A commitment for life: Decades of unraveling the molecular mechanisms behind seed dormancy and germination.

Seeds are unique time capsules that can switch between 2 complex and highly interlinked stages: seed dormancy and germination. Dormancy contributes to the survival of plants because it allows to delay germination to optimal conditions. The switch between dormancy and germination occurs in response to developmental and environmental cues. In this review we provide a comprehensive overview of studies that have helped to unravel the molecular mechanisms underlying dormancy and germination over the last decades. Genetic and physiological studies provided a strong foundation for this field of research and revealed the critical role of the plant hormones abscisic acid and gibberellins in the regulation of dormancy and germination, and later natural variation studies together with quantitative genetics identified previously unknown genetic components that control these processes. Omics technologies like transcriptome, proteome, and translatomics analysis allowed us to mechanistically dissect these processes and identify new components in the regulation of seed dormancy and germination.

The Evolutionary Dynamics of Genetic Incompatibilities Introduced by Duplicated Genes in Arabidopsis thaliana.

Although gene duplications provide genetic backup and allow genomic changes under relaxed selection, they may potentially limit gene flow. When different copies of a duplicated gene are pseudofunctionalized in different genotypes, genetic incompatibilities can arise in their hybrid offspring. Although such cases have been reported after manual crosses, it remains unclear whether they occur in nature and how they affect natural populations. Here, we identified four duplicated-gene based incompatibilities including one previously not reported within an artificial Arabidopsis intercross population. Unexpectedly, however, for each of the genetic incompatibilities we also identified the incompatible alleles in natural populations based on the genomes of 1,135 Arabidopsis accessions published by the 1001 Genomes Project. Using the presence of incompatible allele combinations as phenotypes for GWAS, we mapped genomic regions that included additional gene copies which likely rescue the genetic incompatibility. Reconstructing the geographic origins and evolutionary trajectories of the individual alleles suggested that incompatible alleles frequently coexist, even in geographically closed regions, and that their effects can be overcome by additional gene copies collectively shaping the evolutionary dynamics of duplicated genes during population history.

Construction and analysis of a Noccaea caerulescens TILLING population.

BACKGROUND: Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens. RESULTS: To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus. CONCLUSIONS: The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.

Mutations in EID1 and LNK2 caused light-conditional clock deceleration during tomato domestication.

Circadian period and phase of cultivated tomato (Solanum lycopersicum) were changed during domestication, likely adapting the species to its new agricultural environments. Whereas the delayed circadian phase is mainly caused by allelic variation of EID1, the genetic basis of the long circadian period has remained elusive. Here we show that a partial deletion of the clock gene LNK2 is responsible for the period lengthening in cultivated tomatoes. We use resequencing data to phylogenetically classify hundreds of tomato accessions and investigate the evolution of the eid1 and lnk2 mutations along successive domestication steps. We reveal signatures of selection across the genomic region of LNK2 and different patterns of fixation of the mutant alleles. Strikingly, LNK2 and EID1 are both involved in light input to the circadian clock, indicating that domestication specifically targeted this input pathway. In line with this, we show that the clock deceleration in the cultivated tomato is light-dependent and requires the phytochrome B1 photoreceptor. Such conditional variation in circadian rhythms may be key for latitudinal adaptation in a variety of species, including crop plants and livestock.

Madeiran Arabidopsis thaliana Reveals Ancient Long-Range Colonization and Clarifies Demography in Eurasia.

The study of model organisms on islands may shed light on rare long-range dispersal events, uncover signatures of local evolutionary processes, and inform demographic inference on the mainland. Here, we sequenced the genomes of Arabidopsis thaliana samples from the oceanic island of Madeira. These samples include the most diverged worldwide, likely a result of long isolation on the island. We infer that colonization of Madeira happened between 70 and 85 ka, consistent with a propagule dispersal model (of size ≥10), or with an ecological window of opportunity. This represents a clear example of a natural long-range dispersal event in A. thaliana. Long-term effective population size on the island, rather than the founder effect, had the greatest impact on levels of diversity, and rates of coalescence. Our results uncover a selective sweep signature on the ancestral haplotype of a known translocation in Eurasia, as well as the possible importance of the low phosphorous availability in volcanic soils, and altitude, in shaping early adaptations to the island conditions. Madeiran genomes, sheltered from the complexities of continental demography, help illuminate ancient demographic events in Eurasia. Our data support a model in which two separate lineages of A. thaliana, one originating in Africa and the other from the Caucasus expanded and met in Iberia, resulting in a secondary contact zone there. Although previous studies inferred that the westward expansion of A. thaliana coincided with the spread of human agriculture, our results suggest that it happened much earlier (20-40 ka).

QTL and candidate genes associated with leaf anion concentrations in response to phosphate supply in Arabidopsis thaliana.

BACKGROUND: Phosphorus is often present naturally in the soil as inorganic phosphate, Pi, which bio-availability is limited in many ecosystems due to low soil solubility and mobility. Plants respond to low Pi with a Pi Starvation Response, involving Pi sensing and long-distance signalling. There is extensive cross-talk between Pi homeostasis mechanisms and the homeostasis mechanism for other anions in response to Pi availability. RESULTS: Recombinant Inbred Line (RIL) and Genome Wide Association (GWA) mapping populations, derived from or composed of natural accessions of Arabidopsis thaliana, were grown under sufficient and deficient Pi supply. Significant treatment effects were found for all traits and significant genotype x treatment interactions for the leaf Pi and sulphate concentrations. Using the RIL/QTL population, we identified 24 QTLs for leaf concentrations of Pi and other anions, including a major QTL for leaf sulphate concentration (SUL2) mapped to the bottom of chromosome (Chr) 1. GWA mapping found 188 SNPs to be associated with the measured traits, corresponding to 152 genes. One of these SNPs, associated with leaf Pi concentration, mapped to PP2A-1, a gene encoding an isoform of the catalytic subunit of a protein phosphatase 2A. Of two additional SNPs, associated with phosphate use efficiency (PUE), one mapped to AT5G49780, encoding a leucine-rich repeat protein kinase involved in signal transduction, and the other to SIZ1, a gene encoding a SUMO E3 ligase, and a known regulator of P starvation-dependent responses. One SNP associated with leaf sulphate concentration was found in SULTR2;1, encoding a sulphate transporter, known to enhance sulphate translocation from root to shoot under P deficiency. Finally, one SNP was mapped to FMO GS-OX4, a gene encoding glucosinolate S-oxygenase involved in glucosinolate biosynthesis, which located within the confidence interval of the SUL2 locus. CONCLUSION: We identified several candidate genes with known functions related to anion homeostasis in response to Pi availability. Further molecular studies are needed to confirm and validate these candidate genes and understand their roles in examined traits. Such knowledge will contribute to future breeding for improved crop PUE .

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

Six-Rowed Spike3 (VRS3) Is a Histone Demethylase That Controls Lateral Spikelet Development in Barley.

The complex nature of crop genomes has long prohibited the efficient isolation of agronomically relevant genes. However, recent advances in next-generation sequencing technologies provide new ways to accelerate fine-mapping and gene isolation in crops. We used RNA sequencing of allelic six-rowed spike3 (vrs3) mutants with altered spikelet development for gene identification and functional analysis in barley (Hordeum vulgare). Variant calling in two allelic vrs3 mutants revealed that VRS3 encodes a putative histone Lys demethylase with a conserved zinc finger and Jumonji C and N domain. Sanger sequencing of this candidate gene in independent allelic vrs3 mutants revealed a series of mutations in conserved domains, thus confirming our candidate as the VRS3 gene and suggesting that the row type in barley is determined epigenetically. Global transcriptional profiling in developing shoot apical meristems of vrs3 suggested that VRS3 acts as a transcriptional activator of the row-type genes VRS1 (Hv.HOMEOBOX1) and INTERMEDIUM-C (INT-C; Hv.TEOSINTE BRANCHED1). Comparative transcriptome analysis of the row-type mutants vrs3, vrs4 (Hv.RAMOSA2), and int-c confirmed that all three genes act as transcriptional activators of VRS1 and quantitative variation in the expression levels of VRS1 in these mutants correlated with differences in the number of developed lateral spikelets. The identification of genes and pathways affecting seed number in small grain cereals will enable to further unravel the transcriptional networks controlling this important yield component.

Genes for seed longevity in barley identified by genomic analysis on near isogenic lines.

Genes controlling differences in seed longevity between 2 barley (Hordeum vulgare) accessions were identified by combining quantitative genetics "omics" technologies in near isogenic lines (NILs). The NILs were derived from crosses between the spring barley landraces L94 from Ethiopia and Cebada Capa from Argentina. A combined transcriptome and proteome analysis on mature, nonaged seeds of the 2 parental lines and the L94 NILs by RNA-sequencing and total seed proteomic profiling identified the UDP-glycosyltransferase MLOC_11661.1 as candidate gene for the quantitative trait loci on 2H, and the NADP-dependent malic enzyme (NADP-ME) MLOC_35785.1 as possible downstream target gene. To validate these candidates, they were expressed in Arabidopsis under the control of constitutive promoters to attempt complementing the T-DNA knockout line nadp-me1. Both the NADP-ME MLOC_35785.1 and the UDP-glycosyltransferase MLOC_11661.1 were able to rescue the nadp-me1 seed longevity phenotype. In the case of the UDP-glycosyltransferase, with high accumulation in NILs, only the coding sequence of Cebada Capa had a rescue effect.

Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana.

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17-Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe approximately 5000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The 'American haplotype' was identified from several European localities, potentially due to return migration.

The Footprint of Polygenic Adaptation on Stress-Responsive Cis-Regulatory Divergence in the Arabidopsis Genus.

Adaptation of a complex trait often requires the accumulation of many modifications to finely tune its underpinning molecular components to novel environmental requirements. The investigation of cis-acting regulatory modifications can be used to pinpoint molecular systems partaking in such complex adaptations. Here, we identify cis-acting modifications with the help of an interspecific crossing scheme designed to distinguish modifications derived in each of the two sister species, Arabidopsis halleri and A. lyrata Allele-specific expression levels were assessed in three environmental conditions chosen to reflect interspecific ecological differences: cold exposure, dehydration, and standard conditions. The functions described by Gene Ontology categories enriched in cis-acting mutations are markedly different in A. halleri and A. lyrata, suggesting that polygenic adaptation reshaped distinct polygenic molecular functions in the two species. In the A. halleri lineage, an excess of cis-acting changes affecting metal transport and homeostasis was observed, confirming that the well-known heavy metal tolerance of this species is the result of polygenic selection. In A. lyrata, we find a marked excess of cis-acting changes among genes showing a transcriptional response to cold stress in the outgroup species A. thaliana The adaptive relevance of these changes will have to be validated. We finally observed that polygenic molecular functions enriched in derived cis-acting changes are more constrained at the amino acid level. Using the distribution of cis-acting variation to tackle the polygenic basis of adaptation thus reveals the contribution of mutations of small effect to Darwinian adaptation.

Quantitative trait loci controlling leaf venation in Arabidopsis.

Leaf veins provide the mechanical support and are responsible for the transport of nutrients and water to the plant. High vein density is a prerequisite for plants to have C4 photosynthesis. We investigated the genetic variation and genetic architecture of leaf venation traits within the species Arabidopsis thaliana using natural variation. Leaf venation traits, including leaf vein density (LVD) were analysed in 66 worldwide accessions and 399 lines of the multi-parent advanced generation intercross population. It was shown that there is no correlation between LVD and photosynthesis parameters within A. thaliana. Association mapping was performed for LVD and identified 16 and 17 putative quantitative trait loci (QTLs) in the multi-parent advanced generation intercross and worldwide sets, respectively. There was no overlap between the identified QTLs suggesting that many genes can affect the traits. In addition, linkage mapping was performed using two biparental recombinant inbred line populations. Combining linkage and association mapping revealed seven candidate genes. For one of the candidate genes, RCI2c, we demonstrated its function in leaf venation patterning.

Fine mapping of a major QTL for awn length in barley using a multiparent mapping population.

KEY MESSAGE: Awn length was mapped using a multiparent population derived from cv. Morex and four wild accessions. One QTL was fine mapped and candidate genes were identified in NILs by RNA-seq. Barley awns are photosynthetically active and contribute to grain yield. Awn length is variable among both wild and cultivated barley genotypes and many mutants with alterations in awn length have been identified. Here, we used a multiparent mapping population derived from cv. Morex and four genetically diverse wild barley lines to detect quantitative trait loci (QTLs) for awn length. Twelve QTLs, distributed over the barley genome, were identified with the most significant one located on chromosome arm 7HL (QTL AL7.1). The effect of AL7.1 was confirmed using near isogenic lines (NILs) and fine-mapped in two independent heterogeneous inbred families to a < 0.9 cM interval. With exception of a small effect on grain width, no other traits such as plant height or flowering time were affected by AL7.1. Variant calling on transcripts obtained from RNA sequencing reads in NILs was used to narrow down the list of candidate genes located in the interval. This data may be used for further characterization and unravelling of the mechanisms underlying natural variation in awn length.

Analysis of a plant complex resistance gene locus underlying immune-related hybrid incompatibility and its occurrence in nature.

Mechanisms underlying speciation in plants include detrimental (incompatible) genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler), and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL) RPP1 (Recognition of Peronospora parasitica1)-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3) in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1-R8) genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland). Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

phenoVein-A Tool for Leaf Vein Segmentation and Analysis.

Precise measurements of leaf vein traits are an important aspect of plant phenotyping for ecological and genetic research. Here, we present a powerful and user-friendly image analysis tool named phenoVein. It is dedicated to automated segmenting and analyzing of leaf veins in images acquired with different imaging modalities (microscope, macrophotography, etc.), including options for comfortable manual correction. Advanced image filtering emphasizes veins from the background and compensates for local brightness inhomogeneities. The most important traits being calculated are total vein length, vein density, piecewise vein lengths and widths, areole area, and skeleton graph statistics, like the number of branching or ending points. For the determination of vein widths, a model-based vein edge estimation approach has been implemented. Validation was performed for the measurement of vein length, vein width, and vein density of Arabidopsis (Arabidopsis thaliana), proving the reliability of phenoVein. We demonstrate the power of phenoVein on a set of previously described vein structure mutants of Arabidopsis (hemivenata, ondulata3, and asymmetric leaves2-101) compared with wild-type accessions Columbia-0 and Landsberg erecta-0. phenoVein is freely available as open-source software.

The genetic architecture of freezing tolerance varies across the range of Arabidopsis thaliana.

The capacity to tolerate freezing temperatures limits the geographical distribution of many plants, including several species of agricultural importance. However, the genes involved in freezing tolerance remain largely unknown. Here, we describe the variation in constitutive freezing tolerance that occurs among worldwide accessions of Arabidopsis thaliana. We found that although plants from high latitudes tend to be more freezing tolerant than plants from low latitudes, the environmental factors that shape cold adaptation differ across the species range. Consistent with this, we found that the genetic architecture of freezing tolerance also differs across its range. Conventional genome-wide association studies helped identify a priori and other promising candidate genes. However, simultaneously modelling climate variables and freezing tolerance together pinpointed other excellent a priori candidate genes. This suggests that if the selective factor underlying phenotypic variation is known, multi-trait mixed models may aid in identifying the genes that underlie adaptation.

Arabidopsis semidwarfs evolved from independent mutations in GA20ox1, ortholog to green revolution dwarf alleles in rice and barley.

Understanding the genetic bases of natural variation for developmental and stress-related traits is a major goal of current plant biology. Variation in plant hormone levels and signaling might underlie such phenotypic variation occurring even within the same species. Here we report the genetic and molecular basis of semidwarf individuals found in natural Arabidopsis thaliana populations. Allelism tests demonstrate that independent loss-of-function mutations at GA locus 5 (GA5), which encodes gibberellin 20-oxidase 1 (GA20ox1) involved in the last steps of gibberellin biosynthesis, are found in different populations from southern, western, and northern Europe; central Asia; and Japan. Sequencing of GA5 identified 21 different loss-of-function alleles causing semidwarfness without any obvious general tradeoff affecting plant performance traits. GA5 shows signatures of purifying selection, whereas GA5 loss-of-function alleles can also exhibit patterns of positive selection in specific populations as shown by Fay and Wu's H statistics. These results suggest that antagonistic pleiotropy might underlie the occurrence of GA5 loss-of-function mutations in nature. Furthermore, because GA5 is the ortholog of rice SD1 and barley Sdw1/Denso green revolution genes, this study illustrates the occurrence of conserved adaptive evolution between wild A.thaliana and domesticated plants.

Natural variation for anthocyanin accumulation under high-light and low-temperature stress is attributable to the ENHANCER OF AG-4 2 (HUA2) locus in combination with PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2.

Growing conditions combining high light intensities and low temperatures lead to anthocyanin accumulation in plants. This response was contrasted between two Arabidopsis thaliana accessions, which were used to decipher the genetic and molecular bases underlying the variation of this response. Quantitative trait loci (QTLs) for flowering time (FT) and anthocyanin accumulation under a high-light and low-temperature scenario versus a control environment were mapped. Major QTLs were confirmed using near-isogenic lines. Candidate genes were examined using mutants and gene expression studies as well as transgenic complementation. Several QTLs were found for FT and for anthocyanin content, of which one QTL co-located at the ENHANCER OF AG-4 2 (HUA2) locus. That HUA2 is a regulator of both pathways was confirmed by the analysis of loss-of-function mutants. For a strong expression of anthocyanin, additional allelic variation was detected for the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2 genes which control the anthocyanin pathway. The genetic control of variation for anthocyanin content was dissected in A. thaliana and shown to be affected by a common regulator of flowering and anthocyanin biosynthesis together with anthocyanin-specific regulators.

Quantitative trait loci and candidate genes underlying genotype by environment interaction in the response of Arabidopsis thaliana to drought.

Drought stress was imposed on two sets of Arabidopsis thaliana genotypes grown in sand under short-day conditions and analysed for several shoot and root growth traits. The response to drought was assessed for quantitative trait locus (QTL) mapping in a genetically diverse set of Arabidopsis accessions using genome-wide association (GWA) mapping, and conventional linkage analysis of a recombinant inbred line (RIL) population. Results showed significant genotype by environment interaction (G×E) for all traits in response to different watering regimes. For the RIL population, the observed G×E was reflected in 17 QTL by environment interactions (Q×E), while 17 additional QTLs were mapped not showing Q×E. GWA mapping identified 58 single nucleotide polymorphism (SNPs) associated with loci displaying Q×E and an additional 16 SNPs associated with loci not showing Q×E. Many candidate genes potentially underlying these loci were suggested. The genes for RPS3C and YLS7 were found to contain conserved amino acid differences when comparing Arabidopsis accessions with strongly contrasting drought response phenotypes, further supporting their candidacy. One of these candidate genes co-located with a QTL mapped in the RIL population.

Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in Arabidopsis.

In the Arabidopsis multiparent recombinant inbred line mapping population, a limited number of plants were detected that lacked axillary buds in most of the axils of the cauline (stem) leaves, but formed such buds in almost all rosette axils. Genetic analysis showed that polymorphisms in at least three loci together constitute this phenotype, which only occurs in late-flowering plants. Early flowering is epistatic to two of these loci, called REDUCED SHOOT BRANCHING1 (RSB1) and RSB2, which themselves do not affect flowering time. Map-based cloning and confirmation by transformation with genes from the region where RSB1 was identified by fine-mapping showed that a specific allele of AGAMOUS-Like6 from accession C24 conferred reduced branching in the cauline leaves. Site-directed mutagenesis in the Columbia allele revealed the causal amino acid substitution, which behaved as dominant negative, as was concluded from a loss-of-function mutation that showed the same phenotype in the late-flowering genetic background. This causal allele occurs at a frequency of 15% in the resequenced Arabidopsis thaliana accessions and correlated with reduced stem branching only in late-flowering accessions. The data show the importance of natural variation and epistatic interactions in revealing gene function.