The study of the mechanisms of metal homeostasis in plants is receiving increasing attention. Such knowledge can have important implications: for example, for human health, because it may help improve the nutritional quality of plants; for sustainable crop production, even on micronutrient-deficient soils; and for the future application of phytoremediation in metal-polluted soils. There has been some progress in establishing the molecular basis of metal homeostasis in plants, including the identification of key components (metal transporters and metal chelators) involved in metal uptake, trafficking and sequestration (Clemens, 2001; Mäser et al., 2001; Cobbett & Goldsbrough, 2002). Although most progress is being made in Arabidopsis, the study of metal hyperaccumulators (Brooks et al., 1977; Reeves, 1992), which are characterized by greatly enhanced rates of metal uptake, accumulation and tolerance (Lasat et al., 1996; Shen et al., 1997), can be of great help in unraveling the ways in which plants deal with heavy metals. Eventually this will contribute to a full understanding of the determinants of plant metal accumulation, which is at the moment still ‘a long way ahead’ (Clemens et al., 2002).
Thlaspi caerulescens is a zinc (Zn)/cadmium (Cd)/nickel (Ni) hyperaccumulator species, previously suggested to be a good model species in which to study the mechanisms of heavy metal hyperaccumulation (Assunção et al., 2003a). An important characteristic of T. caerulescens is its natural variation in important traits such as metal accumulation, metal root-to-shoot transport and metal tolerance. Comparison of accessions from different geographical and ecological environments showed a pronounced intraspecific variation for these traits (Meerts & Van Isacker, 1997; Escarréet al., 2000; Schat et al., 2000; Assunção et al., 2003b; Roosens et al., 2003). In general, this variation is of a quantitative nature, probably as a result of the effect of allelic variation at several loci (multigenic), combined with an environmental effect on each locus. This leads to a continuous phenotypic distribution of the trait in a segregating population. A continuous distribution of Zn and Cd accumulation was indeed found for segregating populations derived from T. caerulescens intraspecific crosses (Assunção et al., 2003c; Zha et al., 2004). Such quantitative genetic variation can be exploited to detect and locate the loci contributing to the Zn, Ni or Cd hyperaccumulation or tolerance traits using a so-called quantitative trait loci (QTL) analysis (Alonso-Blanco & Koornneef, 2000).
Thlaspi caerulescens belongs to the Brassicaceae family and shares 88% DNA identity in coding regions with Arabidopsis thaliana (Peer et al. 2003; D. Rigola & M. G. M. Aarts, unpublished results). This close relationship is of importance, as Arabidopsis is a model plant species with a fully sequenced and well-studied genome (AGI, 2000). Comparative genome mapping experiments can highlight the extent to which local gene order, orientation and spacing are conserved between species (Schmidt, 2000). Comparative genetic mapping experiments (for a review see Schmidt et al., 2001) have already revealed extensive conservation of genome organization (colinearity) for species of the Brassicaceae family, both at the macrosynteny and at the microsynteny levels (Kowalski et al., 1994; Cavell et al., 1998; Koch et al., 1999; Acarkan et al., 2000; Lan et al., 2000). This means that the positional information from the Arabidopsis genome can be used as an efficient tool for transferring information and resources to related plant species (Schmidt, 2000) such as T. caerulescens. Ultimately the exploitation of genome colinearity could aid the fine-mapping and subsequent map-based cloning of the genetically identified QTL (Alonso-Blanco & Koornneef, 2000; Borevitz & Chory, 2004) in T. caerulescens.
The aim of the present work was to assemble a genetic linkage map of T. caerulescens based on molecular markers and to map QTL for Zn accumulation. To this end, we used an F3 population derived from a cross between plants of the T. caerulescens accessions Lellingen (LE) and La Calamine (LC). This cross segregates for Zn accumulation, as described in Assunção et al. (2003c). The parent accessions originate from a nonmetalliferous (LE) and a calamine (LC) soil and they have been previously characterized with regard to tolerance, uptake and translocation of Zn, Cd and Ni in hydroponic culture (Assunção et al., 2003b). With respect to Zn, although they are both Zn hyperaccumulators, the LE accession is characterized by a significantly higher Zn accumulation than the LC accession, both in roots and shoots, when compared at the same level of Zn exposure (Assunção et al., 2003b). Additionally, the LC accession, originating from a calamine soil, has been shown to be much more tolerant to Zn than the LE accession, which originates from a nonmetalliferous soil (Assunção et al., 2003b). The F3 population has been genotyped using amplified fragment length polymorphism (AFLP) markers (Vos et al., 1995) to construct an AFLP-based linkage map. Additionally, PCR-based codominant markers, cleaved amplified polymorphic sequences (CAPS) and insertion/deletions (Indels) were developed for the two T. caerulescens accessions (LE and LC). These codominant markers have been used to integrate the parental genetic maps based on AFLP markers. Finally, the genetic linkage map and the root and shoot Zn accumulation phenotypes of the F3 mapping population have been used to map QTL for Zn accumulation.