Construction of a Genetic Linkage Map of Thlaspi Caerulescens And

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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

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).

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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 variation 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. Throughout

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Table 1 List of primers and adapters used to generate the ampliﬁed fragment length Primers/adapters Sequences polymorphism (AFLP) markers EcoRI adapter 5′-CTCGTAGACTGCGTACC-3′ 3′-CATCTGACGCATGGTTAA-5′ E00 (universal primer) GACTGCGTACCAATTC EcoRI + 1 selective nucleotide E01 E00 + A EcoRI + 3 selective nucleotide E32 E00 + AAC E35 E00 + ACA E41 E00 + AGG E45 E00 + ATG PstI adapter 5′-CTCGTAGACTGCGTACATGCA-3′ 3′-CATCTGACGCATGT-5′ P00 (universal primer) GACTGCGTACATGCAG PstI + 0 selective nucleotide P00 P00 PstI + 2 selective nucleotide P14 P00 + AT MseI adapter 5′-GACGATGAGTCCTGAG-3′ 3′-TACTCAGGACTCAT-5′ M00 (universal primer) GATGAGTCCTGAGTAA MseI + 0 selective nucleotide M00 M00 MseI + 2 selective nucleotide M11 M00 + AA M12 M00 + AC M13 M00 + AG M14 M00 + AT M15 M00 + CA M17 M00 + CG M18 M00 + CT M20 M00 + GC M21 M00 + GG M22 M00 + GT M23 M00 + TA M24 M00 + TC M25 M00 + TG MseI +3 selective nucleotide M45 M00 + ATG M47 M00 + CAA M48 M00 + CAC M50 M00 + CAT M51 M00 + CCA M52 M00 + CCC

the experiment the nutrient solutions were replaced twice a PCs selected for use in the F3 mapping population were week and the pots were re-randomized at each solution E32M12, E32M13, E32M14, E32M15, E32M18, E32M20, replacement (Assunção et al., 2003c). E32M22, E32M23, E35M11, E35M13, E35M17, E41M22, E45M20, E45M21, E45M22, E45M24, E45M25, P14M45, P14M47, P14M48, P14M50 and P14M52. AFLP profiles of Genotyping the F1 were also obtained for all the selected PCs. AFLP markers The F3 mapping population was genotyped Segregating markers in the mapping population were using AFLP marker analysis (Vos et al., 1995), which was designated according to the restriction enzymes and the primer performed as described by Qi & Lindhout (1997). Two pairs combination used, and their sizes estimated with reference of restriction enzymes, EcoRI/MseI (E/M) and PstI/MseI (P/ to the SequaMark 10 base ladder (Research Genetics, Hunts- M), were used to generate the restriction fragments for ville, AL, USA). They were scored as dominant markers, using amplification. The restriction enzymes, adaptors and primers the specialized software package for the analysis of DNA used are listed in Table 1. Initially, 58 primer combinations fingerprints AFLP-Quantar® (KeyGene NV, Wageningen, the (PCs), 48 E/M (+3+2) and 10 P/M (+2+3), were tested for Netherlands), and designated according to the AFLP profiles their polymorphism rates between the parental lines LE and of the parent lines (Fig. 1). The AFLP profile of the F1 line LC. Each of the parental lines consisted of four individuals, was used to confirm the reliability of each marker segregating progeny of each of the self-fertilized parents (LE and LC). The in the F3 mapping population.

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each marker detected with the AFLP- Quantar®PRO program, the reliability was checked by comparison with the marker profiles of the parent accessions and the F1, and the codominant scoring of the mapping population was checked. For these codominant markers the letters QP were added to the marker name (Fig. 1). AFLP markers can sometimes be considered as allele markers, when bands scored as different markers actually represent different alleles of the same locus (Alonso-Blanco et al., 1998). Allelic band pairs that could be used as codominant markers were identified, taking into account the following criteria (according to Alonso-Blanco et al., 1998): the two AFLP bands should be derived from different parents, with the same primer combination; in the putative heterozygotes, and the F1, both bands should consistently show weaker intensity than in the lines containing only one band; individuals without any of the alleles do not exist. These pairs of AFLP markers were considered as single locus markers, with their names being composed of both allele names (Fig. 1). Such codominant markers have previously been identified and used in linkage map construction for other species, for example Arabidopsis (Alonso-Blanco et al., 1998), rice (Oriza nativa) (Maheswaran et al., 1997), tomato (Lycopersicon esculentum) (Saliba et al., 2000) or melon (Cucumis melo) (Perin et al., 2002).

Codominant CAPS/Indel markers PCR-based codominant markers were developed for the accessions LE and LC. The markers were based on Expressed Sequence Tags (ESTs), from an EST library of T. caerulescens accession LC (D. Rigola & M. G. M. Aarts, unpublished results), for which homologous genes were found in Arabidopsis and which were evenly distributed over the Arabidopsis genome. Fragments of T. caerulescens genes, represented by the selected ESTs, were amplified from genomic DNA and sequenced in both accessions. An EST was generally considered the true homologue of an Arabidopsis gene if the homology search returned an E-value < e−30 and the identity was 88% or more. The results of this search were also used to locate putative intron positions in T. caerulescens, based on the assumption that their positions are conserved in Arabidopsis and T. caerulescens. For the development of a marker representing Fig. 1 An amplified fragment length polymorphism (AFLP) image the TcZNT1 (Zn transporter) gene (Pence et al., 2000), a obtained with the primer combination E32M12. It includes a set of Lellingen/La Calamine (LE/LC) F3 lines, the F1 line, the LC (calamine ZNT1 cDNA was used. Introns were preferentially targeted accession) and LE (nonmetallicolous accession) parental lines and the for amplification in T. caerulescens. When the Arabidopsis molecular marker (MM). Examples of different types of segregating homologue did not contain an intron or the EST only markers are given: dominant markers, LE-specific (e32m12-192.7- represented a single exon of multiple exons in the Arabidopsis LE) and LC-specific (e32m12-180.8-LC), and codominant markers, homologue (hence an intron could not be targeted for scored with AFLP-Quantar®PRO (KeyGene NV, Wageningen, the Netherlands) (e32m12-215.4-QP) and allelic band pairs (e32m12- amplification), a stretch of EST sequence was targeted for 279.1/277.2). amplification. Search for molecular polymorphisms between the two accessions led to the development of CAPS and Indel markers (Table 2). These PCR-based codominant markers Codominant AFLP markers In order to score codominant were confirmed in both parental lines (the same individuals markers, the AFLP-Quantar®PRO version (KeyGene NV) used to test PCs in the AFLP analysis) and in the F1, before was used. This program can detect codominant markers based being scored in the F3 mapping population. CAPS markers on intensity differences of the corresponding AFLP bands. For were separated on 3% standard electrophoresis grade agarose

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Table 2 List of polymerase chain reaction (PCR)-based codominant markers [cleaved ampliﬁed polymorphic sequences (CAPS)/insertion/ deletions (Indels)], the Arabidopsis homologous gene, primer sequences, approximate marker fragment size (bp) and restriction enzyme used (CAPS) or allelic size difference (Indels)

CAPS/Indel Arabidopsis Fragment Restriction enzyme (CAPS) marker homologue Primer nucleotide sequence size (bp) or allelic size difference (Indels)

CAPS21/22 At2g01610 F 5′-GGTAAGCCAAAGTCATACGACGACG-3′ 490 MnlI R 5′-TCCACGTCTGCACGTTACTCATCTG-3′ CAPS55/56 At4g26050 F 5′-ACGGAATCACGGTGTTGCCG-3′ 480 Trul I R 5′-TGCTTCACTGCTTCCAAACCCTG-3′ CAPS65/66 At5g20830 F 5′-TGTAAGGAAGATCCATCTCACTGGGAC-3′ 250 Trul I R 5′-AAGCCAATACACACCGGTCAATGTC-3′ CAPS75/76 At5g43780 F 5′-CGTTGTCAACATGTCGGTTCCG-3′ 380 HpaI R 5′-TCGCCTCCGATCAACCAGTTTC-3′ CAPS85/86 At2g36540 F 5′-AAAGGCTTTTCTGCTTCAAACACTGTC-3′ 400 DdeI R 5′-TCAGGATGAGAAGAATCGATCATTGG-3′ CAPS89/90 At1g10970 F 5′-CGTGGTTGTGGAAAGAGGGAATG-3′ 360 Trul I R 5′-TGCAATGAGAGGCCTGATCGTG-3′ CAPS95/96 At5g67330 F 5′-AATCGCCGGGTACCGGAAAG-3′ 530 BamHI R 5′-TTACAACTCCAGCCCAGAGAGGAATC-3′ Indel29/30 At2g36830 F 5′-GTGGTAACATCACTCTCCTCCGTGG-3′ 350 14 bp R 5′-AAGCATTTAGCACTCCTACTCCGGC-3′ Indel39/40 At3g19820 F 5′-TGTTCCTCTTTACAAGGTCGGCG-3′ 250 20 bp R 5′-TCCTTGCCTCTTCTCGTACTCGAAC-3′ Indel47/48 At5g21274 F 5′-TCAGAGTTCAAGGAAGCGTTTAGCC-3′ 850 23 bp R 5′-CATCACGGTCCCAAGCTCCTTC-3′

At1g10970 (CAPS89/90) and At5g67330 (CAPS95/96) correspond to the putative zinc (Zn) transporter gene ZIP4 (Grotz et al., 1998) and the metal transporter gene NRAMP4 (Thomine et al., 2000), respectively. F, forward; R, reverse. gels and Indel markers were separated on 2 or 3% Metaphor® Results agarose gels (Cambrex Bio Science Rockland Inc., Rockland, ME, USA). Segregation of zinc accumulation Zinc accumulation, in roots and in shoots, was established in Map construction hydroponically grown plants exposed to 10 µM Zn for 3 wk. A genetic linkage map of the mapping population was For the accumulation of Zn in the roots, the LC and LE accessions constructed based on dominant and codominant AFLP markers exhibited different, slightly overlapping phenotypic frequency and on codominant CAPS/Indel markers. The JoinMap® 3.0 distributions, with LC showing lower Zn accumulation than software package (Plant Research International, Wageningen, LE (Fig. 2a). The frequency distributions of Zn accumulation the Netherlands) (Van Ooijen & Voorrips, 2001) was used for in the shoot exhibited by LC and LE accessions did not linkage grouping and map construction. Kosambi’s mapping overlap, with LC showing lower Zn accumulation than LE function was applied for map-distance calculation (Kosambi, (Fig. 2b). Zinc accumulation in roots and in shoots was also 1944). determined in 71 individuals out of the 81 that constituted the F3 mapping population. The phenotypic frequency distribution for Zn accumulation in both roots and shoots showed QTL analysis segregation of these traits in the F3 population (Fig. 2c,d). The associations between molecular markers and QTL for All but one F3 plant had a root or shoot Zn accumulation Zn accumulation in root and in shoot were detected using the phenotype between the lower and upper limits of the LC or computer program MapQTL® 5 (Kyazma BV, Wageningen, LE phenotypic range. Shoot and root Zn concentrations in the Netherlands) (Van Ooijen, 2004). A logarithm of the odds the F3 population were uncorrelated (r = 0.07). (LOD) score of 3.0 was used as the threshold for detecting QTL (Van Ooijen, 1999). The interval mapping method and Analysis and scoring of molecular markers the multiple-QTL models (MQM) mapping method were used to detect and map QTL. The QTL graphs were prepared Fifty-eight AFLP PCs were tested in the parental lines. The with MapChart (Voorrips, 2002). sizes of fragments generated ranged from about 80 bp to

Fig. 2 Frequency distribution over classes of zinc (Zn) accumulation in roots [classes correspond to 2 µmol Zn g−1 root dry weight (DW)] (a, c) and Zn accumulation in shoots (classes correspond to 5 µmol Zn g−1 shoot DW) (b, d). (a, c) Frequency distributions of Zn concentration in the roots of individuals from the nonmetallicolous [Lellingen (LE)] and calamine [La Calamine (LC)] accessions (a) and of 71 individuals out of the 81 that constitute the F3 mapping population (c). (b, d) Frequency distributions of Zn concentration in shoots of individuals from the LE and LC accessions (b) and of 71 individuals out of the 81 that constitute the F3 mapping population (d). Plants were grown for 3 wk in nutrient solution supplemented with 10 µM Zn.

800 bp, with most fragments smaller than 500 bp. The In addition to the AFLP markers, 10 PCR-based markers average number of well-amplified bands per PC varied were developed, seven of which were CAPS and three of between 30 and 85, with an average of 48 bands, and the which were Indels (Table 2). These codominant markers were average polymorphism rate between LE and LC was 24%. also scored in the mapping population. In total, 337 markers Twenty-two out of the tested 58 PCs were selected for (327 AFLP and 10 CAPS/Indels) were scored. genetic mapping. On average, 15 markers were scored per The CAPS marker caps89/90 corresponds to TcZNT1, a PC, ranging from eight (E35M11) to 34 (E32M12). In T. caerulescens metal transporter gene, homologous to AtZIP4 total, of the 22 PCs, 327 segregating AFLP markers were (ZRT/IRT-like protein) (Grotz et al., 1998), known to mediate identified and scored in the mapping population, of Zn transport and suggested to be involved in Zn hyperaccu- which 133 were LE-specific and 157 were LC-specific, 10 mulation in T. caerulescens (Pence et al., 2000). The CAPS could be scored as codominant using AFLP-Quantar® marker caps95/96 is a homologue of the metal transporter PRO and an additional 27 allelic band pairs were identified AtNRAMP4 (natural resistance-associated macrophage (see Materials and methods) and scored as 27 codominant protein) (Thomine et al., 2000). A T. caerulescens homologue, markers (Fig. 1). In the latter 27 markers, the molecular TcNRAMP4, mediates the transport of Cd, iron, manganese size difference between the two allelic fragments was 1 to and Zn (R. Oomen & S. Thomine, CNRS, Gif-Sur-Yvette, 5 bp. France, personal communication).

Construction of an LE/LC linkage map Discussion A genetic linkage map consisting of seven linkage groups Segregation of zinc accumulation (LGs) with in total 319 markers, including 309 AFLP (of which 37 are codominant) and 10 CAPS/Indels, was To map QTL for Zn accumulation it is necessary that this trait constructed. From the 337 markers scored, 333 were is segregating in the mapping population. We measured both assembled in seven linkage groups with an LOD threshold root and shoot Zn accumulation phenotypes in individuals of grouping value of 5.0. During calculation of map order and the parent accessions and of the segregating F3 progeny, upon distance, an additional 14 markers were omitted because they exposure to 10 µM Zn for 3 wk. These data on root and shoot could not be reliably placed in the linkage groups. A total map Zn accumulation have previously been used to calculate the Zn distance of 449 cM was covered (Fig. 3), corresponding to an accumulation, on a total plant dry weight basis, of individuals of average interval of 1.6 cM between adjacent markers the parent accessions and of the segregating F3 progeny [F3(4)] (absolutely linked markers were excluded in the calculation of in Assunção et al. (2003c). The phenotypic frequency distribu- this average). Linkage groups 1, 2, 4 and 6 have one gap of tions of root and shoot Zn accumulation segregated in the F3 10–15 cM and LG5 has two such intervals. LG6 covers the mapping population, with phenotypic frequency distributions longest genetic distance (80.7 cM) while LG1 covers the being more or less continuous (Fig. 2c,d), suggesting polygenic shortest distance (44.5 cM). or at least digenic inheritance. As we do not have the data to The great majority of the loci (89%) showed genotype calculate heritability levels, it is not possible to estimate the ratios as expected for a segregating F3 population precise numbers of loci involved in the control of these traits. (0.375 : 0.25 : 0.375). For 36 markers this frequency was signiﬁcantly skewed (P < 0.01), with a cluster of eight loci A T. caerulescens genetic linkage map mapping to LG1 between positions 39 and 49 cM largely representing LE alleles, and a cluster of 22 loci mapping to A genetic linkage map with 319 markers was generated (Fig. 3). LG7 between positions 37 and 53 cM with an overrepresen- It consists of seven linkage groups (LGs), corresponding to the tation of LC alleles. The other markers with a disturbed haploid chromosome number of T. caerulescens. As nearly all segregation were not linked to markers with distorted ratios. markers could be included in the map, even at the high LOD value of 5, we conclude the seven linkage groups indeed represent the seven haploid chromosomes of T. caerulescens. With an Mapping QTL average genetic distance between markers of 1.6 cM and with Using the Zn accumulation phenotypic data, we carried out few intervals, no longer than 10–15 cM, between adjacent interval mapping to identify and locate QTL associated with markers, the map covers the T. caerulescens genome very nicely. Zn accumulation in roots and/or shoots. For Zn accumulation Each chromosome contains a more dense cluster of markers, in roots only, we detected two QTL, one in LG3 and another probably representing the putative centromeres. Clustering of in LG5 (Fig. 3). Of several markers linked to these loci, we random genetic markers around the centromere, mainly as a result used markers indel47/48 and e32m14-249.5/250.4 for LG3 of centromeric suppression of recombination, has been reported and LG5, respectively, as cofactors for MQM mapping. Based in maps of several crop species such as barley (Hordeum vulgare), on this analysis, we found the LG3 QTL to have a LOD value tomato and wheat (Triticum aestivum) (Chao et al., 1989; Tanksley of 4.6, explaining 21.7% of the total variance, and the LG5 et al., 1992; Qi et al., 1998). There are two clusters of skewed QTL to have a LOD value of 3.6, explaining 16.6% of the markers, on chromosomes 1 and 7, which show distorted seg- total variance. The trait-enhancing allele for the LG3 QTL regation instead of the expected F3 segregation ratios. This happens originated from the LE parent and the trait-enhancing more frequently in segregating populations [e.g. in Arabidopsis allele for the LG5 QTL originated from the LC parent. lyrata (Yogeeswaran et al., 2005) and Arabidopsis thaliana (Boivin The two cofactors used for MQM analysis of both QTL are et al., 2004)], because of the presence of alleles or combinations codominant markers, which allowed further assessment of the of alleles leading to an unfavourable phenotype. When the joint genotypes for both loci. The analysis of the genotypes of each map was compared with each of the parental maps, in general loci indicated that the trait-enhancing allele LE3 is recessive a similar marker order was found (data not shown), indicating and the trait-enhancing allele LC5 is codominant [one-way that the datasets for the parents are reliable and consistent. analysis of variance (ANOVA) of log-transformed data, Taken together, all our results indicate that we have produced

F2,68 = 11.51 and 10.93, P < 0.001, respectively, followed a robust genetic linkage map for the T. caerulescens genome. by Tukey’s test, to compare means]. For the genotypes of both loci, it appears that the simultaneous presence of QTL for zinc accumulation both trait-enhancing alleles (specially LE3LE3LC5LC5 and LE3LE3LC5LE5 genotypes) has an additive effect on the root A second goal of this research was to identify associations Zn accumulation phenotype (Fig. 4). between molecular markers and the traits of Zn accumulation

www.newphytologist.org © The Authors (2005). Journal compilation © New Phytologist (2005) Research 9 in the roots and shoots. Unfortunately, no significant QTL were found to explain the observed variation for Zn accumulation in shoots in the segregating population, although the phenotypic frequency distribution (Fig. 2d) suggests this trait to be heritable and polygenic. It is possible that the relatively small segregating population (71 individuals) does not allow the detection of significant QTL if there are many loci segregating, each with a relatively small contribution to the trait. However, for Zn accumulation in the roots two QTL were found, each with a substantial LOD score (Fig. 3). These QTL explained, respectively, 21.7 and 16.6% of the total variance and are thus of major importance. Of the two parents, the LE accession showed the highest Zn accumulation in roots and was expected to contribute the Zn accumulation-enhancing alleles. Surprisingly, however, the Fig. 4 Phenotypes of quantitative trait loci (QTL) genotypes. For the trait-enhancing alleles come from different parents. For the locus on LG3, the closely linked marker indel47/48 is used, with QTL on chromosome 3, the LE allele (LE3) gives rise to genotypes LE3LE3 [homozygous for the Lellingen (LE) allele], LE3LC3 higher accumulation, and for the QTL on chromosome 5, it (heterozygous) and LC3LC3 [homozygous for the La Calamine (LC) is the LC allele (LC5) that contributes to higher accumulation. allele]. For the LG5 locus, the closely linked marker e32m14-249.5/ The trait-enhancing allele (LE3) of the QTL on chromosome 250.4 is used, with genotypes LE5LE5 (homozygous for the LE allele), LE5LC5 (heterozygous) and LC5LC5 (homozygous for the LC allele). 3 seems to be recessive, suggesting that a loss-of-function Values represent the means of the concentration of Zn in roots mutation might be involved in the superior Zn accumulation measured in the mapping population and are given as µmol Zn g−1 capacity of the LE accession, relative to the LC accession. The root DW. On each bar the standard error of the mean (SE) and gene responsible for this QTL could be a down-regulator of Zn number of plants with the corresponding genotype (n) are indicated. uptake or, alternatively, promote Zn translocation to the shoot. It seems unlikely, however, that high root Zn concentrations would exclusively result from low rates of translocation to the None of the detected QTL colocalized with TcZNT1 (top shoot, as root and shoot Zn concentrations in the F3 mapping of chromosome 2). The QTL analysis presented here identi- population were uncorrelated (r = 0.07) and the mean shoot fies loci that significantly contribute to the within-species trait Zn concentrations did not differ between the root QTL variability. Although they have high variation in their Zn genotypes (data not shown). accumulation levels, both the LE and the LC accessions The apparent additive effect on the root Zn accumulation hyperaccumulate Zn when compared with a nonaccumulator phenotype of the trait-enhancing alleles (Fig. 4), specially species (Assunção et al., 2003b). It is conceivable that TcZNT1 with the genotypes LE3LE3LC5LC5 and LE3LE3LE5LC5, is of major importance for Zn hyperaccumulation (as sug- would be expected to lead to transgression in the phenotypic gested by Pence et al., 2000 and Assunção et al., 2001), which frequency distribution of the F3 mapping population. This is common to both accessions, but that there is no detectable was not evident; only one individual had a root Zn concen- genetic variation for this locus with respect to Zn accumula- tration higher than the upper limit of the analysed LE tion in this intraspecific cross. This is also in accordance with accession plants (Fig. 2c). However, the phenotypic frequency the very similar mRNA expression of TcZNT1 in LE and LC, distribution of the LE accession was quite broad (Fig. 2a), when grown for 3 wk at 10 µM Zn (Assunção et al., 2001). probably as a result of genetic variation present in the local The best way to further unravel the two identified QTL population from which the LE parent was taken. This is in would be to first identify to which regions of the Arabidopsis accordance with previous findings reported by Molitor et al. genome the T. caerulescens QTL regions correspond and use (2005). The root Zn accumulation phenotype was not deter- this information to either fine-map the region further or mined for the actual LE parental plant, as the analysis is search directly for possible candidate genes based on a pre- destructive. Therefore it is not inconceivable that there are sumed function in metal homeostasis. The QTL on chromo- indeed F3 plants with higher root Zn accumulation than the some 3 (Fig. 3) is closely linked to two T. caerulescens genes original LE parent. which are orthologues of Arabidopsis genes located on the top

Fig. 3 Lellingen/La Calamine (LE/LC) ampliﬁed fragment length polymorphism (AFLP)-based linkage map showing seven linkage groups. Cleaved ampliﬁed polymorphic sequence (CAPS)/insertion/deletion (Indel) codominant markers are shown in bold. AFLP codominant markers are shown in italic. Genetic distance between markers in cM is shown on the left of each linkage group bar. Logarithm of the odds (LOD) score values used for quantitative trait loci (QTL) analysis are indicated on the right of LG3 and LG5. The dashed line indicates a LOD value of 3. The dark bar corresponds to the area of maximum LOD value.

half of chromosome 5 of Arabidopsis: marker indel47/48, Acknowledgements used as a cofactor to further analyse the QTL on LG3, and the We thank Diana Rigola for providing Thlaspi caerulescens EST closely linked marker CAPS65/66 are, respectively, genes data, Petra van den Berg, Fien Meijer and Mattijs Bliek for At5g21274 and At5g20830. This strongly suggests conserva- technical advice and Prof. Dr Maarten Koornneef for critical tion of genome colinearity between T. caerulescens and reading of the manuscript. This research was supported by the Arabidopsis in that region and thus that the T. caerulescens EU project PHYTAC, contract no. QLRT-2001-00429 (to gene underlying the LG3 QTL has an Arabidopsis orthologue AGLA). located on chromosome 5 in the vicinity of At5g20830/ At5g21274. 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b, homozygous for the LC allele; c, homozygous for the LC allele or zinc concentration in the shoots (µmol Zn g−1 shoot DW). The F3 heterozygous; h, heterozygous. individuals (one per F3 line; see the Materials and Methods section) are represented as: ‘LExLC-line nr’. Table S2 Phenotypic data from the Thlaspi caerulescens F3 mapping population. Column A contains the 81 individuals of the F3 mapping population. Column B contains the data on zinc (Zn) concentration This material is available as part of the online article from in the roots (µmol Zn g−1 root DW). Column C contains the data on http://www.blackwell-synergy.com