Download Construction and Analysis of 2 Reciprocal Arabidopsis Introgression

Journal of Heredity 2008:99(4):396–406
doi:10.1093/jhered/esn014
Advance Access publication February 28, 2008
Ó The American Genetic Association. 2008. All rights reserved.
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Construction and Analysis of 2
Reciprocal Arabidopsis Introgression
Line Populations
OTTÓ TÖRJÉK*, RHONDA C. MEYER*, MAIK ZEHNSDORF, MELANIE TELTOW, GEORG STROMPEN,
HANNA WITUCKA-WALL, ANNA BLACHA, AND THOMAS ALTMANN
From the Department of Genetics, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse
24-25, 14476 Potsdam-Golm, Germany (Törjék, Teltow, Strompen, Witucka-Wall, and Altmann); and the Max-PlanckInstitute of Molecular Plant Physiology, Am Muehlenberg 1, 14476 Golm, Germany (Meyer, Zehnsdorf, Blacha, and
Altmann). Ottó Törjék is now at Biological Resource Center, Institute of Plant Biology, Cell Division Cycle and Stress
Adaptation Group, Temesvári krt. 62, PO Box 521, H-6701 Szeged, Hungary.
*These authors contributed equally to the work.
Address correspondence to R. C. Meyer at the address above, or e-mail: [email protected].
Abstract
Two new large reciprocal sets of introgression lines (ILs) were created between the Arabidopsis accessions Col-0 and C24.
In both sets (78 ILs with Col-0 background and 62 ILs with C24 background), the donor segments cover almost the entire
genome with an average substitution size of 18.3 cM. In addition to the basic sets of ILs, further subILs were developed for
2 genomic regions allowing better mapping resolution. SubILs carrying donor segments with candidate genes for flowering
time and reduced fertility were used to demonstrate the usefulness of the reciprocal ILs for quantitative trait loci detection
and fine mapping. For subIL development at high resolution around the reduced fertility locus, we used modified CelIbased assays in one-well format for both marker development and genotyping. This serves as a very flexible and costeffective approach.
Mapping quantitative trait loci (QTL) based on segregating
populations provides only rough positions of QTL (Kearsey
2002). Once QTL have been identified for a trait under
study, it becomes important to characterize and confirm the
contributing individual loci (Koornneef et al. 1997). One of
the most promising ways to do this is the use of
introgression lines (ILs). Lines of an introgression library
have a common recurrent genotype, but different, short,
donor segments from another line giving the ability to focus
precisely on any region of the genome (Eshed and Zamir
1995). These lines have a permanent homozygous genetic
constitution enabling also tests in different environments
and at different time points (Eshed et al. 1992). ILs with
minimized residual genetic variation allow the transformation of the individual QTL into single Mendelian factors.
Using the ILs, it is possible to study nonallelic gene
interactions of QTL regions with the genomic background.
Furthermore, crosses between ILs each bearing nonallelic
individual QTL can be combined to analyze the extent of
interactions (Peleman and Rouppe van der Voort 2003). In
396
addition, creating segregating offspring for the substitution
under study, ILs facilitate fine mapping and may lead toward
gene discovery (Zamir 2001). The usefulness of ILs in finemapping QTL has already been demonstrated in various
plant species (tomato—Frary et al. 2000; Fridman et al.
2000; rice—Takahashi et al. 2001; maize—Salvi et al. 2002;
Arabidopsis—El-Assal et al. 2001; Bentsink et al. 2003). In
Arabidopsis, a publicly available set of chromosome
substitution strains (CSS) (Col-0 chromosomes were
replaced by Ler and Nd) as well as ‘‘stepped aligned inbred
recombinant strains’’ harboring donor segments of increasing length in a single chromosome or ‘‘stepped single
inbred recombinant strains’’ carrying only one crossing-over
event were created by Koumproglou et al. (2002). Recently
Keurentjes et al. (2007) reported the development of
a genome-wide (nonreciprocal) IL population between
Arabidopsis accessions Ler and Cvi.
In high-resolution mapping, trait complexity and number
of identified recombination events, as well as sufficient
marker density are key factors (Peleman et al. 2005). For
Törjék et al. Reciprocal Arabidopsis IL Populations
species with well-analyzed genomes such as Arabidopsis,
large numbers of markers are usually available if standard
combinations of mapping lines (especially Col-0 and Ler) are
used (Cho et al. 1999; Jander et al. 2002; Schmid et al. 2003).
In case of lines with less well-characterized genomes,
additional work may be necessary to develop markers for
high-resolution mapping, requiring further costs and time
for identification of new polymorphisms and establishment
of appropriate techniques for detection.
In the present work, we report the creation of a new
large set of reciprocal ILs between the Arabidopsis
accessions Col-0 and C24 covering the entire genome. For
the top of chromosome IV and the bottom of chromosome
V, additional sets of subILs were created. These latter lines
were used to demonstrate the applicability of these sets of
ILs to locate a major gene for flowering time (subILs for top
of chromosome IV) and to fine map a locus causing reduced
fertility (subILs for top of chromosome V). With respect to
fine mapping, we present a highly efficient and costeffective approach based on CelI endonuclease cleavage in
one-well format, which can be used for both identification
of polymorphisms and genotyping at a high-resolution level.
Materials and Methods
DNA Isolation, Genotyping, and Primer Development
All DNA samples were isolated using the procedure
described in Törjék et al. (2006). Matrix-assisted laser
desorption/ionisation-time of flight (MALDI-ToF) genotyping (analyzed by GAG-Bioscience GmbH, Bremen,
Germany) and SNaPshot analysis were performed as
described in Törjék et al. (2003) and Schmid et al. (2006).
Primers were designed using Oligos 8.8 (Kalendar 2001;
http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm)
or Primer3 (Rozen and Skaletsky 2000). Oligonucleotides
used for IL and subIL development are listed in Table 1.
One-well CelI Assays for Marker Development and
Genotyping
Polymerase chain reaction (PCR) amplification was carried
out in a volume of 12.5 ll containing 15–30 ng genomic
DNA. PCR products were labeled using Cy5.5 deoxycytidine triphosphate, (dCTP; GE Healthcare, Munich,
Germany). Reaction mixtures contained equal amounts of
fluorescently labeled and nonlabeled dCTPs. PCR was
conducted using a thermal cycler (MJ Research, Waltham, MA)
as follows: 94 °C for 2 min followed by 40 polymerization
cycles (94 °C for 10 s, 58 °C for 30 s, 2 °C for 3 min) and
finally extension, denaturation, and reannealing steps (72 °C
for 5 min, 99 °C for 10 s, 70 °C for 20 s, followed by 70 cycles
of 70 °C for 20 s decreasing 0.3 °C per cycle). After PCR
amplification, samples were digested with 1 ll CelI enzyme
(Oleykowski et al. 1998) for 30 min at 45 °C. The digestion
was stopped with 2 ll 0.5 M ethylenediaminetetraacetic acid
(EDTA). Samples were precipitated with 15 ll isopropanol,
washed with 20 ll of 70% ethanol and finally resuspended in
4 ll distilled water.
For analysis on the LI-COR 4300 DNA analyzer
(Lincoln, NE), 0.5 ll of a sample were mixed with 2 ll
formamide loading buffer (33% deionized formamide, 10
mM Tris, pH 7.5, 1 mM EDTA, and 0.02% bromphenol
blue) and denatured at 95 °C for 4 min and 4 °C for 5 min.
Samples were loaded onto 100-tooth membrane combs
(LI-COR) then electrophoresed through a polyacrylamide
gel (6.5% KB Plus, LI-COR) in 1 Tris/Borate/EDTA
running buffer at 1500 V, 40 mA, and 40 W.
Plant Materials and SubIL Development
Col-0 was initially obtained from G. Rédei (University of
Missouri-Columbia, USA) and C24 from J. P. Hernalsteens
(Vrije Universiteit Brussels). As a base population for IL
development, we developed 2 sets of reciprocal (82 Col-0 C24/96 C24 Col-0) BC3F1 lines using the single-seed
descent method. The BC3F1 lines were genotyped with a set
of 111 framework markers established for MALDI-ToF
analysis. Based on the genotyping data, we identified lines
with favorable segments to produce substitutions in both
Col-0 and C24 genomic backgrounds using marker-assisted
selection. This work was organized in a stepwise procedure
taking into account the genomic composition of the assayed
lines: 1) 8 (1 segregating region) or 16 (2 unlinked segregating
regions) descendants were analyzed for the relevant marker
positions using SNaPshot assays; 2) if the segregating segment
was large in the analyzed plants, the most suitable lines were
backcrossed to the recurrent parent with the aim to reduce the
size of the donor segment; 3) if an individual plant contained
a heterozygous segment of the appropriate size, the plant was
selfed for creation of homozygous substitution lines, which
were then selected in the next generation; 4) finally lines
carrying homozygous donor substitutions were seed bulk
amplified. The complete set of ILs and their genotype data are
available from the authors on request (by the time of
publication) based on a simple material transfer agreement.
For subIL development, we used the following initial lines:
M63 (BC3F1–C24 background, heterozygous for markers
between MASC04551chr.III and MASC09219chr.III and from
MASC04123chr.IV to MASC04685chr.IV) and N88 (BC3F1–
Col-0 background heterozygous for markers MASC03898chr.III,
MASC02788chr.III and from MASC04123chr.IV to
MASC02668chr.IV) for creation of subILs at the top of
chromosome IV and N70 (BC3F1–Col-0 background
heterozygous
for markers MASC09211chr.V and
MASC04350chr.V) for creation of subILs at the bottom of
chromosome V. During the development of subILs, up to
300 descendants of one segregating line were involved in
marker analysis and ILs were detected up to BC5F4 generation.
Plants were grown in 1:1 mixture of GS 90 soil (Gebrüder
Patzer, Sinntal-Jossa, Germany) and vermiculite, under
a long-day regime (16 h fluorescent light at 20 °C and 60%
relative humidity/8 h dark at 18 °C and 75% relative humidity.
Phenotypic Analyses
Flowering time was recorded as the number of days from
the date of planting until the opening of the first flower.
397
Journal of Heredity 2008:99(4)
Table 1.
Markers used in this study
Set/marker
Chr. pos Primer (5#–3’)
Amplicon Polymorphism
size (bp) (C24/Col)
Remarks
a
Described in Törjék
et al. (2003); Schmid
et al. (2006); Törjék
et al. (2006)
b
MASC04036
103508
MASC07015
145029
uj26uj34
269893
F-CTCCACGGACACCTTGCTCC
R-AGCGAGCTAGCGAAGGAACC
S-TTTTTTCATGATCCGATTCAAC
ACTTCTCA
F-TCGAAACCCTCCGTCGCCAGC
R-AATTGAAGAAGCCGAAAAGACC
S-TTTTTTTTTTTTTTCTTTTCTTCAC
CGGAGAATCACGACA
586
T/C
199
T/A
241/225 241/225
Johanson et al.
(2000)
c
MASC06738, MASC07101,
MASC01090, K1F13/52-53,
MDD7, K9I9/43-44
MDD3
26757349 F-TGCAGCCTAATCTTCTCGTCG
R-CCTTGTTTGCTTTGGTTTCTCG
Described in
Törjék et al. (2006)
305/298 305/298
d
At5g66210b
At5g66210a
SGCSNP18949
26473368 F-TGTCGTTAGGCAGATGCTCAAA
GTTGC
R-GCTGCTCTTGACCTTAGCTGCCA
TTTC
26474228 F-ACTCCCATCGATACCAAGGCCTC
TACC
R-CCCAAGGTTTACGGCTGAAGTC
AGGTT
26492640 F-GGTAAGTCCGAACCGAACAA
1499
CelI
products
—
1498
CelI
products
—
CelI
products
Known SNP in
amplified region
(Col-0/Ler); TAIR/
Polymorphism:
1005368036
18 SNPs and 1
INDEL described
in the amplified
region for
Col-0/Wei-0
—
R-AAGAAATGATTGGGCACGTC
K1L20/19–21
26499030 F-GACTCCAAATTTGGCGGTGG
1350
R-AATTTGTGTTTAATTTCACGGGAC
Not
detected
AT5g66320
26512991 F-CGTAATTGAGTTTCCCCGGTCG
TCTTT
R-ATGTCGCGTGACCATGAAACAA
AACAT
26522800 F-CGGGATACGCTCTCTCTGTC
1749
CelI
products
1115
CelI
products
Known SNP in
amplified region
(Col-0/Ler); TAIR/
Polymorphism:
1005368037
1042
CelI
products
Known SNP in the
amplified region
(Col-0, Cvi-0, Ei-2/
Nd); MASC SNP
DB: MASC01070
SGCSNP18950
R-ACCGCAGGATCTAAAACACG
K1F13/7–8
398
26534822 F-CCCTGTCATGGGATTGATATGA
ACACG
R-GAATGCGACTGCGTTACAAACC
CTCAT
Törjék et al. Reciprocal Arabidopsis IL Populations
Table 1.
Continued
Set/marker
At5g66400
At5g66440
SM97
Amplicon Polymorphism
size (bp) (C24/Col)
Remarks
Chr. pos Primer (5#–3’)
26535341 F-TCAAATTCACCCAATCAACGACG
ATGT
R-TCTCTCCATTTCATAGGCCCAAG
CATC
26548477 F-GGGCACAAGTGGTGTCTACATG
AGGAA
R-TCTGGATTGCCTTCTGGAAATTG
TAGG
26552058 F-TCCACAAGCCCACCTACAATTTC
CATT
R-CAACGTGCAGTTGGTCTCATTCA
CAAA
1523
CelI
products
1463
CelI
products
1361
CelI
products
AFLP in amplified
region (Col-0, Cvi/
C24, Ler, Ws)
a: Framework marker set; b: Additional markers for the top of chromosome IV; c: Additional markers for the bottom of chromosome IV; d:
Oligonucleotides used for CelI-based assays; base positions on chromosomes: MIPS annotation. Version 171102 (Schoof et al. 2002). Chr. Pos.:
chromosome position of SNP or first base of the amplicon, Polym.:Polymorphism; AFLP: amplified fragment length polymorphism.
Fertility was determined visually based on the amount of
pollen produced. Per line 3 flowers were analyzed under the
stereomicroscope. Plants with reduced fertility had flowers
with no or reduced amount of pollen in comparison to the
plants with normal flowers (Törjék et al. 2006).
Data Visualization and Statistical Analyses
Genotypic data were visualized using the software Graphical
GenoTypes (Van Berloo 1999). Statistical analyses were
performed with Genstat for Windows V6.1 (Payne et al.
2002). For comparisons between genotypes, the analysis of
variance procedure AUNBALANCED was used with
genotype as fixed factor. Significant differences were
detected using Fisher’s least significant difference at the
1% level. Means were calculated using PREDICT.
Results and Discussion
Basic Sets of Reciprocal ILs
To obtain starting populations with a reduced amount of
donor segments in individual lines but still covering the
whole genome, we genotyped sets of 82 Col-0 C24 and 96
C24 Col-0 BC3F1 lines using framework single nucleotide
polymorphism (SNP) markers established for MALDI-ToF
analysis. Genotyping of these lines revealed a complete
coverage of the genome with Col-0 and C24 donor
segments in both reciprocal BC3F1 populations. For each
marker position, we observed heterozygous segments in 9–
25 individual lines in Col-0 C24 BC3F1 population and 8–
38 lines in C24 Col-0 BC3F1 population. The overall
proportions of heterozygosity were 18.7% for Col-0 C24
BC3F1 and 19.5% for C24 Col-0 BC3F1, which is higher
(P , 0.001, chi-square test) than the expected 12.5%. Given
the occurrence of heterosis observed in this cross (Meyer
et al. 2004), plants with higher heterozygosity might possess
higher fitness and they could have been unwittingly selected
for single-seed descent breeding. For the marker-assisted
development of homozygous ILs, we selected only lines
with favorable genomic composition (30 C24 Col0 BC3F1 and 37 Col-0 C24 BC3F1) and processed them as
described in Materials and Methods. We obtained 2
homozygous basic sets consisting of 78 ILs with C24 donor
segments in Col-background and 62 ILs with Col0 introgressions in C24 background (Figure 1, Table 2).
Most of the created ILs contain only 1 donor segment. A
second donor segment is present in 13/78 (16.7%) ILs with
Col-0 genomic background and 9/62 (14.5%) ILs with C24
background. ILs containing only 1 donor segment are
suitable for ‘‘standard’’ QTL analyses. Dual ILs may be
useful to separate different types of epistasis between the
introgressed chromosomal segments and the genetic
background (Kusterer et al. 2007). The reciprocal IL sets
cover the entire Col-0 and C24 genomes with overlapping
substitutions, with 2 exceptions: in the set of ILs with C24
donor segments, a gap exists between markers MASC04685
and MASC03275, which are separated by 3731 kb on
chromosome IV; and in the set of ILs with Col-0 donor
segments, there is no overlap for markers MASC03930 and
MASC03765 on chromosome I. Because the region between
these 2 markers was not analyzed with further markers, we
cannot exclude a possible overlap of the donor segments for
the region flanked by these 2 markers. Average donor
segment sizes covered 4.47 (range: 1–14) and 4.98 (range: 1–16)
marker positions in ILs with Col-0 and C24 backgrounds,
respectively. According to an average distance of 3.87 cM
between 2 adjacent markers (Törjék et al. 2006), these
regions correspond to donor segments of 17.30 and 19.27
cM, respectively.
Validation: the New ILs and SubILs Highlight Interaction between
the Floral Regulators FRI and FLC
To demonstrate the applicability of the ILs for (fine-)
mapping of QTL, we developed additional subILs for top of
399
Journal of Heredity 2008:99(4)
Figure 1. Graphical genotypes of the basic sets of ILs. 78 lines (N lines) with C24 substitutions (a) and 62 lines (M lines) with
Col-0 substitutions (b) were created. ILs were developed using 111 framework SNP markers, which were evenly distributed over
the 5 Arabidopsis chromosomes. For a detailed description of the individual lines, see Supplementary Table 1a. The description of
the sets is summarized in Table 2. Genotypes: yellow—homozygous Col-0, green—homozygous C24. The 5 Arabidopsis
chromosomes are indicated with roman numerals. The graphical genotypes were created with Graphical GenoTypes (Van Berloo
1999) using physical genomic positions. An up-to-date, searchable file containing the names and genotypes of all ILs and subILs is
available from the authors upon request.
chromosome IV around the FRIGIDA gene (FRI,
At4g00650), a well-known major determinant of natural
variation in Arabidopsis for flowering time (Johanson et al.
2000). For better mapping resolution, we extended the
framework markers with 2 additional markers (Table 1)
close to the FRI gene. A third additional marker detects
a deletion polymorphism in the gene itself (Johanson et al.
2000). The subILs developed are shown in Figure 2 (11
subILs with Col-0 donor segments: 2a and 8 lines with C24
donor segments: 2b). Flowering times measured in the
genotyped sets of segregating populations (Table 3) revealed
4 categories depending on the genotypic composition of the
lines (Table 4). Segregating sets of lines for genomic regions
above and below the FRI gene (but not for FRI) such as the
400
descendants of M63/9/1/1 (BC4F4), N88/2/7 (BC5F2), and
N88/2/9 (BC5F2) fell into only one category (A or C) of
flowering time depending on genotypic composition (Table 3).
The offspring of lines M63/9 (BC4F2), M63/9/2 (BC4F3),
M63/9/34 (BC4F3), N88/2/1 (BC5F2), N88/2/14 (BC5F2),
and N88/2/1/17 (BC5F3), which segregate for the FRI gene
itself, always showed 2 clearly distinguishable peaks for
flowering time (Table 3). The drastic changes measured in
flowering time underpin the hypothesis that the single-copy
gene FRI is the candidate gene for this genomic region.
Functional FRI alleles cause plants to delay flowering if not
subjected to vernalization. Nonfunctional FRI alleles (such
as the Col-0 allele) cause plants to flower rapidly (NappZinn 1987; Johanson et al. 2000). The transgressive
Törjék et al. Reciprocal Arabidopsis IL Populations
Table 2.
Summary of the basic IL sets
No. of ILs
No. of lines with double substitutions
Total no. of substitutions
Average segment size
Coverage
Col-0 background
(N lines)
C24 background
(M lines)
78
13
91
4.47
A gap between the
markers: MASC04685
and MASC03275
62
8
70
4.98
A missing overlap for
adjacent markers:
MASC03930 and MASC03765
segregation observed (presence of 4 highly significant
groups of flowering time) suggests a strong interaction of
this region with the genomic background. Functional FRI
alleles enhance the expression of Flowering Locus C (FLC,
At5g10140), a flowering repressor located on chromosome
V (Henderson et al. 2003). Werner et al. (2005) reported
differences between FLCCol-0 and FLCC24, with the ‘‘weak’’
allele assigned to C24. An important feature of the Col-0/
C24 IL population introduced here is the existence of 2
reciprocal sets. Using reciprocal ILs allowed us to determine
the epistatic interactions between FRI and FLC alleles
coming from the same source (Col-0/Col-0 or C24/C24) or
from different sources (Col-0/C24 or C24/Col-0). The
genotype-dependent flowering times measured in IL
populations segregating for FRI (Table 4) corroborate
published data that the interaction between FRI and the
FLC plays an important role in the regulation of flowering
time (Shibaike et al. 1999; Caicedo et al. 2004; Le Corre
2005; Shindo et al. 2005) and highlight the advantage of
using 2 reciprocal sets of ILs.
In Arabidopsis, ILs have been used successfully to verify
individual QTL for flowering time (Koumproglou et al.
2002; Werner et al. 2005), carbon stable isotope ratio d13C
(Juenger et al. 2005), root system size (Gerald et al. 2006), or
phytate and phosphate accumulation (Bentsink et al. 2003).
None of these dedicated Arabidopsis IL collections provide
full genome coverage. Populations covering the full genome
have been developed for tomato and rice (Eshed and Zamir
1995; Canady et al. 2005; Xi et al. 2006). Keurentjes et al.
(2007) established a genome-wide (nonreciprocal) IL
population between Arabidopsis accessions Ler and Cvi
and demonstrated the complementarity of RIL and IL
populations for QTL analysis. Other publicly available
genome-wide sets for Arabidopsis are the CSS (Koumproglou
et al. 2002) in which whole Col-0 chromosomes were replaced
by Ler and Nd chromosomes and the SSRLs (Koumproglou
et al. 2002) with smaller substituted segments.
Fine Mapping of Reduced Fertility Locus at Bottom of
Chromosome V
After the successful demonstration of effects and interactions of previously characterized loci, further analyses
were carried out to investigate interacting loci that cause
reduced fertility. Thus, another subIL set targeting the
genomic region associated with reduced fertility in RILs
Total
140
21
161
4.73
Figure 2. Graphical genotypes of subILs developed for the
top of chromosome IV. 11 lines (M lines) with Col-0 substitutions
(a) and 8 lines (N lines) with C24 substitutions (b) were created.
All M lines have an uniform homozygous C24 and all N lines an
uniform homozygous Col-0 genetic background. Genotypes:
yellow—homozygous Col-0, green—homozygous C24. M lines
(a): 1—M63/9/1/2, 2—M63/9/1/6, 3—M63/9/34/8,
4—M63/9/38, 5—M63/9/40, 6—M63/9/51, 7—M63/9/2/5,
8—M63/9/2/8, 9—M63/9/2/12, 10—M63/9/1/1/16,
11—M63/9/1/1/18. N lines (b): 1—N88/2/14/13, 2—N88/
2/14/21, 3—N88/2/1/10, 4—N88/2/1/31, 5—N88/2/9/15,
6—N88/2/7/1, 7—N88/2/7/27, 8—N88/2/1/17/16.
401
Journal of Heredity 2008:99(4)
Table 3. Genotype-dependent flowering times measured in IL populations segregating for FRI and surrounding area: genotypic
composition of the parental lines
Segregating markers and
their physical positions
MASC02820—IV/41642
MASC04036—IV/103508
MASC07015—IV/145029
uj26uj34—IV/269893
MASC04123—IV/300135
MASC04725—IV/1091297
MASC05042—IV/2187167
MASC09212—IV/2905887
MASC04685—IV/4436613
M63/9* M63/9/1/1 M63/9/2* M63/9/34* N88/2/1* N88/2/7 N88/2/9 N88/2/14* N88/2/1/17*
BC4F2
BC4F4
BC4F3
BC4F3
BC5F2
BC5F2
BC5F2
BC5F2
BC5F3
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
a
a
a
a
a
a
a
ab
ab
ab
ab
ab
b
b
b
b
ab
ab
ab
ab
ab
a
a
a
a
ab
ab
ab
ab
ab
ab
a
a
a
ab
ab
ab
a
a
a
a
a
a
a
a
a
a
a
a
a
a
ab
a
a
ab
ab
ab
ab
ab
ab
ab
a
a
ab
ab
ab
ab
ab
ab
ab
Selfing descendants of these lines were used for both subIL development and measurement of flowering time. The genotypes are indicated as follows:
homozygous Col-0 (a); homozygous C24 (b); heterozygous C24/Col-0 (ab). Lines segregating for FRI (uj26uj34) are highlighted with asterisks. Lines with
M prefix have uniform homozygous C24 genomic backgrounds and lines with N prefix homozygous Col-0 background.
(Törjék et al. 2006) between MASC01090 at 26469329 bp
and K1F13/52–53 at 26580794–26581516 bp was created.
Due to a previous lack of known polymorphisms between
Col-0 and C24 in this 112-kb genomic region, we developed
additional markers by adapting assays based on CelI
endonuclease cleavage. The CelI enzyme from leaf celery
(Apium graveolens) has the capacity to cleave all types of single
base-pair mismatches and small insertions/deletions in
heteroduplex DNA molecules (Oleykowski et al. 1998; Till
et al. 2004). The modified CelI-based assays for marker
development were performed in one-well format incorporating Cy5.5 dCTP (Amersham, München, Germany) dyes
for LI-COR gel-detection system (Supplementary Figure 1).
The 10 primer pairs designed for this 112-kb region
amplified on average 1260 bp in the reference Col-0 DNA
(Table 1). All primer pairs were used to amplify Col-0, C24,
and Col-0 C24 F1 DNA samples and after heat
denaturation and reannealing subjected to CelI treatment.
All amplicons lead to cleaved products in heteroduplexes
(data not shown). These 10 primer pairs allowed an average
marker resolution of 8.7 kb in the covered genomic region
and were thereafter used for high-resolution genotyping. To
this end, 23 recombinant descendants of N70-BC3F1
(homozygous Col-0 background, segregating only for the
bottom of chromosome V for the last 2 framework markers:
MASC09211 and MASC04350) were included in the analysis.
Based on the genotypes detected by CelI assays (heterozygous
or homozygous), we created a high-resolution physical map
for the 23 individual plants. The CelI approach does not
distinguish between homozygous Col-0 and homozygous
C24 genotypes; therefore, we determined the genotypes
iteratively, based on the genotype of the adjacent marker,
Table 4. Genotype-dependent flowering times measured in IL populations segregating for FRI and surrounding area: flowering time
measured in the genotyped segregating populations revealed 4 categories depending on the genotypic composition of the lines
Group
Genotypic
composition
Flowering time ±
standard error
No. of analyzed
plants (lines?)
A
C24 background with
FRICol-0/FRICol-0
30.125 ± 0.740a
96
B
C24 background with
FRICol-0/FRIC24 or
FRIC24/FRIC24
Col-0 background with
FRICol-0/FRICol-0
49.2306 ± 0.725b
100
38.416 ± 0.600c
146
Col-0 background with
FRICol-0/FRIC24 or
FRIC24/FRIC24
107.016 ± 0.906d
64
C
D
Homozygous subILs developed and their allocation to
the different flowering time groups
SM(MASC02820–MASC04685)—M63/9/38 BC4F3
SM(MASC02820–MASC04123)—M63/9/2/5 BC4F4
SM(MASC02820–MASC04036)—M63/9/1/1/16 BC4F5
SM(MASC04725–MASC04685)a—M63/9/34/8 BC4F4
SM(MASC04725–MASC04685)b—M63/9/1/2 BC4F4
SN(MASC04725)—N88/2/1/31 BC5F3
SN(MASC02820–MASC07015)a—N88/2/7/1 BC5F3
SN(MASC04685)—N88/2/9/15 BC5F3
SN(MASC02820–MASC07015)b—N88/2/1/17/2 BC5F4
SN(uj26uj34–MASC04685)—N88/2/14/13 BC5F3
SN(MASC02820-MASC04725)—N88/2/1/10 BC5F3
Flowering times are means calculated in analysis of variance and given in days to flowering ± standard error of means. Significantly (least significant
difference at 1% level) different means are indicated by different letters.
402
Törjék et al. Reciprocal Arabidopsis IL Populations
Figure 3. Graphical genotypes of subILs developed for the bottom of chromosome V. Five lines (N lines) with C24
substitutions (a) were created. All N lines have an uniform homozygous Col-0 genetic background. The lines N70/106/20, N70/
118/23, N70/70/11, and N70/110/4 show normal fertility, whereas line N70/106/17 shows reduced fertility. The fertile lines
share a 56-kb segment not covered by C24 introgressions, indicating the location of the gene causing reduced fertility. The lines
were genotyped using CelI. The homozygous Col-0 and C24 genotypes were determined iteratively using an additional check for
genotypes: DNA samples of the subILs were analyzed either unmixed, mixed with Col-0 DNA, or mixed with C24 DNA,
amplified, and CelI cleaved for the markers At5g66210a (b), K1F13/7-8 (c), and At5g66440 (d). Comparing the 3 fingerprints, we
could confirm the genotypes determined in the previous steps. Genotypes: yellow—homozygous Col-0, green—homozygous C24.
N lines (a–d): 1—N70/106/20, 2—N70/118/23, 3—N70/70/11, 4—N70/110/4, 5—N70/106/17, 6—Col C24 F1 (b–d).
The mixed DNA samples and the heterozygous control DNA of Col C24 F1 showing CelI cleavage are indicated with asterisks.
Cleavage products are indicated with arrowheads.
taking advantage of the close physical distances (Supplementary Figure 2). With the high marker resolution present in the
ILs, the average marker resolution of 8.7 kb genomic region
corresponds to 0.032 cM based on the average recombination
frequency of 274.23 kb cM 1 (measured in C24/Col-0 RILs
previously, Törjék et al. 2006). Assuming independent
crossovers and no interference, double crossover (DCO)
events occur at very low frequencies (;10 5 cM 1). Even
allowing for a DCO frequency of 10 3 cM 1 in the mapping
population, the iterative approach could be applied for
genomic regions of up to 82 kb.
For subIL development, 4 BC3F2 lines (N70/70, N70/
106, N70/118, and N70/110) and their selfed progenies
were analyzed using the same assays. This allowed us to
identify and select 5 subILs (Figure 3). The line N70/106/
17 (BC3F3) contains a C24 introgression covering the
entire region under study and has a reduced fertility
phenotype. The fertile BC3F3 lines N70/106/20, N70/
118/23, N70/70/11, und N70/110/4 have differently
placed substitutions, but with a 56-kb gap between
SGCSNP18949 and At5g66440. Use of the subILs
therefore allowed us to confirm the presence of the QTL
causing reduced fertility in the marker interval defined
by the RIL analysis and to rapidly narrow down the
region associated with reduced fertility from initially 112 kb
to 56 kb.
403
Journal of Heredity 2008:99(4)
To verify the results of the initial CelI analyses, the DNA
samples of the subILs were mixed with either Col-0 or C24
reference DNAs. This analysis allowed the detection of all
homozygotes as well as heterozygotes (Figure 3) and
confirmed the genotypes determined in the earlier steps.
Currently, plant endonucleases such as the CelI enzyme
are mostly used for large-scale identification of point
mutations in mutagenized plant populations termed as
Targeted Identification of Local Lesions IN Genomes
(TILLING) (McCallum et al. 2000). In analogy to TILLING, this technique can also be applied to detection and
distinction of natural variation in genes, termed ecoTILLING (Comai et al. 2004; Gilchrist et al. 2006) where
creation of heteroduplexes will be allowed by mixing of
ecotype DNAs to a reference DNA sample. The use of this
procedure in genotyping and mapping is limited because it
allows only the separation of heterozygous from homozygous data points, whereas the homozygous parental genotypes are not directly distinguishable. A further limitation for
this technique are the high costs associated with the
fluorescent primer labeling required for the most efficient
platforms. Over short physical distances where there is only
a very small chance for 2 recombination breakpoints, the
CelI-based assays provide a time- and cost-effective method
for high-resolution genotyping. Using codominant flanking
markers, the distinction between the homozygous parental
CelI genotypes can be inferred from the adjacent marker
genotypes. If necessary, the CelI assays enable an indirect
determination of all 3 genotypes mixing sample DNAs with
both parental lines separately and subjecting the amplicons
of these DNA mixes to CelI treatment. For a small number
of lines, the incorporation of fluorescently labeled dNTPs
(such as Cy5.5 dCTP for LI-COR) into the PCR products,
the costs for detection will only slightly increase in
comparison to the reaction costs without labeling. This
procedure can be applied to any fully sequenced genome,
which thus can be screened rapidly for polymorphisms. If
a polymorphic CelI-based marker was identified, the same
procedure can be used also for high-resolution genotyping
of the relevant plants. Furthermore, this approach can also
be applied to scan genomic regions for mutations using
mixed DNA samples from mutant and wild-type plants.
influence QTL detection differentially in RILs and ILs. QTL
detected in only one type of population should not be
generally dismissed and the 2 approaches be considered
complementary (Keurentjes et al. 2007). Thus, the new
reciprocal IL set complements the large reciprocal RIL set
available for the same combination Col-0/C24 (Törjek et al.
2006).
In addition to the basic sets of ILs, further subILs were
developed for 2 genomic regions allowing better mapping
resolution. These subILs carrying donor segments with
candidate genes for flowering time and reduced fertility were
used to demonstrate the applicability of the ILs. In the latter
case, the previously defined interval of 112 kb harboring the
responsible gene for reduced fertility has been refined to
a 56-kb region. In addition, stable homozygous subILs were
developed around this locus avoiding the reduced fertility
phenotype. During the development of these lines at high
physical resolution, we used a modified CelI-based assays in
one-well format for both marker development and
genotyping. This improved procedure offers a very flexible
and cost-effective approach.
Supplementary Material
Supplementary material can be found at http://www.
jhered.oxfordjournals.org/.
Funding
The Bundesministerium für Bildung und Forschung
(#0312275A) to T.A.; the Deutsche Forschungsgemeinschaft
(AL 387/6-1, 6-2) to T.A. and R.C.M.; the European
Community (QLG2-CT-2001-01097) to T.A.; the MaxPlanck-Society.
Acknowledgments
We thank B. Till and S. Hennikoff for the opportunity to participate in the
TILLING training program (Fred Hutchinson Cancer Research Center,
Seattle, WA, USA).
Conclusion
In this study, we present 2 new large reciprocal sets of ILs
created between the Arabidopsis accessions Col-0 and C24.
In both of the established sets (78 ILs with Col0 background and 62 ILs with C24 background), the donor
segments cover almost the entire genomes with an average
substitution size of 18.3 cM providing an excellent new
genetic resource for confirmation, characterization, and fine
mapping of QTL in Arabidopsis. Because nearly all regions
are covered with overlaps, approximately 2 times higher
deepness than the average substitution size can be achieved
at mapping using these lines. Several factors, for example,
epistatic interactions or opposing effect QTL in close
vicinity (Kroymann and Mitchell-Olds 2005) are known to
404
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Received July 11, 2007
Accepted January 11, 2008
Corresponding Editor: James Hamrick