Research ConstructionBlackwell Publishing Ltd of a genetic linkage map of Thlaspi caerulescens and quantitative trait loci analysis of zinc accumulation Ana G. L. Assunção1*, Bjorn Pieper2, Jaap Vromans3, Pim Lindhout3, Mark G. M. Aarts2 and Henk Schat1 1Institute of Ecological Sciences, Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands; 2Laboratory of Genetics, Wageningen University, Wageningen, the Netherlands; 3Laboratory of Plant Breeding, Wageningen University, Wageningen, the Netherlands; *Present address: Laboratory of Genetics, Wageningen University, Wageningen, the Netherlands Summary Author for correspondence: • Zinc (Zn) hyperaccumulation seems to be a constitutive species-level trait in Ana G. L. Assunção Thlaspi caerulescens. When compared under conditions of equal Zn availability, Tel: +31 317485413 considerable variation in the degree of hyperaccumulation is observed among Fax: +31 317483146 Email: [email protected] accessions originating from different soil types. This variation offers an excellent opportunity for further dissection of the genetics of this trait. Received: 13 September 2005 •A T. caerulescens intraspecific cross was made between a plant from a nonmetal- Accepted: 2 November 2005 licolous accession [Lellingen (LE)], characterized by relatively high Zn accumulation, and a plant from a calamine accession [La Calamine (LC)], characterized by relatively low Zn accumulation. • Zinc accumulation in roots and shoots segregated in the F3 population. This population was used to construct an LE/LC amplified fragment length polymorphism (AFLP)-based genetic linkage map and to map quantitative trait loci (QTL) for Zn accumulation. Two QTL were identified for root Zn accumulation, with the trait-enhancing alleles being derived from each of the parents, and explaining 21.7 and 16.6% of the phenotypic variation observed in the mapping population. • Future development of more markers, based on Arabidopsis orthologous genes localized in the QTL regions, will allow fine-mapping and map-based cloning of the genes underlying the QTL. Key words: amplified fragment length polymorphism (AFLP) markers, genetic map, quantitative trait loci (QTL) analysis, Thlaspi caerulescens, zinc (Zn) hyperaccumulation. New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01631.x © The Authors (2005). Journal compilation © New Phytologist (2005) transporters and metal chelators) involved in metal uptake, Introduction trafficking and sequestration (Clemens, 2001; Mäser et al., 2001; The study of the mechanisms of metal homeostasis in plants Cobbett & Goldsbrough, 2002). Although most progress is being is receiving increasing attention. Such knowledge can have made in Arabidopsis, the study of metal hyperaccumulators important implications: for example, for human health, (Brooks et al., 1977; Reeves, 1992), which are characterized because it may help improve the nutritional quality of plants; by greatly enhanced rates of metal uptake, accumulation and for sustainable crop production, even on micronutrient- tolerance (Lasat et al., 1996; Shen et al., 1997), can be of great deficient soils; and for the future application of phytoremedia- help in unraveling the ways in which plants deal with heavy tion in metal-polluted soils. There has been some progress metals. Eventually this will contribute to a full understanding in establishing the molecular basis of metal homeostasis in of the determinants of plant metal accumulation, which is at plants, including the identification of key components (metal the moment still ‘a long way ahead’ (Clemens et al., 2002). www.newphytologist.org 1 2 Research Thlaspi caerulescens is a zinc (Zn)/cadmium (Cd)/nickel ance, uptake and translocation of Zn, Cd and Ni in hydro- (Ni) hyperaccumulator species, previously suggested to be a ponic culture (Assunção et al., 2003b). With respect to Zn, good model species in which to study the mechanisms of although they are both Zn hyperaccumulators, the LE acces- heavy metal hyperaccumulation (Assunção et al., 2003a). An sion is characterized by a significantly higher Zn accumula- important characteristic of T. caerulescens is its natural varia- tion than the LC accession, both in roots and shoots, when tion in important traits such as metal accumulation, metal compared at the same level of Zn exposure (Assunção et al., root-to-shoot transport and metal tolerance. Comparison of 2003b). Additionally, the LC accession, originating from a accessions from different geographical and ecological environ- calamine soil, has been shown to be much more tolerant to Zn ments showed a pronounced intraspecific variation for these than the LE accession, which originates from a nonmetallifer- traits (Meerts & Van Isacker, 1997; Escarré et al., 2000; Schat ous soil (Assunção et al., 2003b). The F3 population has been et al., 2000; Assunção et al., 2003b; Roosens et al., 2003). In genotyped using amplified fragment length polymorphism general, this variation is of a quantitative nature, probably as (AFLP) markers (Vos et al., 1995) to construct an AFLP-based a result of the effect of allelic variation at several loci (multi- linkage map. Additionally, PCR-based codominant markers, genic), combined with an environmental effect on each locus. cleaved amplified polymorphic sequences (CAPS) and insertion/ This leads to a continuous phenotypic distribution of the trait deletions (Indels) were developed for the two T. caerulescens in a segregating population. A continuous distribution of accessions (LE and LC). These codominant markers have Zn and Cd accumulation was indeed found for segregating been used to integrate the parental genetic maps based on populations derived from T. caerulescens intraspecific crosses AFLP markers. Finally, the genetic linkage map and the (Assunção et al., 2003c; Zha et al., 2004). Such quantitative root and shoot Zn accumulation phenotypes of the F3 genetic variation can be exploited to detect and locate the loci mapping population have been used to map QTL for Zn contributing to the Zn, Ni or Cd hyperaccumulation or accumulation. tolerance traits using a so-called quantitative trait loci (QTL) analysis (Alonso-Blanco & Koornneef, 2000). Materials and methods Thlaspi caerulescens belongs to the Brassicaceae family and shares 88% DNA identity in coding regions with Arabidopsis Plant material thaliana (Peer et al. 2003; D. Rigola & M. G. M. Aarts, unpublished results). This close relationship is of importance, A Thlaspi caerulescens J. & C. Presl F3 population was used for as Arabidopsis is a model plant species with a fully sequenced constructing the linkage map. The F3 mapping population and well-studied genome (AGI, 2000). Comparative genome consisted of 81 individuals (one individual per F3 line) mapping experiments can highlight the extent to which local derived by single-seed descent from self-fertilized F2 plants gene order, orientation and spacing are conserved between originating from a single self-fertilized F1 plant. The F1 plant species (Schmidt, 2000). Comparative genetic mapping was derived from a cross between a plant from the accession experiments (for a review see Schmidt et al., 2001) have Lellingen (LE), originating from a nonmetalliferous site near already revealed extensive conservation of genome organiza- Lellingen, Luxembourg, and a plant from the accession La tion (colinearity) for species of the Brassicaceae family, both at Calamine (LC), originating from a strongly lead (Pb)/Cd/Zn- the macrosynteny and at the microsynteny levels (Kowalski enriched site near La Calamine, Belgium. This cross has been et al., 1994; Cavell et al., 1998; Koch et al., 1999; Acarkan previously described in Assunção et al. (2003c), in which the et al., 2000; Lan et al., 2000). This means that the positional F3 mapping population has been referred to as F3(4). information from the Arabidopsis genome can be used as an efficient tool for transferring information and resources to Plant culture and phenotyping related plant species (Schmidt, 2000) such as T. caerulescens. Ultimately the exploitation of genome colinearity could aid The Zn accumulation phenotype was measured in roots and the fine-mapping and subsequent map-based cloning of the shoots of 71 individuals [71 F3(4) families] out of the 81 that genetically identified QTL (Alonso-Blanco & Koornneef, constitute the F3 mapping population, and in 10–20 plants 2000; Borevitz & Chory, 2004) in T. caerulescens. originating from the LE and LC accessions. These phenotypic The aim of the present work was to assemble a genetic data [µmol Zn g−1 root dry weight (DW) and µmol Zn g−1 linkage map of T. caerulescens based on molecular markers shoot DW] were obtained from Assunção et al. (2003c), and to map QTL for Zn accumulation. To this end, we used where the plant culture methods and Zn accumulation an F3 population derived from a cross between plants of the measurements have been described. In short, seeds were sown T. caerulescens accessions Lellingen (LE) and La Calamine on moist peat and 3-wk-old seedlings were transferred to 1-l (LC). This cross segregates for Zn accumulation, as described pots (one plant per pot), filled with modified half-strength in Assunção et al. (2003c). The parent accessions originate Hoagland’s nutrient solution, supplemented with 10 µM from a nonmetalliferous (LE) and a calamine (LC) soil and ZnSO4. After 3 wk, the plants were harvested and the Zn they have been previously characterized with regard to toler- concentrations in roots and shoots were measured.