome as being under directional selection. Overall, this study demonstrates the power of a genome-wide SNP data set to inform for studies on natural variation, adaptation and evolution of wild populations.

Climate Change and the Integrity of Science

Abstract: We are deeply disturbed by the recent escalation of political assaults on scientists in general and on climate scientists in particular. All citizens should understand some basic scientific facts. There is always some uncertainty associated with scientific conclusions; science never absolutely proves anything. When someone says that society should wait until scientists are absolutely certain before taking any action, it is the same as saying society should never take action. For a problem as potentially catastrophic as climate change, taking no action poses a dangerous risk for our planet.

Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6.

Abstract: MADS-domain transcription factors play pivotal roles in various developmental processes. The lack of simple loss-of-function phenotypes provides impediments to understand the biological function of some of the MADS-box transcription factors. Here we have characterized the potential role of the Arabidopsis thaliana AGAMOUS-LIKE6 (AGL6) gene by fusing full-length coding sequence with transcriptional activator and repressor domains and suggest a role for AGL6 in lateral organ development and flowering. Upon photoperiodic induction of flowering, AGL6 becomes expressed in abaxial and proximal regions of cauline leaf primordia, as well as the cryptic bracts subtending flowers. In developing flowers, AGL6 is detected in the proximal regions of all floral organs and in developing ovules. Converting AGL6 into a strong activator through fusion to the VP16 domain triggers bract outgrowth, implicating AGL6 in the development of bractless flowers in Arabidopsis. In addition, ectopic reproductive structures form on both bracts and flowers in gAGL6::VP16 transgenic plants, which is dependent on B and C class homeotic genes, but independent of LEAFY. Overexpression of both AGL6 and its transcriptional repressor form, AGL6::EAR, causes precocious flowering and terminal flower formation, suggesting that AGL6 suppresses the function of a floral repressor.

STIMPY mediates cytokinin signaling during shoot meristem establishment in Arabidopsis seedlings

Abstract: The establishment of the primary meristems through proliferation after germination is essential for plant post-embryonic development. Cytokinins have long been considered a key regulator of plant cell division. Here we show that cytokinins are essential for early seedling development of Arabidopsis. Loss of cytokinin perception leads to a complete failure of meristem establishment and growth arrest after germination. We also present evidence that cytokinin signaling is involved in activation of the homeobox gene STIMPY (STIP or WOX9) expression in meristematic tissues, which is essential for maintaining the meristematic fate. Cytokinin-independent STIP expression is able to partially compensate for the shoot apical meristem growth defects in mutants that cannot sense cytokinin. These findings identify a new branch of the cytokinin signaling network, linking cytokinin to the process of meristem and seedling establishment.

AGRONOMICS1: A New Resource for Arabidopsis Transcriptome Profiling

Abstract: Transcriptome profiling has become a routine tool in biology. For Arabidopsis (Arabidopsis thaliana), the Affymetrix ATH1 expression array is most commonly used, but it lacks about one-third of all annotated genes present in the reference strain. An alternative are tiling arrays, but previous designs have not allowed the simultaneous analysis of both strands on a single array. We introduce AGRONOMICS1, a new Affymetrix Arabidopsis microarray that contains the complete paths of both genome strands, with on average one 25mer probe per 35-bp genome sequence window. In addition, the new AGRONOMICS1 array contains all perfect match probes from the original ATH1 array, allowing for seamless integration of the very large existing ATH1 knowledge base. The AGRONOMICS1 array can be used for diverse functional genomics applications such as reliable expression profiling of more than 30,000 genes, detection of alternative splicing, and chromatin immunoprecipitation coupled to microarrays (ChIP-chip). Here, we describe the design of the array and compare its performance with that of the ATH1 array. We find results from both microarrays to be of similar quality, but AGRONOMICS1 arrays yield robust expression information for many more genes, as expected. Analysis of the ATH1 probes on AGRONOMICS1 arrays produces results that closely mirror those of ATH1 arrays. Finally, the AGRONOMICS1 array is shown to be useful for ChIP-chip experiments. We show that heterochromatic H3K9me2 is strongly confined to the gene body of target genes in euchromatic chromosome regions, suggesting that spreading of heterochromatin is limited outside of pericentromeric regions.

Ten years of genetics and genomics: what have we achieved and where are we heading?

Abstract: To celebrate the first 10 years of Nature Reviews Genetics, we asked eight leading researchers for their views on the key developments in genetics and genomics in the past decade and the prospects for the future. Their responses highlight the incredible changes that the field has seen, from the explosion of genomic data and the many possibilities it has opened up to the ability to reprogramme adult cells to pluripotency. The way ahead looks similarly exciting as we address questions such as how cells function as systems and how complex interactions among genetics, epigenetics and the environment combine to shape phenotypes.

Global effects of the small RNA biogenesis machinery on the Arabidopsis thaliana transcriptome

Abstract: In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation.

Directed gene silencing with artificial microRNAs.

Abstract: The characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertion collections provide a large resource of potential null alleles that can often be easily accessed through convenient Web sites (e.g., http://signal.salk.edu ). They are, however, not available for nonmodel species, require stacking for knockout of redundant homologs, and do not easily allow for partial or regulated loss of gene function, which is particularly useful when null alleles are lethal. Transgene approaches that employ directed gene silencing can substitute for null alleles and also enable refined studies of gene function, e.g., by tissue-specific and inducible gene-silencing. This chapter describes the generation and application of artificial microRNAs (amiRNAs) as a gene silencing tool in a wide variety of different plant species.

Transient assays for the analysis of miRNA processing and function.

Abstract: Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Agroinfiltration has been used successfully in many different applications, including the analysis of small RNAs. We describe here a protocol for analysis of miRNA processing using agroinfiltration of N. benthamiana leaves.

Identification of a spontaneous frame shift mutation in a nonreference Arabidopsis accession using whole genome sequencing.

Abstract: Short-read sequencing technologies support the facile de novo identification of spontaneous and chemically induced mutations in the Arabidopsis ( Arabidopsis thaliana ) reference genome ([Schneeberger et al., 2009b][1]; [Ossowski et al., 2010][2]). Here, we show that short-read sequencing is also

Natural Diversity in Flowering Responses of Arabidopsis thaliana Caused by Variation in a Tandem Gene Array

Abstract: Tandemly arrayed genes that belong to gene families characterize genomes of many organisms. Gene duplication and subsequent relaxation of selection can lead to the establishment of paralogous cluster members that may evolve along different trajectories. Here, we report on the structural variation in MADS AFFECTING FLOWERING 2 (MAF2) gene, one member of the tandemly duplicated cluster of MADS-box-containing transcription factors in Arabidopsis thaliana. The altered gene structure at the MAF2 locus is present as a moderate-frequency polymorphism in Arabidopsis and leads to the extensive diversity in transcript patterns due to alternative splicing. Rearrangements at the MAF2 locus are associated with an early flowering phenotype in BC5 lines. The lack of suppression of flowering time in a MAF2-insertion line expressing the MAF2-specific artificial miRNA suggests that these MAF2 variants are behaving as loss-of-function alleles. The variation in gene architecture is also associated with segregation distortion, which may have facilitated the spread and the establishment of the corresponding alleles throughout the Eurasian range of the A. thaliana population.

Arabidopsis and relatives as models for the study of genetic and genomic incompatibilities

Abstract: The past few years have seen considerable advances in speciation research, but whether drift or adaptation is more likely to lead to genetic incompatibilities remains unknown. Some of the answers will probably come from not only studying incompatibilities between well-established species, but also from investigating incipient speciation events, to learn more about speciation as an evolutionary process. The genus Arabidopsis, which includes the widely used Arabidopsis thaliana, provides a useful set of model species for studying many aspects of population divergence. The genus contains both self-incompatible and incompatible species, providing a platform for studying the impact of mating system changes on genetic differentiation. Another important path to plant speciation is via formation of polyploids, and this can be investigated in the young allotetraploid species A. arenosa. Finally, there are many cases of intraspecific incompatibilities in A. thaliana, and recent progress has been made in discovering the genes underlying both F(1) and F(2) breakdown. In the near future, all these studies will be greatly empowered by complete genome sequences not only for all members of this relatively small genus, but also for many different individuals within each species.

The use of class iib restriction endonucleases in 2nd generation sequencing applications

Abstract: The present invention relates to a method for genotyping DNA molecules contained in at least one DNA sample. The method includes: (a) digesting the DNA molecules contained in at least one DNA sample with a class IIB restriction endonuclease to generate DNA fragments; (b) optionally separating DNA fragments comprising the recognition site for the class IIB restriction endonuclease from the remaining DNA fragments; (c) attaching at least one adaptor DNA to the 5′ and/or 3′ end of one or both strands of the DNA fragments comprising the recognition site for the class IIB restriction endonuclease obtained in a) or separated in b) to form adaptor-fragment constructs; (d) determining the sequence of at least a fraction of the DNA fragments obtained in c); and (e) assigning genotypes to the at least one DNA sample analyzed based on the sequence data obtained in d). The present invention further relates to method for determining the position of DNA molecules comprised in a DNA library within the DNA sequence represented by the DNA library or within a known DNA sequence and for establishing a cross-reference between individual DNA molecules and their location in an at least three dimensional matrix.