QTL-rzepak,

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Theor Appl Genet (2010) 120:921–931
DOI 10.1007/s00122-009-1221-0
ORIGINAL PAPER
Extent and structure of linkage disequilibrium in canola quality
winter rapeseed (Brassica napus L.)
Wolfgang Ecke
•
Rosemarie Clemens
•
Nora Honsdorf
•
Heiko C. Becker
Received: 27 May 2009 / Accepted: 12 November 2009 / Published online: 2 December 2009
The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract Linkage disequilibrium was investigated in
canola quality winter rapeseed to analyze (1) the prospects
for whole-genome association analyses and (2) the impact
of the recent breeding history of rapeseed on linkage dis-
equilibrium. A total of 845 mapped AFLP markers with
allele frequencies C0.1 were used for the analysis of
linkage disequilibrium in a population of 85 canola quality
winter rapeseed genotypes. A low overall level of linkage
disequilibrium was found with a mean r
2
of only 0.027
over all 356,590 possible marker pairs. At a significance
threshold of P = 2.8 9 10
-7
, which was derived by a
Bonferroni correction from a global a-level of 0.1, only
0.78% of the marker pairs were in significant linkage dis-
equilibrium. Among physically linked marker pairs, the
level of linkage disequilibrium was about five times higher
with more than 10% of marker pairs in significant linkage
disequilibrium. Linkage disequilibrium decayed rapidly
with distance between linked markers with high levels of
linkage disequilibrium extending only for about 2 cM.
Owing to the rapid decay of linkage disequilibrium with
distance association analyses in canola quality rapeseed
will have a significantly higher resolution than QTL anal-
yses in segregating populations by interval mapping, but
much larger number of markers will be necessary to cover
the whole genome. A major impact of the recent breeding
history of rapeseed on linkage disequilibrium could not be
observed.
Introduction
QTL mapping in segregating populations derived from
biparental crosses has become a common tool in the
analysis of quantitative traits in plants. Although this
approach allows the identification of loci contributing to a
quantitative trait and the estimation of their effects, it has
some inherent disadvantages. First, mapping populations
have to be developed specifically for the QTL mapping. In
addition, only the allelic diversity sampled in the two
parents of the cross can be analyzed, that is, QTL where
both parents have the same allele cannot be detected.
Furthermore, due to the limited number of recombination
events available in a segregating population, the resolution
is somewhat limited, resulting in confidence intervals for
the QTL positions in the range of several cM up to several
tens of cM (van Ooijen
1992
; Darvasi et al.
1993
).
An alternative approach could be association analysis or
linkage disequilibrium (LD) mapping using natural popu-
lations or, in the case of crop plants, collections of varieties
and breeding lines. Owing to the higher number of
recombination events in such a material, a higher resolu-
tion could be achieved than in segregating populations
(Ewens and Spielman
2001
; Jannink et al.
2001
). In addi-
tion, the allelic diversity would not be limited to the
diversity occurring between two parental lines. Further-
more, this approach could be easily integrated into the
breeding process for new crop varieties. For example,
Kraakman et al. (
2004
) used data from official Danish
variety trials and mapped AFLP markers to localize QTL
Communicated by M. Kearsey.
Electronic supplementary material The online version of this
article (doi:
contains supplementary
material, which is available to authorized users.
W. Ecke (
&
)
R. Clemens
N. Honsdorf
H. C. Becker
Department of Crop Sciences, Georg-August-University
G ¨ttingen, Von-Siebold-Str. 8, 37075 G ¨ttingen, Germany
e-mail: wecke@gwdg.de
123
922
Theor Appl Genet (2010) 120:921–931
for yield and yield stability in modern two-row spring
barley cultivars.
Association analysis is based on the linkage disequilib-
rium between linked loci and is strongly dependent on the
extent and structure of the linkage disequilibrium in the
population analyzed. Linkage disequilibrium, the non-ran-
dom association of alleles at different loci, is created by
mutation, admixture between genetically distinct popula-
tions, selection and genetic drift, and decays by genetic
recombination (Flint-Garcia et al.
2003
). Accordingly, the
linkage disequilibrium in a population is dependent on the
population history and the mating system of the species.
Linkage disequilibrium has been analyzed in a number of
plant species, either globally, using molecular markers, or
locally by sequencing specific genomic segments of up to
several hundred kilobytes. In maize, an allogamous spe-
cies, Tenaillon et al. (
2001
) observed a rapid decay of
linkage disequilibrium within 100–200 bp in a genetically
broad material of inbred lines and exotic landraces. On the
other hand, in Arabidopsis thaliana, an autogamous spe-
cies, linkage disequilibrium extended over about 1 cM or
250 kb in a global population of 20 accessions from dif-
ferent parts of the world and the decay of linkage dis-
equilibrium with distance was even lower in local
populations (Nordborg et al.
2002
). In sugar cane, where
elite varieties are propagated clonally, Jannoo et al. (
1999
)
observed linkage disequilibrium between RFLP markers to
extend over up to 10 cM. Nevertheless, analyzing only
inbred lines of maize Remington et al. (
2001
) found link-
age disequilibrium extending over 1.5 kb and Rafalski
(
2002
) reported that linkage disequilibrium in elite germ-
plasm of maize extends over more than 100 kb. Analyzing
linkage disequilibrium with SSR markers in the flint and
dent germplasm groups used in maize hybrid breeding in
Europe Stich et al. (
2005
) observed very high levels of
linkage disequilibrium with 55 and 48% of the linked and
unlinked marker pairs, respectively, in significant LD in the
flint germplasm group and an average length of LD blocks
of 26 cM. The levels of linkage disequilibrium were even
higher in the dent germplasm group. Conversely, Kim et al.
(
2007
) observed a rapid decay of linkage disequilibrium
within 10 kb in a sample of 19 A. thaliana accession. The
results from the different populations indicate that popu-
lation history may be more important than the mating
system in determining the level of linkage disequilibrium
in a population. A strong influence of population history
was also observed in barley and soybean (Caldwell et al.
2006
; Hyten et al.
2007
). In both crop plants, linkage dis-
equilibrium extends farthest in elite breeding materials
while decaying the most rapidly in wild relatives with
landraces taking an intermediate position. When comparing
SSR and AFLP markers, Stich et al. (
2006
) also found a
strong influence of the marker type on the detection of
linkage disequilibrium. The level of linkage disequilibrium
detected in European maize inbred lines was much higher
with SSR markers than with AFLP markers, presumably
because the former distinguish between more alleles than
the latter.
Rapeseed is a partially allogamous species that is bred
like an autogamous species with controlled crosses fol-
lowed by several generations of selfing to develop new
varieties. It gained its current importance as a major oil
crop in temperate regions only after two rounds of intense
selection for two new quality traits: zero erucic acid and
low glucosinolate content, which were initially introduced
into the breeding material from one donor genotype each in
the 1960s and 1970s, respectively. Current elite breeding
materials produce seed oil free from erucic acid and a meal
low in glucosinolates—a quality termed ‘canola’—and are
supposed to be derived from a limited number of crosses
between the original genotypes with these quality traits and
breeding lines of that time (Becker et al.
1999
). Accord-
ingly, the introduction of the two traits may have consti-
tuted a genetic bottleneck in the breeding history of
rapeseed that, together with the following intense selection
for the new traits, could have had a major impact on the
level and structure of linkage disequilibrium in current
canola quality rapeseed materials.
In rapeseed, QTL mapping in segregating populations is
well established and has been used in a number of studies
to analyze quality traits such as oil content (Ecke et al.
1995
; Zhao et al.
2005
; Delourme et al.
2006
; Qiu et al.
2006
; Zhao et al.
2006
), glucosinolate content (Toroser
et al.
1995
; Uzunova et al.
1995
), tocopherol content
(Marwede et al.
2005
), phytosterol and sinapate ester
content (Amar et al.
2008
), and the fatty acid composition
of the seed oil (Thormann et al.
1996
; Zhao et al.
2008
)as
well as disease resistances such as blackleg (Pilet et al.
1998
) or heterosis (Radoev et al.
2008
). So far, no study
has been published on the application of association anal-
ysis in rapeseed or about linkage disequilibrium in rape-
seed populations. The objective of this study was to
determine the extent and structure of linkage disequilib-
rium in canola quality winter rapeseed to (1) analyze the
prospects for association analysis in current elite breeding
materials of this crop plant and (2) to elucidate the impact
the introduction of the ‘canola’ quality has had on the
linkage disequilibrium in this material.
Materials and methods
Plant materials
Linkage disequilibrium was analyzed in a set of 85
Northern European canola quality winter rapeseed varieties
123
Theor Appl Genet (2010) 120:921–931
923
and breeding lines (Table
1
), further called LD population.
For the analysis, one individual plant per variety was used.
For genetic mapping, a mapping population of 94 doubled
haploid lines derived from one F
1
plant of a cross between
the winter rapeseed variety ‘Express’ and a resynthesized
rapeseed, ‘R53’, was used. This population had already
been used to develop a genetic map in rapeseed comprised
mainly of SSR markers (Radoev et al.
2008
).
DNA preparation and AFLP analysis
Table 1 Origin of the 85 canola quality varieties and breeding lines
used in the analysis of linkage disequilibrium in rapeseed
Variety
Breeder
Variety
Breeder
DNA was prepared from 0.1 g of leaf material of 3 weeks
old greenhouse grown plants using Nucleon PhytoPure
extraction kits (RPN8510, GE Healthcare Bio-Sciences
AB, Uppsala, Sweden) following the manufacturer’s
instructions.
The EcoRI primers used in AFLP analysis were labeled
with one of the following four fluorescent dyes: (6, 5) FAM,
NED, VIC, or PET (Applied Biosystems, Darmstadt,
Germany). AFLP analyses were carried out following the
protocol of Vos et al. (
1995
) modified for multiplexing in
the PCR according to F. Kopisch-Obuch (personal
communication): 250 ng DNA were digested in 30 llRL
buffer (10 mM Tris–acetate, 10 mM Mg–acetate, 50 mM
K–acetate, 5 mM DTT, pH 7.5) with 4 U EcoRI
(Fermentas, St. Leon-Rot, Germany) and 4 U MseI(New
England Biolabs, Frankfurt, Germany) for 1.5 h at 37.
After adding 10 ll of a mix containing 5 pmol EcoRI
adapter, 50 pmol MseI adapter, 1 mM ATP and 1 U
T4-DNA ligase (Promega, Mannheim, Germany) in RL
buffer, DNA and adapters were ligated in a time series
of different temperatures (3 h 10 min 37, 3 min 33.5,
3 min 30, 4 min 26 and finally 15 min 22). The final
restriction–ligation product (RL) was diluted 1:5 with
HPLC grade water. For pre-amplification, 8 ll of the
diluted RL was added to 12 ll of a reaction mixture, giv-
ing final concentrations of 19 Taq buffer (Solis Biodyne,
Tartu, Estonia, Reaction buffer B), 3.125 mM MgCl
2
,
0.45 mM dNTPs, 10 pmol EcoRI?1 primer, 9 pmol
MseI?1 primer and 2.5 U Taq DNA polymerase (FIREPol,
Solis Biodyne). The pre-amplification was carried out in a
Biometra T1 Thermocycler (Biometra GmbH, G
¨
ttingen,
Germany) with the following program: 94 for 30 s, 20
cycles of 94 for 30 s, 56 for 30 s and 72 for 2 min, and a
final 5 min at 72. The pre-amplification product was
diluted 1:10 with HPLC grade water. The final AFLP
amplification used 6 ll of the diluted pre-amplification
product in a total reaction volume of 20 ll containing 19
Taq buffer, 0.36 mM dNTPs, 3.125 mM MgCl
2
,1UTaq
polymerase, 7 pmol MseI?3 primer, 2 pmol of (6, 5)FAM
labeled EcoRI?3 primer, 2 pmol of VIC labeled EcoRI?3
primer, 4 pmol of NED labeled EcoRI?3 primer,
and 6 pmol of PET labeled EcoRI?3 primer. The protocol
for the Thermocycler was as follows: 1 cycle of 94 for
1 min, 65 for 30 s, and 72 for 2 min, 12 cycles of 94
for 30 s, 64.2 for 30 s and 72 for 2 min, 25 cycles of 94
for 30 s, 56 for 30 s and 72 for 2 min, and finally 72 for
5 min.
Alesi
KWS
Magnum
Syngenta
Remy
KWS
Madrigal
Syngenta
Robust
KWS
Laser
Syngenta
Alaska
KWS
Fortis
Syngenta
Pirola
KWS
Smart
Syngenta
Adder
KWS
Roxet
Syngenta
Milena
KWS
NK Bravour
Syngenta
Allure
KWS
NK Fair
Syngenta
Agalon
KWS
Aviso
SW Seed
K615
KWS
Sansibar
SW Seed
KW1519
KWS
SWGospel
SW Seed
Picasso
KWS
Verona
SW Seed
Lord
KWS
Tenor
SW Seed
KW3077
KWS
Expert
SW Seed
Rodeo
KWS
Musette
SW Seed
Rapid
Limagrain-Nickerson
Kvintett
SW Seed
Boston
Limagrain-Nickerson
Falstaff
SW Seed
Escort
Limagrain-Nickerson
SW Sinatra
SW Seed
Montego
Limagrain-Nickerson
Viking
NPZ
Ontario
Limagrain-Nickerson
Aragon
NPZ
Pacific
Limagrain-Nickerson
Aurum
NPZ
Savannah
Limagrain-Nickerson
Lorenz
NPZ
Missouri
Limagrain-Nickerson
Baros
NPZ
Manitoba
Limagrain-Nickerson
Rasmus
NPZ
Ladoga
Limagrain-Nickerson
Gefion
NPZ
Atlantic
Limagrain-Nickerson
Nugget
NPZ
Cooper
Limagrain-Nickerson
Zephir
NPZ
Licapo
DSV
SLM 0413
NPZ
Capitol
DSV
SLM 0512
NPZ
Idol
DSV
LSF 0519
NPZ
Vivol
DSV
HSL 1032
NPZ
Bristol
DSV
Campari
NPZ
Lirajet
DSV
Caramba
NPZ
Lisabeth
DSV
Express 617
NPZ
Lipid
DSV
Prince
NPZ
Lipton
DSV
Wotan
NPZ
Lisek
DSV
Amor
Petersen/Raps GbR
Contact
DSV
Orlando
Saaten Union
Columbus
DSV
Pollen
Adrien Momont
Lion
DSV
Ascona
SW Seed
Oase
DSV
Duell
Raps GbR
Apex
Syngenta
Jessica
–
Recital
Syngenta
KWS KWS SAAT AG; DSV Deutsche Saatveredelung AG; NPZ Nord-
deutsche Pflanzenzucht Hans-Georg Lembke KG
123
924
Theor Appl Genet (2010) 120:921–931
The AFLP products were separated on an ABI PRISM
3100 Genetic Analyser (Applied Biosystems) using 50-cm
capillary arrays and GeneScan-500 LIZ size standard
(Applied Biosystems). GeneMapper v3.7 software
(Applied Biosystems) was used for a semi-automatic
marker scoring. Since in GeneMapper v3.7’s output, AFLP
primer combinations are written as markers and the actual
AFLP markers as alleles of these markers a Perl script,
‘Extract_marker’, was developed to transform GeneMap-
per’s output into a marker matrix. The primer combinations
used, the labels of the EcoRI primers and the numbers of
markers identified with the different primer combinations
are listed in Table S1.
analyses were carried out in Microsoft Office Excel 2007.
The threshold for declaring linkage disequilibrium between
two markers significant was derived by a Bonferroni cor-
rection from a global a-level of 0.1, resulting in a per test
threshold of P = 2.8 9 10
-7
.
Results
Marker analysis and map construction
By using 132 primer combinations 2161 AFLP markers
could be scored in the mapping population. In the LD
population, 1,463 of these markers were also polymorphic
and 898 showed allele frequencies equal to or larger than
0.1. Of the markers with allele frequencies C0.1 in the LD
population 845 could be mapped in the mapping popula-
tion. The AFLP markers were mapped within a framework
of 167 markers from the earlier map that had been estab-
lished in the mapping population by Radoev et al. (
2008
).
After the initial map construction, the markers were dis-
tributed across 21 linkage groups. By mapping some
additional markers from the full set of 2,161 markers, the
map could be consolidated in 19 linkage groups, a number
corresponding to the 19 chromosomes of the haploid
rapeseed genome. Based on map alignments using the SSR
markers from the earlier map, 18 of the linkage groups
could be named according to the N-nomenclature of
rapeseed linkage groups. The last linkage group was des-
ignated as N8 by exclusion. The final map (Table
2
) has a
length of 2,473 cM and comprises 1,032 markers distrib-
uted across 551 map positions. Included are 865 new AFLP
markers that cover 2,345 cM (95%) of the total map.
Individual linkage groups range in length from 77 to
242 cM, holding between 27 and 132 markers. The full
map is listed in Table S2.
Genetic mapping
Genetic mapping of the AFLP markers was based on a
framework map previously established in the mapping
population by Radoev et al. (
2008
). Using the program
MAPMAKER/EXP V.3.0b, the new markers were assigned
to linkage groups by the ‘near’ command with an LOD
threshold of 4.0 and a maximum recombination frequency
of 0.4. Linkage groups were then reanalyzed using the
‘order’ command. Finally, markers that could not be placed
by the ‘order’ command were manually placed using the
‘try’ command. Double crossovers were identified using
MAPMAKER’s ‘genotype’ command and were rechecked
in the trace files and, if necessary, corrected, followed by a
remapping of the affected markers. Markers with high
numbers of double crossovers and markers with strongly
disturbed segregations where one class was represented by
fewer than 25 genotypes were excluded from the mapping.
Linkage groups were named according to the N-nomen-
clature proposed by Parkin et al. (
1995
). Recently, a new
nomenclature was proposed by the Steering Committee of
the Multinational Brassica Genome Project (
. In this nomenclature, A1–A10 correspond to
N1–N10 and C1–C10 to N11–N19.
Levels of linkage disequilibrium
Analysis of linkage disequilibrium
Linkage disequilibrium in canola quality winter rapeseed
was analyzed using pairwise combinations of the 845
AFLP markers with allele frequencies C0.1 in the LD
population. With a mean r
2
value of only 0.027 over all
356,590 possible pairwise combinations, the overall level
of linkage disequilibrium in the rapeseed genome is very
low (Table
3
). This conclusion is reinforced by the obser-
vation that only 0.78% of marker pairs are in significant
LD. With a mean r
2
of 0.122 linkage disequilibrium among
physically linked marker pairs, that is pairs where both
markers are on the same linkage group, is nearly five times
higher than the overall mean. Furthermore, 11.58% of these
marker pairs are in significant LD and with a count of
2,658 represent the vast majority of marker pairs in
For the analysis of linkage disequilibrium, only markers
with allele frequencies in the LD population of 0.1 or larger
were used. This discrimination against rare alleles is jus-
tifiable because the information from them is neither useful
in the analysis of linkage disequilibrium nor in association
analysis. R
2
values of linkage disequilibrium for all pair-
wise marker combinations and the corresponding signifi-
cance levels (P values) were calculated using the program
TASSEL V.2.0.1 (Zhang et al.
2006
). Recombination fre-
quencies between marker pairs were calculated by a Perl
script and added to the corresponding rows of the LD table
generated by TASSEL. All further statistical and graphical
123
Theor Appl Genet (2010) 120:921–931
925
Table 2 Summary of the
genetic map with the AFLP
markers used in the analysis of
LD in rapeseed
Linkage
group
No. of
markers
No. of
map pos.
Length
(cM)
a
New AFLP
markers
Framework
markers
b
N1
33
25
118
26
7
N2
28
21
141
20
8
N3
46
26
122
39
7
N4
32
19
78
27
5
N5
50
30
174
43
7
N6
54
26
135
45
9
N7
41
17
77
37
4
N8
27
16
77
24
3
N9
65
26
133
55
10
N10
37
21
83
27
10
N11
132
49
132
122
10
N12
52
30
161
45
7
N13
82
52
242
67
15
N14
63
28
147
53
10
N15
51
30
142
39
12
a
Recombination frequencies
were transformed to
centimorgan according to the
Kosambi mapping function
b
Markers from the map of
Radoev et al. (
2008
)
N16
44
28
130
35
9
N17
54
34
124
43
11
N18
74
32
140
62
12
N19
67
41
117
56
11
Sum
1032
551
2473
865
167
Table 3 Number of marker pairs and average level of LD (mean r
2
) in different classes of marker pairs
Class
a
All pairs in the class
Pairs in significant LD at P = 2.8 9 10
-7
n (%)
b
Mean r
2
n (%)
c
Mean r
2
All
356,590 (100.00)
0.027
2,775 (0.78)
0.722
Linked
22,951 (6.44)
0.122
2,658 (11.58)
0.729
Unlinked
333,639 (93.56)
0.020
117 (0.04)
0.544
a
All: all marker pairs, linked: pairs of markers from the same linkage group, unlinked: pairs where the markers are on different linkage groups
b
Percentage from all (356,590) marker pairs
c
Percentage from all marker pairs in the class
significant LD indicating that the major determinant of
linkage disequilibrium in the rapeseed genome is genetic
linkage. Accordingly, only 117 of the unlinked marker
pairs are in significant LD and at 0.544, the mean r
2
of
distance (Fig.
1
b). Closely linked marker pairs at recom-
bination frequencies of 0–2% show high levels of linkage
disequilibrium with mean r
2
values ranging from 0.566 to
0.374, but at a recombination rate of 5%, the mean r
2
is
already down to 0.1 and at high distances, it is not sig-
nificantly different from the overall mean of 0.027. Like-
wise, the fraction of marker pairs in significant LD decays
from 65 to 48% for closely linked marker pairs to 6% at a
recombination rate of 5% and 1–3% at intermediate
recombination rates (6–20%). With the exception of two
marker pairs at 24 and 27%, no marker pairs in significant
LD are found at higher recombination rates.
The rapid decay of linkage disequilibrium is also
apparent when looking at the distribution of linkage dis-
equilibrium across individual linkage groups (Fig.
2
a).
Colors indicative of high LD are close to the diagonal
these marker pairs is still lower than the mean r
2
of 0.729
of the linked marker pairs in significant LD.
Structure of linkage disequilibrium
To investigate the structure of linkage disequilibrium in the
rapeseed genome, the dependency of linkage disequilib-
rium on distance was analyzed among the physically linked
marker pairs. The number of marker pairs at recombination
rates from 0 to 50% ranges from 126 to 1,554 (Fig.
1
a)
providing a solid base for this analysis. Among the linked
marker pairs, linkage disequilibrium decays rapidly with
123
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