- Top of page
- Materials and Methods
- Supporting Information
Throughout a species' range, populations adapt to local conditions. Understanding the genetic basis of such local adaptation has been a major goal in evolutionary biology for over a century. The last decade has witnessed profound advances in molecular genetics approaches for the identification of genomic regions associated with adaptive traits, allowing for the rigorous assessment of this long-standing question (Barton & Keightley, 2002).
Plants have developed sophisticated physiological, developmental and genetic mechanisms to optimize the time of flowering (Bastow & Dean, 2003). Because the transition from vegetative growth to flowering is crucial for plant reproductive success, plants integrate different environmental cues to achieve a flowering response that is adapted to local conditions. The observation that the flowering time of many plant populations varies with latitude or altitude suggests that this trait contributes to geographic adaptation (Lacey, 1988; Kalisz & Wardle, 1994; Olsson & Ågren, 2002). Hence, an understanding of the pathways and genetic mechanisms that contribute to the natural variation in flowering time is a central goal of many studies in plant evolutionary genomics.
The model plant Arabidopsis thaliana (hereafter referred to as Arabidopsis) serves as a workhorse for the study of plant molecular biology. This species is distributed widely throughout northern latitudes and exhibits substantial natural variation in flowering time. Two different approaches have been used to characterize the life history and flowering behavior of Arabidopsis (Nordborg & Bergelson, 1999). The ecological criterion is based on the timing of seed germination and flowering; winter annuals germinate in the fall, overwinter as rosettes and flower in late spring, whereas summer annuals germinate in the spring and flower in mid to late summer. Based on this criterion, Arabidopsis has a winter annual life history throughout most of its range, although summer annuals have also been described (Napp-Zinn, 1985; Pigliucci, 2003; Koornneef et al., 2004; Shindo et al., 2007). Climatic data from the southern edge of the native range indicate that, although soil temperatures very rarely fall below freezing, they do fall below 4°C (Ågren & Schemske, 2012), which is a sufficient temperature for a vernalization response (Nordborg & Bergelson, 1999; Shindo et al., 2005).
By contrast, the physiological criterion classifies plants as winter annuals if vernalization, that is, a period of cold temperature at the seed or rosette stage, is required for flowering, and as summer annuals if plants can flower without vernalization. These categories are also referred to as ‘early’ or ‘late’ flowering, respectively (Clarke et al., 1995; Gazzani et al., 2003), because the vegetative phase is shorter in plants that do not require vernalization. Some populations flower without vernalization in the laboratory, and would therefore be classified as summer annuals by the physiological criterion, but are winter annuals under the ecological criterion (D. W. Schemske & J. Ågren, pers. comm.). We suggest that the ecological criterion for the classification of life history should be adopted universally, because it best reflects the actual timing of growth and flowering in the field.
The genetic basis of flowering time in Arabidopsis has received considerable attention (Simpson & Dean, 2002; Sung & Amasino, 2004; Amasino, 2010). The analysis of Arabidopsis mutants has characterized multiple genetic pathways that regulate the transition from vegetative to reproductive growth. The photoperiod and vernalization pathways regulate the response to environmental signals, whereas the autonomous and gibberellin pathways respond to endogenous signals and are functionally independent from environmental cues (Simpson & Dean, 2002). These pathways form a complex network of > 100 genes that regulate flowering. However, only a few genes identified from mutant screens have also been associated with the natural variation in flowering time. Of these, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are thought to be major regulators of flowering in natural populations (Burn et al., 1993; Lee et al., 1993; Clarke & Dean, 1994; Lempe et al., 2005). FLC, a transcription factor encoding a MADs-box protein, represses the expression of other transcription factors promoting flowering (Michaels & Amasino, 1999). Functional alleles at FRI delay flowering by activating the strong expression of FLC (Johanson et al., 2000). Vernalization represses FLC expression by epigenetic modification, reducing its sensitivity to FRI, and thus promotes flowering (Amasino, 2004).
The molecular analysis of natural accessions has revealed considerable allelic variation for FRI and FLC, and many mutations are known that cause inactivation of either FRI or FLC. Such mutants are often significantly associated with early flowering (Johanson et al., 2000; Le Corre et al., 2002; Gazzani et al., 2003; Lempe et al., 2005). Over 70% of early-flowering accessions contain loss-of-function alleles at the FRI locus (Shindo et al., 2005). In a geographic survey of the relationship between flowering time and FRI, active FRI alleles were associated with a latitudinal cline in flowering, but no such cline was detected in accessions with nonfunctional FRI (Stinchcombe et al., 2004). In the presence of functional FRI alleles, FLC allelic variation contributes to a latitudinal cline in flowering, and FLC expression is most highly correlated with flowering time variation (Caicedo et al., 2004; Lempe et al., 2005; Shindo et al., 2005). These results suggest that FRI and FLC are targets of natural selection (Stinchcombe et al., 2004; Izawa, 2007). Although FRI and FLC are important determinants of natural variation in flowering time for some Arabidopsis populations, extensive molecular and genetic analysis of Arabidopsis accessions has indicated that other loci also contribute to flowering time (Lempe et al., 2005; Werner et al., 2005a,b; Li et al., 2006).
Quantitative trait locus (QTL) mapping has proven to be a powerful method for the identification of the genetic basis of quantitative trait variation (Tanksley, 1993; Barton & Keightley, 2002; Mitchell-Olds & Schmitt, 2006), and numerous QTL studies have been conducted examining flowering time variation in Arabidopsis. The majority of these studies include the mapping of populations derived from one or both of the laboratory strains Columbia (Col) and Landsberg erecta (Ler), both of which could be classified as summer annuals based on a physiological criterion alone (Lister & Dean, 1993). Knowing where in the life cycle variation in flowering time is manifest is a critical step in assessing the mechanisms responsible for population differences in flowering time. For example, in plants requiring vernalization, the developmental and physiological pathways that contribute to the variation in flowering time among populations may differ from those of populations that do not require vernalization. It is thus important to understand the life histories of populations grown under conditions of their native environments.
Recently, Ågren & Schemske (2012) have presented the results of a multi-year reciprocal transplant study between Arabidopsis populations originating from Sweden and Italy – the geographic limits of the native distribution. This experiment is the first of its kind involving Arabidopsis (Lowry, 2012), and has demonstrated strong adaptive differentiation of these populations to their source environment. In this study, freezing tolerance and flowering time were identified as putative adaptive traits conferring local adaptation. At the Italian field site, the Italy population flowered 33 and 50 d before Sweden in the 2 yr of study; at the Swedish field site, the Italy population flowered 3 d before Sweden in both years (Ågren & Schemske, 2012). Here, we report the results of extensive genetic mapping experiments designed to examine the genetic basis of variation in flowering time differences between these populations. Given the winter annual life history of the study populations, we employed a two-tiered approach to dissect the genetic basis of flowering time. We first searched for the QTLs required for plants to flower in the absence of vernalization, and then identified the QTLs that contribute to variation in flowering following vernalization. In this way, we are able to decouple flowering time variation from a vernalization requirement.
Our study addressed the following questions. What are the number, location and magnitude of effect for QTLs contributing to flowering time? What are the genetic regions responsible for differences in vernalization response and do these contribute to flowering time variation after vernalization? Do QTLs co-localize with FRI and FLC as seen in other studies? Do candidate genes underlying QTLs differ between parental populations in coding region sequence?