Download Construction of a genetic linkage map of Thlaspi

Research
Construction of a genetic linkage map of Thlaspi
caerulescens and quantitative trait loci analysis of zinc
accumulation
Blackwell Publishing Ltd
Ana G. L. Assunção1*, Bjorn Pieper2, Jaap Vromans3, Pim Lindhout3, Mark G. M. Aarts2 and Henk Schat1
1
Institute 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:
Ana G. L. Assunção
Tel: +31 317485413
Fax: +31 317483146
Email: [email protected]
Received: 13 September 2005
Accepted: 2 November 2005
• Zinc (Zn) hyperaccumulation seems to be a constitutive species-level trait in
Thlaspi caerulescens. When compared under conditions of equal Zn availability,
considerable variation in the degree of hyperaccumulation is observed among
accessions originating from different soil types. This variation offers an excellent
opportunity for further dissection of the genetics of this trait.
• A T. caerulescens intraspecific cross was made between a plant from a nonmetallicolous 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)
Introduction
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 micronutrientdeficient 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
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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).
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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; Escarré et 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 toler-
ance, 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.
Materials and methods
Plant material
A Thlaspi caerulescens J. & C. Presl F3 population was used for
constructing the linkage map. The F3 mapping population
consisted of 81 individuals (one individual per F3 line)
derived by single-seed descent from self-fertilized F2 plants
originating from a single self-fertilized F1 plant. The F1 plant
was derived from a cross between a plant from the accession
Lellingen (LE), originating from a nonmetalliferous site near
Lellingen, Luxembourg, and a plant from the accession La
Calamine (LC), originating from a strongly lead (Pb)/Cd/Znenriched site near La Calamine, Belgium. This cross has been
previously described in Assunção et al. (2003c), in which the
F3 mapping population has been referred to as F3(4).
Plant culture and phenotyping
The Zn accumulation phenotype was measured in roots and
shoots of 71 individuals [71 F3(4) families] out of the 81 that
constitute the F3 mapping population, and in 10–20 plants
originating from the LE and LC accessions. These phenotypic
data [µmol Zn g−1 root dry weight (DW) and µmol Zn g−1
shoot DW] were obtained from Assunção et al. (2003c),
where the plant culture methods and Zn accumulation
measurements have been described. In short, seeds were sown
on moist peat and 3-wk-old seedlings were transferred to 1-l
pots (one plant per pot), filled with modified half-strength
Hoagland’s nutrient solution, supplemented with 10 µM
ZnSO4. After 3 wk, the plants were harvested and the Zn
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
polymorphism (AFLP) markers
Primers/adapters
Sequences
EcoRI adapter
5′-CTCGTAGACTGCGTACC-3′
3′-CATCTGACGCATGGT TAA-5′
E00 (universal primer)
EcoRI + 1 selective nucleotide E01
EcoRI + 3 selective nucleotide E32
E35
E41
E45
GACTGCGTACCAAT TC
E00 + A
E00 + AAC
E00 + ACA
E00 + AGG
E00 + ATG
PstI adapter
5′-CTCGTAGACTGCGTACATGCA-3′
3′-CATCTGACGCATGT-5′
P00 (universal primer)
PstI + 0 selective nucleotide P00
PstI + 2 selective nucleotide P14
GACTGCGTACATGCAG
P00
P00 + AT
MseI adapter
5′-GACGATGAGTCCTGAG-3′
3′-TACTCAGGACTCAT-5′
M00 (universal primer)
MseI + 0 selective nucleotide M00
MseI + 2 selective nucleotide M11
M12
M13
M14
M15
M17
M18
M20
M21
M22
M23
M24
M25
MseI +3 selective nucleotide M45
M47
M48
M50
M51
M52
GATGAGTCCTGAGTAA
M00
M00 + AA
M00 + AC
M00 + AG
M00 + AT
M00 + CA
M00 + CG
M00 + CT
M00 + GC
M00 + GG
M00 + GT
M00 + TA
M00 + TC
M00 + TG
M00 + ATG
M00 + CAA
M00 + CAC
M00 + CAT
M00 + CCA
M00 + CCC
the experiment the nutrient solutions were replaced twice a
week and the pots were re-randomized at each solution
replacement (Assunção et al., 2003c).
Genotyping
AFLP markers The F3 mapping population was genotyped
using AFLP marker analysis (Vos et al., 1995), which was
performed as described by Qi & Lindhout (1997). Two pairs
of restriction enzymes, EcoRI/MseI (E/M) and PstI/MseI (P/
M), were used to generate the restriction fragments for
amplification. The restriction enzymes, adaptors and primers
used are listed in Table 1. Initially, 58 primer combinations
(PCs), 48 E/M (+3+2) and 10 P/M (+2+3), were tested for
their polymorphism rates between the parental lines LE and
LC. Each of the parental lines consisted of four individuals,
progeny of each of the self-fertilized parents (LE and LC). The
PCs selected for use in the F3 mapping population were
E32M12, E32M13, E32M14, E32M15, E32M18, E32M20,
E32M22, E32M23, E35M11, E35M13, E35M17, E41M22,
E45M20, E45M21, E45M22, E45M24, E45M25, P14M45,
P14M47, P14M48, P14M50 and P14M52. AFLP profiles of
the F1 were also obtained for all the selected PCs.
Segregating markers in the mapping population were
designated according to the restriction enzymes and the primer
combination used, and their sizes estimated with reference
to the SequaMark 10 base ladder (Research Genetics, Huntsville, AL, USA). They were scored as dominant markers, using
the specialized software package for the analysis of DNA
fingerprints AFLP-Quantar® (KeyGene NV, Wageningen, the
Netherlands), and designated according to the AFLP profiles
of the parent lines (Fig. 1). The AFLP profile of the F1 line
was used to confirm the reliability of each marker segregating
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).
Fig. 1 An amplified fragment length polymorphism (AFLP) image
obtained with the primer combination E32M12. It includes a set of
Lellingen/La Calamine (LE/LC) F3 lines, the F1 line, the LC (calamine
accession) and LE (nonmetallicolous accession) parental lines and the
molecular marker (MM). Examples of different types of segregating
markers are given: dominant markers, LE-specific (e32m12-192.7LE) and LC-specific (e32m12-180.8-LC), and codominant markers,
scored with AFLP-Quantar®PRO (KeyGene NV, Wageningen, the
Netherlands) (e32m12-215.4-QP) and allelic band pairs (e32m12279.1/277.2).
Codominant AFLP markers In order to score codominant
markers, the AFLP-Quantar®PRO version (KeyGene NV)
was used. This program can detect codominant markers based
on intensity differences of the corresponding AFLP bands. For
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
the TcZNT1 (Zn transporter) gene (Pence et al., 2000), a
ZNT1 cDNA was used. Introns were preferentially targeted
for amplification in T. caerulescens. When the Arabidopsis
homologue did not contain an intron or the EST only
represented a single exon of multiple exons in the Arabidopsis
homologue (hence an intron could not be targeted for
amplification), a stretch of EST sequence was targeted for
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
were confirmed in both parental lines (the same individuals
used to test PCs in the AFLP analysis) and in the F1, before
being scored in the F3 mapping population. CAPS markers
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
marker
Arabidopsis
homologue
CAPS21/22
At2g01610
CAPS55/56
At4g26050
CAPS65/66
At5g20830
CAPS75/76
At5g43780
CAPS85/86
At2g36540
CAPS89/90
At1g10970
CAPS95/96
At5g67330
Indel29/30
At2g36830
Indel39/40
At3g19820
Indel47/48
At5g21274
Primer nucleotide sequence
F 5′-GGTAAGCCAAAGTCATACGACGACG-3′
R 5′-TCCACGTCTGCACGT TACTCATCTG-3′
F 5′-ACGGAATCACGGTGT TGCCG-3′
R 5′-TGCTTCACTGCT TCCAAACCCTG-3′
F 5′-TGTAAGGAAGATCCATCTCACTGGGAC-3′
R 5′-AAGCCAATACACACCGGTCAATGTC-3′
F 5′-CGTTGTCAACATGTCGGT TCCG-3′
R 5′-TCGCCTCCGATCAACCAGT T TC-3′
F 5′-AAAGGCTTT TCTGCT TCAAACACTGTC-3′
R 5′-TCAGGATGAGAAGAATCGATCAT TGG-3′
F 5′-CGTGGTTGTGGAAAGAGGGAATG-3′
R 5′-TGCAATGAGAGGCCTGATCGTG-3′
F 5′-AATCGCCGGGTACCGGAAAG-3′
R 5′-TTACAACTCCAGCCCAGAGAGGAATC-3′
F 5′-GTGGTAACATCACTCTCCTCCGTGG-3′
R 5′-AAGCATTTAGCACTCCTACTCCGGC-3′
F 5′-TGTTCCTCTTTACAAGGTCGGCG-3′
R 5′-TCCTTGCCTCTTCTCGTACTCGAAC-3′
F 5′-TCAGAGTTCAAGGAAGCGT T TAGCC-3′
R 5′-CATCACGGTCCCAAGCTCCT TC-3′
Fragment
size (bp)
Restriction enzyme (CAPS)
or allelic size difference (Indels)
490
MnlI
480
Trul I
250
Trul I
380
HpaI
400
DdeI
360
Trul I
530
BamHI
350
14 bp
250
20 bp
850
23 bp
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®
agarose gels (Cambrex Bio Science Rockland Inc., Rockland,
ME, USA).
Map construction
A genetic linkage map of the mapping population was
constructed based on dominant and codominant AFLP markers
and on codominant CAPS/Indel markers. The JoinMap® 3.0
software package (Plant Research International, Wageningen,
the Netherlands) (Van Ooijen & Voorrips, 2001) was used for
linkage grouping and map construction. Kosambi’s mapping
function was applied for map-distance calculation (Kosambi,
1944).
QTL analysis
The associations between molecular markers and QTL for
Zn accumulation in root and in shoot were detected using the
computer program MapQTL® 5 (Kyazma BV, Wageningen,
the Netherlands) (Van Ooijen, 2004). A logarithm of the odds
(LOD) score of 3.0 was used as the threshold for detecting
QTL (Van Ooijen, 1999). The interval mapping method and
the multiple-QTL models (MQM) mapping method were
used to detect and map QTL. The QTL graphs were prepared
with MapChart (Voorrips, 2002).
Results
Segregation of zinc accumulation
Zinc accumulation, in roots and in shoots, was established in
hydroponically grown plants exposed to 10 µM Zn for 3 wk.
For the accumulation of Zn in the roots, the LC and LE accessions
exhibited different, slightly overlapping phenotypic frequency
distributions, with LC showing lower Zn accumulation than
LE (Fig. 2a). The frequency distributions of Zn accumulation
in the shoot exhibited by LC and LE accessions did not
overlap, with LC showing lower Zn accumulation than LE
(Fig. 2b). Zinc accumulation in roots and in shoots was also
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
segregation of these traits in the F3 population (Fig. 2c,d).
All but one F3 plant had a root or shoot Zn accumulation
phenotype between the lower and upper limits of the LC or
LE phenotypic range. Shoot and root Zn concentrations in
the F3 population were uncorrelated (r = 0.07).
Analysis and scoring of molecular markers
Fifty-eight AFLP PCs were tested in the parental lines. The
sizes of fragments generated ranged from about 80 bp to
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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
average number of well-amplified bands per PC varied
between 30 and 85, with an average of 48 bands, and the
average polymorphism rate between LE and LC was 24%.
Twenty-two out of the tested 58 PCs were selected for
genetic mapping. On average, 15 markers were scored per
PC, ranging from eight (E35M11) to 34 (E32M12). In
total, of the 22 PCs, 327 segregating AFLP markers were
identified and scored in the mapping population, of
which 133 were LE-specific and 157 were LC-specific, 10
could be scored as codominant using AFLP-Quantar®
PRO and an additional 27 allelic band pairs were identified
(see Materials and methods) and scored as 27 codominant
markers (Fig. 1). In the latter 27 markers, the molecular
size difference between the two allelic fragments was 1 to
5 bp.
In addition to the AFLP markers, 10 PCR-based markers
were developed, seven of which were CAPS and three of
which were Indels (Table 2). These codominant markers were
also scored in the mapping population. In total, 337 markers
(327 AFLP and 10 CAPS/Indels) were scored.
The CAPS marker caps89/90 corresponds to TcZNT1, a
T. caerulescens metal transporter gene, homologous to AtZIP4
(ZRT/IRT-like protein) (Grotz et al., 1998), known to mediate
Zn transport and suggested to be involved in Zn hyperaccumulation in T. caerulescens (Pence et al., 2000). The CAPS
marker caps95/96 is a homologue of the metal transporter
AtNRAMP4 (natural resistance-associated macrophage
protein) (Thomine et al., 2000). A T. caerulescens homologue,
TcNRAMP4, mediates the transport of Cd, iron, manganese
and Zn (R. Oomen & S. Thomine, CNRS, Gif-Sur-Yvette,
France, personal communication).
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Construction of an LE/LC linkage map
A genetic linkage map consisting of seven linkage groups
(LGs) with in total 319 markers, including 309 AFLP
(of which 37 are codominant) and 10 CAPS/Indels, was
constructed. From the 337 markers scored, 333 were
assembled in seven linkage groups with an LOD threshold
grouping value of 5.0. During calculation of map order and
distance, an additional 14 markers were omitted because they
could not be reliably placed in the linkage groups. A total map
distance of 449 cM was covered (Fig. 3), corresponding to an
average interval of 1.6 cM between adjacent markers
(absolutely linked markers were excluded in the calculation of
this average). Linkage groups 1, 2, 4 and 6 have one gap of
10–15 cM and LG5 has two such intervals. LG6 covers the
longest genetic distance (80.7 cM) while LG1 covers the
shortest distance (44.5 cM).
The great majority of the loci (89%) showed genotype
ratios as expected for a segregating F3 population
(0.375 : 0.25 : 0.375). For 36 markers this frequency was
significantly skewed (P < 0.01), with a cluster of eight loci
mapping to LG1 between positions 39 and 49 cM largely
representing LE alleles, and a cluster of 22 loci mapping to
LG7 between positions 37 and 53 cM with an overrepresentation of LC alleles. The other markers with a disturbed
segregation were not linked to markers with distorted ratios.
Mapping QTL
Using the Zn accumulation phenotypic data, we carried out
interval mapping to identify and locate QTL associated with
Zn accumulation in roots and/or shoots. For Zn accumulation
in roots only, we detected two QTL, one in LG3 and another
in LG5 (Fig. 3). Of several markers linked to these loci, we
used markers indel47/48 and e32m14-249.5/250.4 for LG3
and LG5, respectively, as cofactors for MQM mapping. Based
on this analysis, we found the LG3 QTL to have a LOD value
of 4.6, explaining 21.7% of the total variance, and the LG5
QTL to have a LOD value of 3.6, explaining 16.6% of the
total variance. The trait-enhancing allele for the LG3 QTL
originated from the LE parent and the trait-enhancing
allele for the LG5 QTL originated from the LC parent.
The two cofactors used for MQM analysis of both QTL are
codominant markers, which allowed further assessment of the
genotypes for both loci. The analysis of the genotypes of each
loci indicated that the trait-enhancing allele LE3 is recessive
and the trait-enhancing allele LC5 is codominant [one-way
analysis of variance (ANOVA) of log-transformed data,
F2,68 = 11.51 and 10.93, P < 0.001, respectively, followed
by Tukey’s test, to compare means]. For the genotypes of
both loci, it appears that the simultaneous presence of
both trait-enhancing alleles (specially LE3LE3LC5LC5 and
LE3LE3LC5LE5 genotypes) has an additive effect on the root
Zn accumulation phenotype (Fig. 4).
Discussion
Segregation of zinc accumulation
To map QTL for Zn accumulation it is necessary that this trait
is segregating in the mapping population. We measured both
root and shoot Zn accumulation phenotypes in individuals of
the parent accessions and of the segregating F3 progeny, upon
exposure to 10 µM Zn for 3 wk. These data on root and shoot
Zn accumulation have previously been used to calculate the Zn
accumulation, on a total plant dry weight basis, of individuals of
the parent accessions and of the segregating F3 progeny [F3(4)]
in Assunção et al. (2003c). The phenotypic frequency distributions of root and shoot Zn accumulation segregated in the F3
mapping population, with phenotypic frequency distributions
being more or less continuous (Fig. 2c,d), suggesting polygenic
or at least digenic inheritance. As we do not have the data to
calculate heritability levels, it is not possible to estimate the
precise numbers of loci involved in the control of these traits.
A T. caerulescens genetic linkage map
A genetic linkage map with 319 markers was generated (Fig. 3).
It consists of seven linkage groups (LGs), corresponding to the
haploid chromosome number of T. caerulescens. As nearly all
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
average genetic distance between markers of 1.6 cM and with
few intervals, no longer than 10–15 cM, between adjacent
markers, the map covers the T. caerulescens genome very nicely.
Each chromosome contains a more dense cluster of markers,
probably representing the putative centromeres. Clustering of
random genetic markers around the centromere, mainly as a result
of centromeric suppression of recombination, has been reported
in maps of several crop species such as barley (Hordeum vulgare),
tomato and wheat (Triticum aestivum) (Chao et al., 1989; Tanksley
et al., 1992; Qi et al., 1998). There are two clusters of skewed
markers, on chromosomes 1 and 7, which show distorted segregation instead of the expected F3 segregation ratios. This happens
more frequently in segregating populations [e.g. in Arabidopsis
lyrata (Yogeeswaran et al., 2005) and Arabidopsis thaliana (Boivin
et al., 2004)], because of the presence of alleles or combinations
of alleles leading to an unfavourable phenotype. When the joint
map was compared with each of the parental maps, in general
a similar marker order was found (data not shown), indicating
that the datasets for the parents are reliable and consistent.
Taken together, all our results indicate that we have produced
a robust genetic linkage map for the T. caerulescens genome.
QTL for zinc accumulation
A second goal of this research was to identify associations
between molecular markers and the traits of Zn accumulation
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www.newphytologist.org © The Authors (2005). Journal compilation © New Phytologist (2005)
Research
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
trait-enhancing alleles come from different parents. For the
QTL on chromosome 3, the LE allele (LE3) gives rise to
higher accumulation, and for the QTL on chromosome 5, it
is the LC allele (LC5) that contributes to higher accumulation.
The trait-enhancing allele (LE3) of the QTL on chromosome
3 seems to be recessive, suggesting that a loss-of-function
mutation might be involved in the superior Zn accumulation
capacity of the LE accession, relative to the LC accession. The
gene responsible for this QTL could be a down-regulator of Zn
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
shoot, as root and shoot Zn concentrations in the F3 mapping
population were uncorrelated (r = 0.07) and the mean shoot
Zn concentrations did not differ between the root QTL
genotypes (data not shown).
The apparent additive effect on the root Zn accumulation
phenotype of the trait-enhancing alleles (Fig. 4), specially
with the genotypes LE3LE3LC5LC5 and LE3LE3LE5LC5,
would be expected to lead to transgression in the phenotypic
frequency distribution of the F3 mapping population. This
was not evident; only one individual had a root Zn concentration higher than the upper limit of the analysed LE
accession plants (Fig. 2c). However, the phenotypic frequency
distribution of the LE accession was quite broad (Fig. 2a),
probably as a result of genetic variation present in the local
population from which the LE parent was taken. This is in
accordance with previous findings reported by Molitor et al.
(2005). The root Zn accumulation phenotype was not determined for the actual LE parental plant, as the analysis is
destructive. Therefore it is not inconceivable that there are
indeed F3 plants with higher root Zn accumulation than the
original LE parent.
Fig. 4 Phenotypes of quantitative trait loci (QTL) genotypes. For the
locus on LG3, the closely linked marker indel47/48 is used, with
genotypes LE3LE3 [homozygous for the Lellingen (LE) allele], LE3LC3
(heterozygous) and LC3LC3 [homozygous for the La Calamine (LC)
allele]. For the LG5 locus, the closely linked marker e32m14-249.5/
250.4 is used, with genotypes LE5LE5 (homozygous for the LE allele),
LE5LC5 (heterozygous) and LC5LC5 (homozygous for the LC allele).
Values represent the means of the concentration of Zn in roots
measured in the mapping population and are given as µmol Zn g−1
root DW. On each bar the standard error of the mean (SE) and
number of plants with the corresponding genotype (n) are indicated.
None of the detected QTL colocalized with TcZNT1 (top
of chromosome 2). The QTL analysis presented here identifies loci that significantly contribute to the within-species trait
variability. Although they have high variation in their Zn
accumulation levels, both the LE and the LC accessions
hyperaccumulate Zn when compared with a nonaccumulator
species (Assunção et al., 2003b). It is conceivable that TcZNT1
is of major importance for Zn hyperaccumulation (as suggested by Pence et al., 2000 and Assunção et al., 2001), which
is common to both accessions, but that there is no detectable
genetic variation for this locus with respect to Zn accumulation in this intraspecific cross. This is also in accordance with
the very similar mRNA expression of TcZNT1 in LE and LC,
when grown for 3 wk at 10 µM Zn (Assunção et al., 2001).
The best way to further unravel the two identified QTL
would be to first identify to which regions of the Arabidopsis
genome the T. caerulescens QTL regions correspond and use
this information to either fine-map the region further or
search directly for possible candidate genes based on a presumed function in metal homeostasis. The QTL on chromosome 3 (Fig. 3) is closely linked to two T. caerulescens genes
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.
© The Authors (2005). Journal compilation © New Phytologist (2005) www.newphytologist.org
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half of chromosome 5 of Arabidopsis: marker indel47/48,
used as a cofactor to further analyse the QTL on LG3, and the
closely linked marker CAPS65/66 are, respectively, genes
At5g21274 and At5g20830. This strongly suggests conservation of genome colinearity between T. caerulescens and
Arabidopsis in that region and thus that the T. caerulescens
gene underlying the LG3 QTL has an Arabidopsis orthologue
located on chromosome 5 in the vicinity of At5g20830/
At5g21274. In this region of chromosome 5 there are three
genes possibly involved in metal homeostasis, namely
AtFRO4 (At5g23980) (ferric reductase oxidase, FRO) and
AtFRO5 (At5g23990), both encoding ferric-chelate reductases,
and AtYSL2 (At5g24380) (yellow-stripe like, YSL), encoding
a yellow-striped leaf-like protein. AtFRO4 and AtFRO5 have
not been studied in much detail, but they share homology to
AtFRO2, which is a root cell plasma membrane ferric reductase involved in iron uptake (Robinson et al., 1999). It seems
unlikely, however, that these genes would be directly involved
in Zn accumulation. AtYSL2 encodes a protein demonstrated
to facilitate transport of nicotianamine-chelated iron and
copper. In yeast this protein was not found to complement a
Zn uptake mutant (DiDonato et al., 2004).
For the QTL on chromosome 5 of T. caerulescens (Fig. 3),
there is no closely linked marker corresponding to a region of
the Arabidopsis genome. The only markers linking the two
genomes are indel29/30 and CAPS85/86, both residing on
the lower half of chromosome 2 of Arabidopsis at At2g36830
and At2g36540, respectively. These markers are around
20 cM distant from the most likely QTL position. As there is
no additional information on colinearity between Arabidopsis
and T. caerulescens or any other closely related species, this
may well correspond to a region on another chromosome of
Arabidopsis.
In this study we have constructed a reliable and robust
genetic map for a segregating F3 population of a so far genetically uncharacterized plant species. Such a map is very useful
for the further analysis of natural genetic variation for Zn
accumulation found among wild accessions of T. caerulescens.
Although we used only a limited set of markers corresponding
to orthologous genes in both T. caerulescens and Arabidopsis,
it was possible to make realistic suggestions about which
region of the Arabidopsis genome could harbour an orthologue
of the T. caerulescens gene underlying one of the identified
QTL. This is not conclusive evidence that there is indeed such
an orthologue, as T. caerulescens contains genes not found to
have an Arabidopsis orthologue (D. Rigola & M. G. M.
Aarts, unpublished results). However, this is a suitable starting
point to focus on for further fine-mapping of the region in
order to limit the number of putative QTL candidate genes.
In the future, fine-mapping of the QTL regions in
T. caerulescens and local saturation of the T. caerulescens map
with more Arabidopsis ‘anchors’ will provide the required
information that can eventually lead to the cloning of the
QTL genes.
Acknowledgements
We thank Diana Rigola for providing Thlaspi caerulescens EST
data, Petra van den Berg, Fien Meijer and Mattijs Bliek for
technical advice and Prof. Dr Maarten Koornneef for critical
reading of the manuscript. This research was supported by the
EU project PHYTAC, contract no. QLRT-2001-00429 (to
AGLA).
References
Acarkan A, Rossberg M, Koch M, Schmidt R. 2000. Comparative genome
analysis reveals extensive conservation of genome organisation for
Arabidopsis thaliana and Capsella rubella. Plant Journal 23: 55–62.
Alonso-Blanco C, Koornneef M. 2000. Naturally occurring variation in
Arabidopsis: an underexploited resource for plant genetics. Trends in Plant
Science 5: 22–29.
Alonso-Blanco C, Peeters AJM, Koornneef M, Lister C, Dean C, Van den
Bosch N, Pot J, Kuiper MTR. 1998. Development of an AFLP based
linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and
construction of a Ler/Cvi recombinant inbred line population. Plant
Journal 14: 259–271.
Arabidopsis Genome Initiative (AGI). 2000. Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana. Nature 408:
796–815.
Assunção AGL, Da Costa Martins P, De Folter S, Vooijs R, Schat H, Aarts
MGM. 2001. Elevated expression of metal transporter genes in three
accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant, Cell
& Environment 24: 217–226.
Assunção AGL, Schat H, Aarts MGM. 2003a. Thlaspi caerulescens, an
attractive model species to study heavy metal hyperaccumulation in plants.
New Phytologist 159: 351–360.
Assunção AGL, Ten Bookum WM, Nelissen HJM, Vooijs R, Schat H,
Ernst WHO. 2003b. Differential metal-specific tolerance and
accumulation patterns among Thlaspi caerulescens populations originating
from different soil types. New Phytologist 159: 411–419.
Assunção AGL, Ten Bookum WM, Nelissen HJM, Vooijs R, Schat H,
Ernst WHO. 2003c. A co-segregation analysis of zinc (Zn) accumulation
and Zn tolerance in the Zn hyperaccumulator Thlaspi caerulescens. New
Phytologist 159: 383–390.
Boivin K, Acarkan A, Mbulu R-S, Clarenz O, Schmidt R. 2004. The
Arabidopsis genome sequence as a tool for genome analysis in Brassicaceae.
A comparison of the Arabidopsis and Capsella rubella genomes. Plant
Physiology 135: 735–744.
Borevitz JO, Chory J. 2004. Genomic tools for QTL analysis and gene
discovery. Current Opinion in Plant Biology 7: 132–136.
Brooks RR, Lee J, Reeves RD, Jaffrré T. 1977. Detection of nickeliferous
rocks by analysis of herbarium specimens of indicator plants. Journal of
Geochemical Exploration 7: 49–57.
Cavell A, Lydiate D, Parkin I, Dean C, Trick M. 1998. A 30 centimorgan
segment of Arabidopsis thaliana chromosome 4 has six collinear
homologues within the Brassica napus genome. Genome 41: 62–69.
Chao S, Sharp PJ, Worland AJ, Warham EJ, Koebner RMD, Gale MD.
1989. RFLP-based genetic maps of wheat homoeologous group-7
chromosomes. Theoretical and Applied Genetics 78: 495–504.
Clemens S. 2001. Molecular mechanisms of plant metal tolerance and
homeostasis. Planta 212: 475–486.
Clemens S, Palmgren MG, Krämer U. 2002. A long way ahead:
understanding and engineering plant metal accumulation. Trends in Plant
Science 7: 309–315.
Cobbett C, Goldsbrough P. 2002. Phytochelatins and metallothioneins:
Roles in heavy metal detoxification and homeostasis. Annual Review of
Plant Biology 53: 159–182.
www.newphytologist.org © The Authors (2005). Journal compilation © New Phytologist (2005)
Research
DiDonato RJ Jr, Roberts LA, Sanderson T, Eisley RB, Walker EL. 2004.
Arabidopsis. Yellow Stripe-Like2 (YSL2): a metal-regulated gene encoding
a plasma membrane transporter of nicotianamine-metal complexes. Plant
Journal 39: 403–414.
Escarré J, Lefèbvre C, Gruber W, Leblanc M, Lepart J, Rivière Y, Delay B.
2000. Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from
metalliferous and nonmetalliferous sites in the Mediterranean area:
implications for phytoextraction. New Phytologist 145: 429–437.
Grotz N, Fox TC, Connolly E, Park W, Guerinot ML, Eide D. 1998.
Identification of a family of zinc transporter genes from Arabidopsis that
respond to zinc deficiency. Proceedings of the National Academy of Sciences,
USA 95: 7220–7224.
Koch M, Bishop J, Mitchell-Olds T. 1999. Molecular systematics and
evolution of Arabidopsis and Arabis. Plant Biology 1: 529–537.
Kosambi DD. 1944. The estimation of map distance from recombination
values. Annals of Eugenics 12: 172–175.
Kowalski SP, Lan T-H, Feldmann KA, Paterson AH. 1994. Comparative
mapping of Arabidopsis thaliana and Brassica oleracea chromosomes reveals
islands of conserved organization. Genetics 138: 499–510.
Lan T-H, Delmonte TA, Reischmann KP, Hyman J, Kowalski SP. 2000.
An EST-enriched comparative map of Brassica oleracea and Arabidopsis
thaliana. Genome Research 10: 776 –788.
Lasat MM, Baker AJM, Kochian LV. 1996. Physiological characterization
of root Zn2+ absorption and translocation to shoots in Zn
hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiology
112: 1715–1722.
Maheswaran M, Subudhi PK, Nandi S, Xu JC, Parco A, Yang DC, Huang N.
1997. Polymorphism, distribution, and segregation of AFLP markers in
a doubled haploid rice population. Theoretical and Applied Genetics 94:
39–45.
Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN,
Amtmann A, Maathuis FJ, Sanders D, Harper JF, Tchieu J, Gribskov M,
Persans MW, Salt DE, Kim SA, Guerinot ML. 2001. Phylogenetic
relationships within cation transporter families of Arabidopsis. Plant
Physiology 126: 1646–1667.
Meerts P, Van Isacker N. 1997. Heavy metal tolerance and accumulation in
metallicolous and non-metallicolous populations of Thlaspi caerulescens
from continental Europe. Plant Ecology 133: 221–231.
Molitor M, Dechamps C, Gruber W, Meerts P. 2005. Thlaspi caerulescens
on nonmetalliferous soil in Luxembourg: ecological niche and genetic
variation in mineral element composition. New Phytologist 165:
503–512.
Peer WA, Mamoudian M, Lahner B, Reeves RD, Murphy AS, Salt DE.
2003. Identifying model metal hyperaccumulating plants: germplasm
analysis of 20 Brassicaceae accessions from a wide geographical area. New
Phytologist 159: 421–430.
Pence NS, Larsen PB, Ebbs SD, Letham DLD, Lasat MM, Garvin DF,
Eide D, Kochian LV. 2000. The molecular physiology of heavy metal
transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proceedings
of the National Academy of Sciences, USA 97: 4956–4960.
Perin C, Hagen LS, Conto VD, Katzir N, Danin Poleg Y, Portnoy V,
Baudracco Arnas S, Chadoeuf J, Dogimont C, Pitrat M. 2002. A
reference map of Cucumis melo based on two recombinant inbred line
populations. Theoretical and Applied Genetics 104: 1017–1034.
Qi X, Lindhout P. 1997. Development of AFLP markers in barley. Molecular
and General Genetics 254: 330–336.
Qi X, Stam P, Lindhout P. 1998. Use of locus-specific AFLP markers to
construct a high-density molecular map in barley. Theoretical and Applied
Genetics 96: 376–384.
Reeves RD. 1992. The hyperaccumulation of nickel by serpentine plants. In:
Baker AJM, Proctor J, Reeves RD, eds. The vegetation of Ultramafic
(Serpentine) soils. Proceedings of the First International Conference on
Serpentine Ecology. Andover, UK: Intercept, 253–277.
Robinson NJ, Procter CM, Connolly EL, Guerinot ML. 1999.
A ferric-chelate reductase for iron uptake from soils. Nature 397: 694–697.
Roosens N, Verbruggen N, Meerts P, Ximénez-Embún P, Smith JAC.
2003. Natural variation in cadmium tolerance and its relationship to metal
hyperaccumulation for seven populations of Thlaspi caerulescens from
western Europe. Plant, Cell & Environment 26: 1657–1672.
Saliba-Colombani V, Causse M, Gervais L, Philouze J. 2000. Efficiency of
RFLP, RAPD, and AFLP markers for the construction of an intraspecific
map of the tomato genome. Genome 43: 29–40.
Schat H, Llugany M, Bernhard R. 2000. Metal-specific patterns of
tolerance, uptake, and transport of heavy metals in hyperaccumulating and
non-hyperaccumulating metallophytes. In: Terry N, Banuelos G, eds.
Phytoremediation of contaminated soils and water. Boca Raton, FL, USA:
CRC Press, 171–188.
Schmidt R. 2000. Synteny: recent advances and future prospects. Current
Opinion in Plant Biology 3: 97–102.
Schmidt R, Acarkan A, Boivin K. 2001. Comparative structural genomics
in the Brassicaceae family. Plant Physiology 39: 253–262.
Shen ZG, Zhao FJ, McGrath SP. 1997. Uptake and transport of zinc in the
hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator
Thlaspi ochroleucum. Plant, Cell & Environment 20: 898–906.
Tanksley SD, Ganal MW, Prince JP, De Vicente MC, Bonierbale MW,
Broun P, Fulton TM, Giovannoni JJ, Grandillo S, Martin GB,
Messeguer R, Miller JC, Miller L, Paterson AH, Pineda O, Röder MS,
Wing RA, Wu W, Young ND. 1992. High-density molecular linkage
maps of tomato and potato genomes. Genetics 132: 1141–1160.
Thomine S, Wang RC, Ward JM, Crawford NM, Schroeder JI. 2000.
Cadmium and iron transport by members of a plant metal transporter
family in Arabidopsis with homology to Nramp genes. Proceedings of the
National Academy of Sciences, USA 97: 4991–4996.
Van Ooijen JW. 1999. LOD significance thresholds for QTL analysis in
experimental populations of diploid species. Heredity 83: 613–624.
Van Ooijen JW. 2004. MapQTL® 5, software for the mapping of quantitative
trait loci in experimental populations. Wageningen, the Netherlands:
Kyazma B.V.
Van Ooijen JW, Voorrips RE. 2001. JoinMap® 3.0, software for the
calculation of genetic linkage maps. Wageningen, the Netherlands: Plant
Research International.
Voorrips RE. 2002. MapChart: Software for the graphical presentation of
linkage maps and QTLs. Journal of Heredity 93: 77–78.
Vos P, Hogers R, Bleeker R, Reijans M, Van de Lee T, Hornes M,
Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. 1995. AFLP:
a new technique for DNA fingerprinting. Nucleic Acids Research 23:
4407–4414.
Yogeeswaran K, Frary A, York TL, Amenta A, Lesser AH, Nasrallah JB,
Tanksley SD, Nasrallah ME. 2005. Comparative genome analyses of
Arabidopsis spp. Inferring chromosomal rearrangement events in the
evolutionary history of A. thaliana. Genome Research 15: 505–515.
Zha HG, Jiang RF, Zhao FJ, Vooijs R, Schat H, Barker JHA, McGrath S.
2004. Co-segregation analysis of cadmium and zinc accumulation in
Thlaspi caerulescens interecotypic crosses. New Phytologist 163: 299 –
312.
Supplementary material
The following supplementary material is available for this
article online.
Table S1 Genotypic data from the Thlaspi caerulescens F3 mapping
population. It contains the scorings of each of the 337 molecular
markers (rows) for each of the 81 individuals of the F3 mapping
population (columns). The F3 individuals (one per F3 line; see the
Materials and Methods section) are represented as: ‘LExLC-line nr’.
The interpretation of the genotypic data is as follows: a, homozygous
for the LE allele; d, homozygous for the LE allele or heterozygous;
© The Authors (2005). Journal compilation © New Phytologist (2005) www.newphytologist.org
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b, homozygous for the LC allele; c, homozygous for the LC allele or
heterozygous; h, heterozygous.
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
in the roots (µmol Zn g−1 root DW). Column C contains the data on
zinc concentration in the shoots (µmol Zn g−1 shoot DW). The F3
individuals (one per F3 line; see the Materials and Methods section)
are represented as: ‘LExLC-line nr’.
This material is available as part of the online article from
http://www.blackwell-synergy.com
www.newphytologist.org © The Authors (2005). Journal compilation © New Phytologist (2005)