Extracting genotype information of Arabidopsis thaliana recombinant inbred lines from transcript profiles established with high-density oligonucleotide arrays.

KEY MESSAGE: Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A. thaliana accessions. Individual probes of the GeneChip® that show sequence polymorphisms between different A. thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A. thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A. thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Genetic dissection of metabolite variation in Arabidopsis seeds: evidence for mQTL hotspots and a master regulatory locus of seed metabolism.

To gain insight into genetic factors controlling seed metabolic composition and its relationship to major seed properties, an Arabidopsis recombinant inbred line (RIL) population, derived from accessions Col-0 and C24, was studied using an MS-based metabolic profiling approach. Relative intensities of 311 polar primary metabolites were used to identify associated genomic loci and to elucidate their interactions by quantitative trait locus (QTL) mapping. A total of 786 metabolic QTLs (mQTLs) were unequally distributed across the genome, forming several hotspots. For the branched-chain amino acid leucine, mQTLs and candidate genes were elucidated in detail. Correlation studies displayed links between metabolite levels, seed protein content, and seed weight. Principal component analysis revealed a clustering of samples, with PC1 mapping to a region on the short arm of chromosome IV. The overlap of this region with mQTL hotspots indicates the presence of a potential master regulatory locus of seed metabolism. As a result of database queries, a series of candidate regulatory genes, including bZIP10, were identified within this region. Depending on the search conditions, metabolic pathway-derived candidate genes for 40-61% of tested mQTLs could be determined, providing an extensive basis for further identification and characterization of hitherto unknown genes causal for natural variation of Arabidopsis seed metabolism.

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.

Natural variation in biogenesis efficiency of individual Arabidopsis thaliana microRNAs.

Like protein-coding genes, loci that produce microRNAs (miRNAs) are generally considered to be under purifying selection, consistent with miRNA polymorphisms being able to cause disease. Nevertheless, it has been hypothesized that variation in miRNA genes may contribute to phenotypic diversity. Here we demonstrate that a naturally occurring polymorphism in the MIR164A gene affects leaf shape and shoot architecture in Arabidopsis thaliana, with the effects being modified by additional loci in the genome. A single base pair substitution in the miRNA complementary sequence alters the predicted stability of the miRNA:miRNA(∗) duplex. It thereby greatly reduces miRNA accumulation, probably because it interferes with precursor processing. We demonstrate that this is not a rare exception and that natural strains of Arabidopsis thaliana harbor dozens of similar polymorphisms that affect processing of a wide range of miRNA precursors. Our results suggest that natural variation in miRNA biogenesis resulting from cis mutations is a common contributor to phenotypic variation in plants.

QTL analysis of early stage heterosis for biomass in Arabidopsis.

The main objective of this study was to identify genomic regions involved in biomass heterosis using QTL, generation means, and mode-of-inheritance classification analyses. In a modified North Carolina Design III we backcrossed 429 recombinant inbred line and 140 introgression line populations to the two parental accessions, C24 and Col-0, whose F (1) hybrid exhibited 44% heterosis for biomass. Mid-parent heterosis in the RILs ranged from -31 to 99% for dry weight and from -58 to 143% for leaf area. We detected ten genomic positions involved in biomass heterosis at an early developmental stage, individually explaining between 2.4 and 15.7% of the phenotypic variation. While overdominant gene action was prevalent in heterotic QTL, our results suggest that a combination of dominance, overdominance and epistasis is involved in biomass heterosis in this Arabidopsis cross.