Although multiple environmental cues regulate the transition to flowering in Arabidopsis thaliana, previous studies have suggested that wild A. thaliana accessions fall primarily into two classes, distinguished by their requirement for vernalization (extended winter-like temperatures), which enables rapid flowering under long days. Much of the difference in vernalization response is apparently due to variation at two epistatically acting loci, FRI and FLC. We present the response of over 150 wild accessions to three different environmental variables. In long days, FLC is among those genes whose expression is most highly correlated with flowering. In short days, FRI and FLC are less important, although their contribution is still significant. In addition, there is considerable variation not only in vernalization response, but also in the response to differences in day length or ambient growth temperature. The identification of accessions that flower relatively early or late in specific environments suggests that many of the flowering-time pathways identified by mutagenesis, such as those that respond to day length, contribute to flowering-time variation in the wild. In contrast to differences in vernalization requirement, which are mainly mediated by FRI and FLC, it seems that variation in these other pathways is due to allelic effects at several different loci.

BackgroundEven when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL) mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs).Methodology/Principal FindingsWe present two new AI-RIL populations of Arabidopsis thaliana genotyped with an average intermarker distance of 600 kb. The advanced intercrossing design led to expansion of the genetic map in the two populations, which contain recombination events corresponding to 50 kb/cM in an F2 population. We used the AI-RILs to map QTL for light response and flowering time, and to identify segregation distortion in one of the AI-RIL populations due to a negative epistatic interaction between two genomic regions.Conclusions/SignificanceThe two new AI-RIL populations, EstC and KendC, derived from crosses of Columbia (Col) to Estland (Est-1) and Kendallville (Kend-L) provide an excellent resource for high precision QTL mapping. Moreover, because they have been genotyped with over 100 common markers, they are also excellent material for comparative QTL mapping.

The MADS-AFFECTING FLOWERING 2 (MAF2) gene of Arabidopsis thaliana has been characterized as a repressor of flowering. The molecular basis of MAF2 gene function and role of alternative MAF2 transcripts in flowering time modulation is not understood. MAF2 splice variant expression was quantified in cold-acclimated plants by quantitative RT-PCR. Cold influenced the abundance of splice variants and prompted a functional study of splice forms. Individual variants were overexpressed in the Col background and were assayed for their ability to delay flowering. Overexpression of MAF2 variants 2 and 4 had limited effect on flowering time. Overexpression of MAF2 splice variant 1 resulted in early flowering and affected the expression of the endogenous MAF2 gene and its paralogues, confounding functional assessment. In the Ll-2 Arabidopsis accession, a MAF2, MAF3, MAF4 and FLC null line, MAF2 var1 was consistent in its effect on reproductive delay under ambient and reduced temperatures, indicating that it acts as a repressor of flowering.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9982-2) contains supplementary material, which is available to authorized users.

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Functional characterization of regulatory genes governing flowering time is a research priority for breeding earliness in crop Brassicas. Highly polyploid genomes of Brassicas pose challenges in unraveling homeolog gene function. In Arabidopsis, five MIR172 paralogs control flowering time and floral organ identity by down-regulating AP2 and AP2-like genes. The impact of homeolog diversification on MIR172 loci, however, needs to be examined in morphologically diverse Brassicas. Herein, we analyze fractionation status and phylogeny of MIR172 and target AP2 from Brassicas and compare functionality of MIR172 variants representing distinct sub-genomes and progenitor genomes. Copy number analysis revealed higher retention of MIR172 loci relative to AP2 in diploid and amphi-diploid Brassica species. Dendrogram of 87 MIR172 sequences from Brassicaceae showed five major clusters corresponding to MIR172a-MIR172e which further separated into sub-genome and progenitor genome specific clades. Similar groupings were observed in the phylogeny of 11 Brassica AP2 and AP2-like genes. Over-expression of a pair of natural variants for each of MIR172b, MIR172d and MIR172e representing sub-genomes, progenitor genomes and species of Brassicas displayed floral acceleration in all transgenic lines indicating a strong selection pressure on MIR172. All gain-of-function lines, except 35S::MIR172e and 35S::MIR172e' displayed floral organ defects implying altered target spectrum of MIR172e relative to MIR172b and MIR172d. Expression of MIR172e caused marginal earliness in flowering time in B. juncea. In conclusion, this study demonstrates tightly preserved role of homeologs and natural variants of MIR172 family in mediating flowering in Brassicas and suggests their deployment for introgression of early flowering trait.

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SOC1, a MADS-box type II transcription factor, integrates environmental and endogenous cues to promote flowering in angiosperms. Recent reports implicating SOC1 in roles beyond floral transition prompted functional characterization of SOC1 in polyploid rapeseed mustard genomes. Gene characterization in Brassicas necessitates analysis of composite homeolog function. While insertional mutagenesis is untenable in Brassicas owing to gene redundancy, gain-of-function approach entails serial characterization of individual homeologs. Herein, we demonstrate modulated floral promotive effects in natural variants of Brassica SOC1 and provide lateral branching as a probable outcome of polyploidy-induced gene diversification. Ectopic expression of two B genome specific SOC1 variants in Arabidopsis thaliana resulted in differential floral acceleration and manifestation of multiple vegetative rosettes. Characterization of composite homeolog function in B. juncea via introgression of Brassica SOC1 specific artificial miRNA, designed to target homeologs, also exhibited modifications in floral transition and lateral branching. Comprehensive analysis of field performance of B. juncea transgenics displayed altered fitness across 11 agronomic traits. Crucially, reduced SOC1 levels directly impacted two developmental traits, namely, flowering time and number of lateral branches which in turn influenced several dependent agronomic traits. While delayed flowering and crop maturity resulted in altered fatty acid composition with higher SFA and lower PUFA in transgenics relative to controls, reduction in overall count of lateral branches caused a concomitant decrease in silique count which ultimately impacted total seed yield in transgenics. Statistical analysis revealed number of secondary branches as the most critical trait influencing seed yield. Based on our findings, we propose enhancing levels Brassica SOC1, a key target, for achieving earliness in flowering, improved seed yield and oil quality, and studying trait trade-offs.