A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2.

Variation of flowering time is found in the natural populations of many plant species. The underlying genetic variation, mostly of a quantitative nature, is presumed to reflect adaptations to different environments contributing to reproductive success. Analysis of natural variation for flowering time in Arabidopsis thaliana has identified several quantitative trait loci (QTL), which have yet to be characterized at the molecular level. A major environmental factor that determines flowering time is photoperiod or day length, the length of the light period, which changes across the year differently with geographical latitude. We identified the EDI locus as a QTL partly accounting for the difference in flowering response to the photoperiod between two Arabidopsis accessions: the laboratory strain Landsberg erecta (Ler), originating in Northern Europe, and Cvi, collected in the tropical Cape Verde Islands. Positional cloning of the EDI QTL showed it to be a novel allele of CRY2, encoding the blue-light photoreceptor cryptochrome-2 that has previously been shown to promote flowering in long-day (LD) photoperiods. We show that the unique EDI flowering phenotype results from a single amino-acid substitution that reduces the light-induced downregulation of CRY2 in plants grown under short photoperiods, leading to early flowering.     

Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2

Most agronomic traits of importance, whether physiological (such as nutrient use efficiency) or developmental (such as flowering time), are controlled simultaneously by multiple genes and their interactions with the environment. Here, we show that variation in sulfate content between wild Arabidopsis thaliana accessions Bay-0 and Shahdara is controlled by a major quantitative trait locus that results in a strong interaction with nitrogen availability in the soil. Combining genetic and biochemical results and using a candidate gene approach, we have cloned the underlying gene, showing how a single-amino acid substitution in a key enzyme of the assimilatory sulfate reduction pathway, adenosine 5'-phosphosulfate reductase, is responsible for a decrease in enzyme activity, leading to sulfate accumulation in the plant. This work illustrates the potential of natural variation as a source of new alleles of known genes, which can aid in the study of gene function and metabolic pathway regulation. Our new insights on sulfate assimilation may have an impact on sulfur fertilizer use and stress defense improvement.     

A thermosensory pathway controlling flowering time in Arabidopsis thaliana

Onset of flowering is controlled by environmental signals such as light and temperature. Molecular-genetic studies in Arabidopsis thaliana have focused on daily light duration, or photoperiod, and transient exposure to winter-like temperatures, or vernalization. Yet ambient growth temperature, which is strongly affected by current changes in global climate, has been largely ignored. Here, we show that genes of the autonomous pathway, previously thought only to act independently of the environment as regulators of the floral repressor FLC (ref. 1), are centrally involved in mediating the effects of ambient temperature. In contrast to wild-type plants and those mutant in other pathways, autonomous-pathway mutants flower at the same time regardless of ambient temperature. In contrast, the exaggerated temperature response of cryptochrome-2 mutants is caused by temperature-dependent redundancy with the phytochrome A photoreceptor. As with vernalization and photoperiod, ambient temperature ultimately affects expression of the floral pathway integrator FT.     

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.     

Natural variation at Strubbelig Receptor Kinase 3 drives immune-triggered incompatibilities between Arabidopsis thaliana accessions

Accumulation of genetic incompatibilities within species can lead to reproductive isolation and, potentially, speciation. In this study, we show that allelic variation at SRF3 (Strubbelig Receptor Family 3), encoding a receptor-like kinase, conditions the occurrence of incompatibility between Arabidopsis thaliana accessions. The geographical distribution of SRF3 alleles reveals that allelic forms causing epistatic incompatibility with a Landsberg erecta allele at the RPP1 resistance locus are present in A. thaliana accessions in central Asia. Incompatible SRF3 alleles condition for an enhanced early immune response to pathogens as compared to the resistance-dampening effect of compatible SRF3 forms in isogenic backgrounds. Variation in disease susceptibility suggests a basis for the molecular patterns of a recent selective sweep detected at the SRF3 locus in central Asian populations.     

A mixed-model approach for genome-wide association studies of correlated traits in structured populations

Genome-wide association studies (GWAS) are a standard approach for studying the genetics of natural variation. A major concern in GWAS is the need to account for the complicated dependence structure of the data, both between loci as well as between individuals. Mixed models have emerged as a general and flexible approach for correcting for population structure in GWAS. Here, we extend this linear mixed-model approach to carry out GWAS of correlated phenotypes, deriving a fully parameterized multi-trait mixed model (MTMM) that considers both the within-trait and between-trait variance components simultaneously for multiple traits. We apply this to data from a human cohort for correlated blood lipid traits from the Northern Finland Birth Cohort 1966 and show greatly increased power to detect pleiotropic loci that affect more than one blood lipid trait. We also apply this approach to an Arabidopsis thaliana data set for flowering measurements in two different locations, identifying loci whose effect depends on the environment.     

The genetics of plant metabolism

Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography-time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.     

Dwarf8 polymorphisms associate with variation in flowering time.

Historically, association tests have been used extensively in medical genetics, but have had virtually no application in plant genetics. One obstacle to their application is the structured populations often found in crop plants, which may lead to nonfunctional, spurious associations. In this study, statistical methods to account for population structure were extended for use with quantitative variation and applied to our evaluation of maize flowering time. Mutagenesis and quantitative trait locus (QTL) studies suggested that the maize gene Dwarf8 might affect the quantitative variation of maize flowering time and plant height. The wheat orthologs of this gene contributed to the increased yields seen in the 'Green Revolution' varieties. We used association approaches to evaluate Dwarf8 sequence polymorphisms from 92 maize inbred lines. Population structure was estimated using a Bayesian analysis of 141 simple sequence repeat (SSR) loci. Our results indicate that a suite of polymorphisms associate with differences in flowering time, which include a deletion that may alter a key domain in the coding region. The distribution of nonsynonymous polymorphisms suggests that Dwarf8 has been a target of selection.     

Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1

Vernalization is the process by which sensing a prolonged exposure to winter cold leads to competence to flower in the spring. In winter annual Arabidopsis thaliana accessions, flowering is suppressed in the fall by expression of the potent floral repressor FLOWERING LOCUS C (FLC). Vernalization promotes flowering via epigenetic repression of FLC. Repression is accompanied by a series of histone modifications of FLC chromatin that include dimethylation of histone H3 at Lys9 (H3K9) and Lys27 (H3K27). Here, we report that A. thaliana LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) is necessary to maintain the epigenetically repressed state of FLC upon return to warm conditions typical of spring. LHP1 is enriched at FLC chromatin after prolonged exposure to cold, and LHP1 activity is needed to maintain the increased levels of H3K9 dimethylation at FLC chromatin that are characteristic of the vernalized state.     

A complete BAC-based physical map of the Arabidopsis thaliana genome.

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.     

A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening

A major component in the regulatory network controlling fruit ripening is likely to be the gene at the tomato Colorless non-ripening (Cnr) locus. The Cnr mutation results in colorless fruits with a substantial loss of cell-to-cell adhesion. The nature of the mutation and the identity of the Cnr gene were previously unknown. Using positional cloning and virus-induced gene silencing, here we demonstrate that an SBP-box (SQUAMOSA promoter binding protein-like) gene resides at the Cnr locus. Furthermore, the Cnr phenotype results from a spontaneous epigenetic change in the SBP-box promoter. The discovery that Cnr is an epimutation was unexpected, as very few spontaneous epimutations have been described in plants. This study demonstrates that an SBP-box gene is critical for normal ripening and highlights the likely importance of epialleles in plant development and the generation of natural variation.     

Whole-genome sequencing of multiple Arabidopsis thaliana populations

The plant Arabidopsis thaliana occurs naturally in many different habitats throughout Eurasia. As a foundation for identifying genetic variation contributing to adaptation to diverse environments, a 1001 Genomes Project to sequence geographically diverse A. thaliana strains has been initiated. Here we present the first phase of this project, based on population-scale sequencing of 80 strains drawn from eight regions throughout the species' native range. We describe the majority of common small-scale polymorphisms as well as many larger insertions and deletions in the A. thaliana pan-genome, their effects on gene function, and the patterns of local and global linkage among these variants. The action of processes other than spontaneous mutation is identified by comparing the spectrum of mutations that have accumulated since A. thaliana diverged from its closest relative 10 million years ago with the spectrum observed in the laboratory. Recent species-wide selective sweeps are rare, and potentially deleterious mutations are more common in marginal populations.     

Root tip contact with low-phosphate media reprograms plant root architecture

Plant roots are able to sense soil nutrient availability. In order to acquire heterogeneously distributed water and minerals, they optimize their root architecture. One poorly understood plant response to soil phosphate (P(i)) deficiency is a reduction in primary root growth with an increase in the number and length of lateral roots. Here we show that physical contact of the Arabidopsis thaliana primary root tip with low-P(i) medium is necessary and sufficient to arrest root growth. We further show that loss-of-function mutations in Low Phosphate Root1 (LPR1) and its close paralog LPR2 strongly reduce this inhibition. LPR1 was previously mapped as a major quantitative trait locus (QTL); the molecular origin of this QTL is explained by the differential allelic expression of LPR1 in the root cap. These results provide strong evidence for the involvement of the root cap in sensing nutrient deficiency, responding to it, or both. LPR1 and LPR2 encode multicopper oxidases (MCOs), highlighting the essential role of MCOs for plant development.     

Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus

We previously mapped the type 2 diabetes mellitus-2 locus (T2dm2), which affects fasting insulin levels, to distal chromosome 19 in a leptin-deficient obese F2 intercross derived from C57BL/6 (B6) and BTBR T+ tf/J (BTBR) mice. Introgression of a 7-Mb segment of the B6 chromosome 19 into the BTBR background (strain 1339A) replicated the reduced insulin linked to T2dm2. The 1339A mice have markedly impaired insulin secretion in vivo and disrupted islet morphology. We used subcongenic strains derived from 1339A to localize the T2dm2 quantitative trait locus (QTL) to a 242-kb segment comprising the promoter, first exon and most of the first intron of the Sorcs1 gene. This was the only gene in the 1339A strain for which we detected amino acid substitutions and expression level differences between mice carrying B6 and BTBR alleles of this insert, thereby identifying variation within the Sorcs1 gene as underlying the phenotype associated with the T2dm2 locus. SorCS1 binds platelet-derived growth factor, a growth factor crucial for pericyte recruitment to the microvasculature, and may thus have a role in expanding or maintaining the islet vasculature. Our identification of the Sorcs1 gene provides insight into the pathway underlying the pathophysiology of obesity-induced type 2 diabetes mellitus.     

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.     

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.