Download Theoretical and Applied Genetics

Theor Appl Genet (2012) 124:505–513
DOI 10.1007/s00122-011-1724-3
ORIGINAL PAPER
Genetic mapping of the Leptosphaeria maculans avirulence gene
corresponding to the LepR1 resistance gene of Brassica napus
Kaveh Ghanbarnia • Derek J. Lydiate • S. Roger Rimmer
Genyi Li • H. Randy Kutcher • Nicholas J. Larkan •
Peter B. E. McVetty • W. G. Dilantha Fernando
•
Received: 18 December 2010 / Accepted: 7 October 2011 / Published online: 30 October 2011
Ó Springer-Verlag 2011
Abstract AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to
Brassica LepR1, a plant gene controlling dominant, racespecific resistance to this pathogen. An in vitro cross
between the virulent L. maculans isolate, 87-41, and the
avirulent isolate, 99-56, was performed in order to map
the AvrLepR1 gene. The disease reactions of the 94 of the
resulting F1 progenies were tested on the canola line ddm12-6s-1, which carries LepR1. There were 44 avirulent
progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-126s-1 is controlled by a single gene. Tetrad analysis also
indicated a 1:1 segregation ratio. The AvrLepR1 gene was
positioned on a genetic map of L. maculans relative to 259
sequence-related amplified polymorphism (SRAP) markers,
two cloned avirulence genes (AvrLm1 and AvrLm4-7) and
the mating type locus (MAT1). The genetic map consisted of
36 linkage groups, ranging in size from 13.1 to 163.7 cM,
Communicated by H. Becker.
K. Ghanbarnia G. Li P. B. E. McVetty W. G. D. Fernando (&)
Department of Plant Science, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada
e-mail: [email protected]
Present Address:
K. Ghanbarnia D. J. Lydiate S. R. Rimmer N. J. Larkan
Agriculture and Agri-Food Canada, Saskatoon Research Centre,
Saskatoon, SK S7N 0X2, Canada
Present Address:
H. R. Kutcher
Crop Development Centre, College of Agriculture
and Bioresources, University of Saskatchewan,
3C02 Agriculture Building, 51 Campus Drive,
Saskatoon, SK S7N 5A8, Canada
and spanned a total of 2,076.4 cM. The AvrLepR1 locus was
mapped to linkage group 4, in the 13.1 cM interval flanked
by the SRAP markers SBG49-110 and FT161-223. The
AvrLm4-7 locus was also positioned on linkage group 4,
close to but distinct from the AvrLepR1 locus, in the 5.4 cM
interval flanked by FT161-223 and P1314-300. This work
will make possible the further characterization and mapbased cloning of AvrLepR1. A combination of genetic
mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence
genes that have been characterized previously.
Introduction
Blackleg disease of canola and other Brassica crops is
caused by the haploid ascomycete pathogen Leptosphaeria
maculans (Sun et al. 2007; Fitt et al. 2006). The pathogen
has been reported in North America (Canada, USA and
Mexico), most countries in Europe, Australia, Brazil,
Argentina, and a few countries in Africa (Fitt et al. 2006).
Despite ongoing resistance breeding, financial losses to this
disease range from 22 to 300 million dollars per year in
canola-producing regions in the world (Fitt et al. 2006).
Two types of genetic resistance to L. maculans have
been described in Brassica species; qualitative resistance
(that is usually assayed at the seedling stage) and quantitative resistance (that is usually studied in adult-plants)
(Ansan-Melayah et al. 1998; Pilet et al. 1998; Dion et al.
1995; Ferreria et al. 1995; Rimmer 2006). Qualitative or
race-specific resistance follows the gene-for-gene model
for plant–pathogen interactions (Flor 1971) and includes
the genes Rlm1, Rlm2 and Rlm4 (Ansan-Melayah et al.
1998), Rlm3, Rlm5, Rlm6, and Rlm8 (Balesdent et al.
2002), Rlm7 and Rlm9 (Delorume et al. 2004), LepR1 and
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LepR2 (Yu et al. 2005), and LepR3 (Li and Cowling 2003).
The genetic control of quantitative resistance is currently
more poorly defined but appears to play a significant role in
the protection of Brassica crops (Rimmer 2005).
Genetic studies in L. maculans have identified nine
avirulence gene specificities, AvrLm1 to 9, corresponding to
nine resistance genes of Brassica hosts. Most of these genes
are contained within two genetic clusters, the AvrLm1-2-6
cluster (Balesdent et al. 2002) and the AvrLm3-4-7-9 cluster
(Balesdent et al. 2005) while AvrLm5 and AvrLm8 are not
linked to any of the other genes (Balesdent et al. 2002).
Three of the avirulence genes have been cloned; AvrLm1
(Gout et al. 2006), AvrLm6 (Fudal et al. 2007) and the dual
avirulence gene, AvrLm4-7 (Parlange et al. 2009), that is
recognized by two distinct resistance genes from Brassica
napus, Rlm4 and Rlm7. The proteins predicted from the
coding sequences of all three genes are small and secreted.
The products of AvrLm6 (Fudal et al. 2007) and AvrLm4-7
(Parlange et al. 2009) are both cysteine-rich, while the
protein encoded by the AvrLm1 gene is predicted to have a
paucity of disulphide bridges (Gout et al. 2006).
The LepR1 gene for resistance to L. maculans was
derived from Brassica rapa subsp. sylvestris and was
transferred to canola using two independent crossing strategies as bridges; resynthesis of B. napus (Crouch 1994) and
the formation of AAC allotriploids with backcrossing to B.
napus (S. R. Rimmer, unpublished). LepR1 controls racespecific interactions with L. maculans and was effective
against most isolates tested, including ones collected from
Canada, Australia and Europe (Yu et al. 2005). Yu et al.
(2005) used a population of B. napus double haploid (DH)
lines segregating for LepR1 to map the gene to chromosome
2 of the Brassica A-genome (B. napus linkage group N2
corresponds to Brassica chromosome A2).
A rare L. maculans isolate, 87-41, that is virulent on B.
napus plants carrying LepR1 was identified by Yu et al.
(2005). This made it theoretically possible to determine the
genetic location in the L. maculans genome of AvrLepR1, the
avirulence gene with which LepR1 interacts. In this report, we
describe the genetic mapping of AvrLepR1 relative to markerloci defined by sequence-related amplified polymorphism
(SRAP) markers (Li and Quiros 2001) and the specificity
testing of AvrLepR1 to establish its relationship to the AvrLm3, AvrLm4, AvrLm7 and AvrLm9 genes which all mapped
to a similar region of the L. maculans genome.
Materials and methods
B. napus lines and L. maculans isolates
The B. napus line ddm-12-6s-1 (S. R. Rimmer, unpublished), carries LepR1 and is a BC2S2 selection from a cross
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Theor Appl Genet (2012) 124:505–513
between double haploid B. napus DH12075 (Mayerhofer
et al. 2005) and B. rapa subsp. sylvestris (Crouch 1994),
backcrossed to N–o-1, a DH line derived from ‘Westar’
(Sharpe et al. 1995). A distinct selection from the ‘Westar’
canola variety, which was described by Chen and Fernando
(2006), was used as a positive control for infection by
L. maculans. The L. maculans isolate, 87-41 (Yu et al.
2005), was expected to be virulent on both ddm-12-6s-1
and ‘Westar’, while isolates WA-74, 99-49 and 99-56 (Yu
et al. 2005) were expected to be avirulent on ddm-12-6s-1
but virulent on ‘Westar’. All four isolates were obtained
from the collection of Dr. S. R. Rimmer. The following
B. napus differential lines and cultivars, 22-1-1, ‘Falcon’,
23-1-1 and ‘Darmor’, which harbored Rlm3, Rlm4, Rlm7
and Rlm9, respectively (Balesdent et al. 2005), were used
to differentiate the corresponding Avr genes in the
L. maculans parental isolates and progeny.
Inoculum preparation and virulence phenotyping
The preparation of pycnidiospores and the inoculation of
B. napus cotyledons were performed as described previously (Ferreria et al. 1995). Each L. maculans isolate was
tested on 12 seedlings of the ddm-12-6s-1 tester line and 12
seedlings of ‘Westar’ as a highly susceptible control. The
disease reactions were scored 12, 14 and 21 days after
inoculation and rated using the 0–9 scale described by
Williams (1985).
Crossing isolates and tetrad analysis
L. maculans isolates were crossed by pairing single-pycnidiospore isolates as described by Mengistu et al. (1995)
and tetrad analysis was performed following the protocol
of Gall et al. (1994). Toothpicks bearing pseudothecia were
stored in vials with silica gel after air-drying.
Sequence-related amplified polymorphism markers
Sequence-related amplified polymorphism (SRAP) markers were generated using the methods described by Li and
Quiros (2001). Each amplification reaction used L. maculans template DNA, one fluorescently labeled primer and
one unlabelled primer. PCR amplifications were carried out
using a programmable thermal controller (BIO RAD thermal cycler gradient, BIO RAD, California, USA). Four
reaction products, each using a distinct fluorescent dye,
were typically pooled before size separation and detection
of the fluorescently labeled DNA amplification products
using an ABI 3100 Genetic Analyzer (ABI, California).
The four distinguishable dyes used to label samples were
FC1 (Blue), PM88 (Red), SA7 (Green) and SA12 (Yellow).
The LIZ 500 (Orange) fluorescent dye was used to label
Theor Appl Genet (2012) 124:505–513
Table 1 Nucleotide sequences of primer pairs used to amplify
fragments of AvrLm1 (1/2), a region of the L. maculans genome
15 cM removed from AvrLm4-7 (3/4) and SNPs primers (5/6 and 7/8)
designed to preferentially amplify specific alleles at the AvrLm4-7
locus
Primer
number
Sequence (50 to 30 )
1a
TCTAGCTCCCCAGCTACCAAGAAC
a
2
GAACCACGACATCGGCTAATATTTTAG
3b
CTGGTAAGAACTGTACCGTAGACTC
4b
TAATGTTCCATCCAGGAAAACTAGA
5c
GGGACAAGTGCCTTGAGGATAGCT
6c
7d
GCTTATTTAACAATCAAGTTGTTTACTCCTATTTT
CATTCAGGGTCATGGTCTAAACCAGT
8d
TCAAGGCACTTGTCCCACAAAC
507
(Table 1) were designed from the AvrLm1 gene sequence
(EMBL database, accession number AM084345) and the
BAC 28G8 sequence (EMBL database, accession number
AM998637), respectively. These sequences were positioned on the same chromosome as the AvrLm4-7 gene,
*15 cM distant from it (Parlange et al. 2009). The
AvrLm4-7 locus of L. maculans was amplified using the
AVRint-UP/-Lo primer pair described by Parlange et al.
(2009). The PCR-amplification products of AvrLm4-7
variants were cloned using TOPO TA cloning kits (Invitrogen, USA) and submitted for contract sequencing. The
primer 5/6 amplified the 99-56 allele at nucleotide 212 of
the AvrLm4-7 gene (SNP212) while the primer pair 7/8
amplified the 87-41 allele at nucleotide 241 (SNP241)
(Table 1).
a
Primer pair 1 and 2 amplified a 333 bp fragment in isolate 87-41
and produced no amplification product from isolate 99-56
Genetic map construction
b
Primer pair 3 and 4 amplified a 491 bp fragment in isolate 87-41
and produced no amplification product from isolate 99-56
c
Primer pair 5 and 6 amplified a 220 bp fragment in isolate 99-56
and produced no amplification product from isolate 87-41
d
Primer pair 7 and 8 amplified a 291 bp fragment in isolate 87-41
and produced no amplification product from isolate 99-56
the DNA fragments of the internal size standard. All five
dyes were supplied by ABI (ABI, California). Genomic
DNA was extracted from parents and F1 progeny as
described by Pongam et al. (1998).
Linkage analysis was performed using the computer program JoinMap version 3.0 (Van Ooijen and Voorrips
2001). Minimum LOD (logs of the odds ratios of linkage
vs. no linkage) scores of 4.0 (maximum recombination
fraction of 0.4) were used to group loci. Recombination
frequencies were converted to centi-Morgan (cM) map
distances using Kosambi’s mapping function (Kosambi
1944).
Results
Molecular markers for the L. maculans mating type
locus and the avirulence genes AvrLm1 and AvrLm4-7
Selecting the parental isolates for mapping AvrLepR1
The primer pairs used to amplify the two allelic variants of
the mating type locus were described by Cozijnsen and
Howlett (2003). The diagnostic primer pairs 1/2 and 3/4
An experimental system for the genetic mapping of AvrLepR1 required a pair of L. maculans isolates exhibiting
contrasting abilities to infect a B. napus line carrying
Fig. 1 The aerial portions of four seedlings of B. napus line ddm-126s-1, which carries the LepR1 gene for resistance to L. maculans (the
fungus that causes blackleg disease in canola). One cotyledon of each
seedling has been wounded and inoculated with L. maculans
pycnidiospores as described by Williams (1985). The two seedlings
on the right were inoculated with the avirulent isolate 99-56 which
carries the common, active form of the AvrLepR1 gene and which
elicited a rapid resistance reaction from line ddm-12-6s-1, corresponding to a disease rating of 1 (Williams 1985). The two seedlings
on the left were inoculated with the virulent isolate 87-41 which
carries the uncommon, inactive form of the avirulence gene
(avrLepR1) and which produced a conspicuously successful infection
on line ddm-12-6s-1, corresponding to a disease rating of 8 (Williams
1985)
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Table 2 Virulence/avirulence phenotypes of four L. maculans isolates (87-41, WA-74, 99-43 and 99-56) used to inoculate B. napus cotyledons
of a susceptible control line (‘Westar’) and a plant line carrying LepR1 (ddm-12-6s-1)
Isolates
Replication 1
Replication 2
Mean rating
on Westar
Mean rating on
ddm-12-6s-1
Lowest/highest rating
on ddm-12-6s-1
Mean rating
on Westar
Mean rating
on ddm-12-6s-1
Lowest/highest rating
on ddm-12-6s-1
87-41
7.5
7.5
5/9
7.6
7.6
5/9
WA-74
7.9
4.7
3/7
8.1
4.9
3/7
99-43
8.1
5.6
3/7
8.1
5.3
3/7
99-56
9
1.6
1/5
9
2.1
1/5
‘Westar’ (the susceptible control). The population of
L. maculans isolates segregated for virulence (50 isolates)
and avirulence (44 isolates) and the segregation ratio
approximated to the 1:1 ratio (v2 = 0.38, P = 0.66)
expected for genetic control of the phenotype by a single
gene (Fig. 2). The 1:1 segregation ratio was supported by
the analysis of complete sets of ascospores from two asci,
each segregated 1:1 avirulent/virulent. All 94 progenies
were virulent on ‘Westar’.
45
Number of progeny
40
35
30
25
20
15
10
5
0
0-0.9
1-1.9
2-2.9
3-3.9
4-4.9
5-5.9
6-6.9
7-7.9
8-8.9
Disease rating
Fig. 2 The frequency distribution of disease ratings within the L.
maculans population of 94 single-ascospore isolates derived from the
87-41 (virulent) 9 99-56 (avirulent) cross. The population was
segregating for virulence on B. napus ddm-12-6s-1, controlled by
AvrLepR1, and the disease ratings were scored 14 days postinoculation using the 0–9 rating scale described by Williams (1985)
LepR1. B. napus lines ddm-12-6s-1, carrying LepR1, and
‘Westar’ were used to test the virulence of the four L.
maculans isolates. As expected, isolate 87-41 was virulent
on both genotypes (Fig. 1; Table 2). Of the three isolates
expected to be avirulent, WA-74 and 99-43 produced
intermediate reactions on ddm-12-6s-1 while 99-56 was
obviously avirulent (Fig. 1; Table 2). All four L. maculans
isolates were highly virulent on the ‘Westar’ positive
control (Table 2). Repeating the whole experiment yielded
essentially identical results (Table 2). The four L. maculans isolates were tested with primers diagnostic for the
mating type locus. Isolates 87-41, WA-74 and 99-43 yielded the amplification products expected for the MAT1-1
mating type while 99-56 yielded those expected for the
MAT1-2 mating type.
L. maculans mating and virulence/avirulence
phenotyping of the resulting recombinants
Based on the above results, 87-41 and 99-56 were crossed
to produce a population segregating for AvrLepR1. A total
of 94 single-ascospore progenies were isolated from the
L. maculans cross and each isolate was tested for virulence
on B. napus line ddm-12-6s-1 (that carries LepR1) and
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Identifying SRAP markers informative
for the L. maculans cross
SRAP primer pairs were screened using DNA of the two
parental isolates (87-41 and 99-56) separately. Informative
primer pairs that amplified one or more DNA fragments that
were specific to one or the other of the parental isolates were
selected for further use. In addition, bulked segregant analysis (BSA; Michelmore et al. 1991) employing contrasting
bulked samples, one containing equal amounts of DNA from
12 avirulent isolates and the other containing equal amounts
of DNA from 12 virulent isolates, was used to identify SRAP
markers closely linked to AvrLepR1. Most primer pairs
produced 30-40 amplification products with lengths ranging
from 50 to 600 base pairs (bp). The average number of
informative amplification products per primer pair was 1.1,
with the most informative primer pair producing six products
that were polymorphic between the two parents.
The segregation patterns of 283 informative SRAP
polymorphisms were scored in the 94 isolates of the L.
maculans mapping population. The informative DNA
fragments were scored as either present or absent. Of the
283 loci assayed with SRAP markers, 254 loci (*90%)
had balanced 1:1 segregation ratios (P [ 0.05) while the
remaining 29 loci (*10%) exhibited distorted segregation
ratios (P \ 0.05). This indicated that the 94 single-ascospore progenies represented a fairly balanced population
with only an approximately twofold over representation of
loci with distorted segregation ratios. Of the 29 distorted
loci (P \ 0.05), only four were highly distorted (P \ 0.01)
and of these only one had a probability \0.001.
Theor Appl Genet (2012) 124:505–513
Genetic linkage analysis and mapping the AvrLepR1
avirulence gene of L. maculans
Genetic linkage analysis was carried out on the 283 SRAPdefined marker-loci. This established 36 linkage groups
each containing at least two loci and together representing
259 loci (Fig. 3) with 24 marker-loci remaining unlinked.
The linkage groups spanned 2076.4 cM of L. maculans
genome with individual linkage groups ranging from 13.1
to 164.8 cM in length (Fig. 3).
The segregation pattern of the molecular markers specific for the L. maculans mating type gene positioned the
mating type locus (MAT) 15.8 cM from the top of linkage
group 7 (Fig. 3). PCR amplifications with primers specific
for AvrLm1 (Table 1) yielded a 333 bp fragment using
DNA from isolate 87-41 and produced no amplification
product from isolate 99-56 DNA confirming the presence
of AvrLm1 in isolate 87-41 and its absence from isolate
99-56. Segregation for the AvrLm1 marker positioned the
gene 61.3 cM from the top of linkage group 13 (Fig. 3).
Segregation for virulence/avirulence on B. napus line
ddm-12-6s-1 identified the L. maculans locus (AvrLepR1)
that controls avirulence against the host’s LepR1 gene. The
L. maculans AvrLepR1 gene was located 10.1 cM from the
top of linkage group 4 in the 13.1 cM interval flanked by,
SBG49-110 and FT161-223 (Fig. 3). Linkage group 4 was
164.8 cM in length and carried 21 SRAP-defined loci. It
also carried the 28G8-490 and P1314-300 marker-loci that
are diagnostic of the chromosome carrying the AvrLm4-7
gene. This suggested that AvrLepR1 could be a new
member of the AvrLm3-4-7-9 genetic cluster (Balesdent
et al. 2005; Van de Wouw et al. 2008) or even a new allele
of the AvrLm4-7 gene. Both the parental isolates (87-41
and 99-56) were avirulent on B. napus plants carrying
either Rlm4 or Rlm7, indicating that each isolate carried a
functional AvrLm4-7 gene and that the AvrLm4-7 locus
could not be mapped in the segregating population based
on the segregation of differential disease reactions. In order
to test the possibility that the (functionally indistinguishable) alleles of AvrLm4-7 in isolates 87-41 and 99-56 could
be distinguished at the level of precise nucleotide
sequence, the AvrLm4-7 locus of each parental isolate was
amplified (using the AVRint-UP/-Lo primer pair: Parlange
et al. 2009), cloned and sequenced with fivefold replication. A comparison of the sequences of the two parental
alleles and the published sequence of AvrLm4-7 gene
(Parlange et al. 2009) revealed that the 99-56 allele carried
SNP212 (a substitution of A for C at nucleotide 212 of the
amplification product) while the 87-41 allele carried
SNP241 (a substitution of C for G at nucleotide 241 of the
amplification product). Genetic markers based on the differential PCR-amplification of these SNP’s using primer
pairs 5/6 and 7/8 (Table 1) allowed segregation at the
509
AvrLm4-7 locus to be scored in the mapping population
and the AvrLm4-7 locus to be positioned 6.8 cM below the
AvrLepR1 locus on linkage group 4 (Fig. 4) and in the
5.4 cM interval flanked by FT161-223 and P1314-300. To
confirm this result, the AvrLm4-7 gene was amplified,
cloned and sequenced in six recombinant isolates carrying
crossovers between the FT161-223 and P1314-300 markerloci flanking to AvrLm4-7. The AvrLm4-7 gene regions
from six non-recombinant isolates, three avirulent and
three virulent, were also cloned and sequenced as controls.
In every case, the gene-sequence analysis of the AvrLm4-7
alleles confirmed the earlier analysis based on SNP
markers.
AvrLepR1 has a specificity distinct from those
of AvrLm3, AvrLm4, AvrLm7 and AvrLm9
With AvrLepR1 possibly mapping to the AvrLm3-4-7-9
genetic cluster, it was considered prudent to investigate
whether AvrLepR1 interacted with Rlm3, Rlm4, Rlm7 or
Rlm9. The interactions of each parent of the L. maculans
mapping population (87-41 and 99-56), together with a
random sample of 30 of the single-ascospore progenies,
were tested with a set of six B. napus lines. The lines 22-11 (Rlm3), cv. ‘Falcon’ (Rlm4), 23-1-1 (Rlm7) and cv.
‘Darmor’ (Rlm9) were each reported to carry a single
distinct gene for resistance to L. maculans, while ddm-126s-1 carried LepR1 and cv. ‘Westar’ was used as the susceptible control. The results are summarized in Table 3.
The two parental isolates and the 30 progenies all interacted identically with line 22-1-1, cv. ‘Falcon’, line 23-1-1,
cv. ‘Darmor’ and ‘Westar’ (the isolates were either uniformly virulent or uniformly avirulent: Table 3). Only on
ddm-12-6s-1 did the L. maculans isolates exhibit variation.
Isolate 87-41 and 15 of the recombinants were virulent on
ddm-12-6s-1, while isolate 99-56 and the remaining 15
recombinants were avirulent on this line. These results
indicated that the LepR1—AvrLepR1 gene interaction
was distinct from those involving Rlm3, Rlm4, Rlm7 or
Rlm9.
Discussion
LepR1 is a new gene for resistance to L. maculans that was
introduced into amphidiploid B. napus (canola) recently
from the related diploid species, B. rapa, and that is harbored by the B. napus line ddm-12-6s-1 (Yu et al. 2005).
Several gene-for-gene interactions involving specific
L. maculans avirulence genes and the corresponding
resistance genes of the B. napus host have already been
described (Ansan-Melayah et al. 1995; Balesdent et al.
2001, 2005). The genetic mapping of AvrLepR1 positioned
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2
1
16.7
23.7
25.8
26.7
31.1
S12PM32-214
FT194-398
PT207-332
FBG48-376
PBG5-231
PPM110-102
PPM104-101
PT197-309
FT194-516
FBG48-225
S12PM88-358
PT6-237
PT6-398
PT151-409
PT6-442
FPM88-309
18.1
29.5
34.5
42.9
48.1
50.4
52.3
53.4
55.0
56.2
57.5
58.5
59.3
60.4
63.6
69.4
4
0.0
S12T145-503
9.7
FT161-136
PT140-510
PBG75-285
FBG68-226
25.7
S12T142-204
S12ODD2-466
PT168-437
11
0.0
PT166-189
PT45-217
50.5
51.6
7.7
S12T41-57
S12PM113-365
37.0
PT86-501
10
0.0
SBG73-161
7.1
10.7
FT175-482
PT13-256
55.0
FT10-350
16.2
ST122-267
66.4
PT207-547
0.0
SBG49-110
10.1
13.1
16.9
18.5
18.6
AvrLepR1
FT161-223
AvrLm4-7
P1314-300
FT158-199
PT82-480
33.9
PT204-392
SPM96-281
S12PM103-194
99.0
SPM87-542
PT83-472
42.6
50.5
50.9
51.4
53.8
60.4
FT198-122
FT56-480
SPM29-156
106.8
FT59-286
ST139-260
S12T124-417
S12T132-139
FBG11-274
FBG11-346
0.0
5.4
8.6
20
0.0
6.4
7.6
10.1
ST139-391
FT137-143
99.2
FT180-472 102.8
PT56-234
18.2
ST127-255
FBG41-304
0.0
PPM29-198 22.6
PT183-323
22
S12T138-529
PPM58-227
ST133-378
SPM58-533
SBG73-396
0.0
2.3
7.1
8.5
13.1
ST190-347
34.8
S12PM14-161
41.5
44.6
ST17-341
SBG56-383
SPM45-216
56.1
23
FT126-111
FT49-111
FT122-109
PT199-294
41.5
47.5
49.4
55.0
0.0
PT65-320
12.0
FT161-194
24.9
FT160-274
43.7
S12ODD2-81
37.2
ST183-481
24
0.0
FT204-470
ST84-124
PPM108-480
106.4
FBG69-114
ST159-220
36.5
FT204-155
55.2
57.3
60.1
61.4
64.9
PBG75-401
PT135-262
PT94-488
PT194-400
PPM16-224
83.3
FT204-487
17
FT204-342
0.0
FBG18-114
105.4
S12BG56-438
107.6
ST161-328
0.0
FBG12-237
FT198-226 15.2
FBG75-295 19.7
24.9
PT83-485
ST151-109
ST158-337
S12T4-372
0.0
9.7
11.7
SPM101-136
SPM95-137
23.1
26.5
31.2
S12T72-183
S12T34-342
ST180-357
S12PM95-239
FBG75-151
FT198-143
FT21-177
ST39-215
FT203-347
FT150-145
8.5
13.0
13.9
18.9
26.7
29.6
30.6
FT124-237
49.1
11.3
S12PM111-383 32.9
16
FT128-368
22.1
PT157-332
45.3
50.1
FBG69-258
SBG73-88
33.2
37.6
PT6-64
ST148-366
56.8
ST154-459
66.3
SBG68-330
51.2
56.1
57.1
61.3
FBG94-290
ST12T27-135
FODD3-371
AvrLm1
74.6
PT160-294
85.1
FT128-127
9.0
ST127-276
FPM29-78
FBG68-214
FT139-147
FBG41-123
FBG5-311
FT128-449
0.0
PBG17-361
6.1
FT188-164
0.0
4.1
38.4
33.9
FT199-176
33
PBG55-355
6.2
8.5
FPM76-123
SPM58-170
15.0
PT190-142
PT135-199
ST135-124
13.4
PPM16-394
19.7
S12T56-110
67.0
0.0
ST4-516
6.5
FT200-353
22.2
FPM73-206
50.6
ST85-307
58.7
58.9
ST56-342
SBG39-248
S12T59-298
PT14-386
29
0.0
S12PM76-447
28
FT204-392
18.9
27
26
PT59-449
30
SBG73-297
ST145-77
28.1
29.3
35
ST122-473
SPM88-269
S12T168-374
FT106-268
34
0.0
0.0
19
SPM20-306
FT189-201
FBG41-346
ST137-115
140.5
18
39.9
0.0
28.6
30.5
31.6
31.8
33.1
0.0
0.0
25
27.3
17.3
PPM58-281
86.1
89.9
21.7
25.4
S12PM111-167
FT160-136
27.6
12.0
14.0
16.4
FBG55-302
FBG39-123
PT39-524
13
0.0
32
PT204-92
FBG53-361 61.4
67.0
70.0
FPM113-139
0.0
FT161-387
FPM29-351
FT38-322
ST57-308
Mat
FPM100-289
FBG69-130
FBG69-97
0.0
9.8
14.7
15.5
15.8
15.9
19.0
22.0
0.0
PPM103-98
85.0
92.5
31
0.0
62.7
PT194-309
ST56-144
ST179-403 131.5
PT192-145
138.0
ST165-81
PT106-135
PPM29-543 138.2
PT126-92
SPM117-369
FBG55-269
14
ST126-427
FT82-247
FT57-467
0.0
SBG20-393 4.7
FT197-329
FPM14-140
5.6
134.6
138.2
140.6
146.2
149.5
S12PM29-308 152.2
154.7
160.7
164.8
FT59-329
21
FT174-348
FT194-372
PT60-406
FT174-217
PT163-228
FBG76-369
PT157-423
PT59-345
SPM117-85
PT56-172
PT178-249
SPM88-314
PT183-353
115.5
12
77.0
FT146-330
PT157-238
PT14-313
PPM32-441
PPM37-100
PT153-279
8
7
0.0
6.7
11.1
16.5
17.9
20.5
21.9
22.2
25.4
29.6
37.8
15
FBG53-318
PT150-506
57.2
59.4
11.7
14.8
18.1
19.5
23.5
27.6
87.3
91.5
106.2
ST85-183
35.8
PT153-284
67.8
FBG53-246
ST86-137
22.3
24.9
0.0
28G8-490
S12T41-490
ST155-381
PPM63-440 49.1
PT65-499
52.9
ST159-321
40.1
42.0
S12ODD2-342 42.5
45.1
46.2
FBG75-105
54.1
19.2
22.6
6
5
0.0
SPM104-459
PPM104-415
76.5
77.9
9
3
ST100-105
0.0
SPM53-233
0.0
ST155-474
ST132-362
26.6
ST158-171
FBG39-319
15.3
PPM32-73
PT56-434
39.4
36
0.0
0.0
2.4
0.0
PBG17-226
PPM58-110
39.9
24.2
FT57-221
32.8
FT198-293
PT13-289
Fig. 3 Genetic map of L. maculans based on 264 loci associated into
36 linkage groups (LG’s). The majority of loci, 259, were detected
using sequence-related amplified polymorphism (SRAP) markers.
The mating type locus (MAT1) on LG7, AvrLm1 on LG11 and
AvrLm4-7 on LG4, were each assayed using specially designed PCRbased markers. The AvrLepR1 locus was mapped on LG4. The locus
names are shown to the right of each LG and genetic distances from
the top in cM are indicated on the left of each LG
the gene on linkage group 4 close to, or part of, the
AvrLm3-4-7-9 cluster reported by Balesdent et al. (2005).
A new genetic map of L. maculans, based on a population of 94 single-ascospore progenies and segregating for
virulence on B. napus line ddm-126s-1, was developed.
The population was assayed with a set of informative PCR-
based markers (predominantly SRAP markers) and 288
polymorphic loci were identified. Genetic linkage analysis
established 36 linkage groups spanning a total of
2,076.4 cM (Fig. 3) with 24 marker-loci remaining
unlinked. The relatively large number of linkage groups,
36, compared to 21 groups in the L. maculans genetic map
123
Theor Appl Genet (2012) 124:505–513
Fig. 4 A large-scale view of
the top of linkage group 4
showing the relative positions of
AvrLepR1 and AvrLm4-7. The
locus names are shown to the
right of the LG and the genetic
distances from the top in cM are
indicated on the left of the LG
511
0.0
SBG49-110
10.1
13.1
16.9
18.5
18.6
AvrLepR1
FT161-223
AvrLm4-7
P1314-300
FT158-199
40.2
42.0
42.5
45.1
46.2
54.2
28G8-490
S12T41-490
ST155-381
PPM63-440
PT65-499
ST159-321
This marker clustering is a general feature of many PCRbased genetic markers and has been noted in genetic maps
of a range of fungi, including, Mycosphaerella graminicola
(Kema et al. 2002), Cochliobolus sativus (Zhong et al.
2002) and Blumeria graminis (Pedersen et al. 2002).
Twenty-nine or 10% of the SRAP marker-loci exhibited
segregation distortion to the degree expected to occur
naturally in only 5% of the markers (v2 = 16.9,
P \ 0.001). The elevated level of segregation distortion
observed in the SRAP marker-loci could have been the
result of a small degree of inadvertent selection having
been imposed on the population. Alternatively, it could
have been caused by systematic scoring errors at a small
number of loci (*14). The SRAP markers are dominant
and thus prone to errors caused by failed amplification
reactions. Further interrogation of the scoring and mapping
data revealed that the 29 loci exhibiting segregation distortion (P \ 0.05) were not randomly distributed but were
rather clustered on 14 of the 36 linkage group and that only
2 of the distorted loci were not linked to another marker
(LOD score \4.0).
The established physical size of the L. maculans genome
is 45 Mbp (Rouxel et al. 2011). The 36 linkage groups of
the new genetic map span a total of 2,076.4 cM suggesting
a physical to genetic ratio of 22 kb per cM. However, the
map is incomplete so this initial figure is certainly an overestimate of the average physical size per cM for the genome. An earlier genetic investigation of L. maculans based
on 443 loci produced a map that encompassed only
2,129 cM (Kuhn et al. 2006). Since this earlier map might
be expected to represent a more complete coverage of the
genome, it is possible that the meiotic divisions that produced the single-spore isolates on which the new map is
based experienced an elevated recombination frequency
relative to those which gave rise to the earlier population.
The current investigation located the AvrLepR1 locus on
linkage group 4 of the new genetic map of the L. maculans
genome. With a map composed largely of SRAP markers,
it was not possible to use comparative mapping to position
developed by Cozijnsen et al. (2000) and 15 groups (containing 5 or more markers) reported by Kuhn et al. (2006)
and the high proportion of unlinked loci (24 out of 288, 8%
of the loci) suggest that the marker coverage of the
L. maculans genome was incomplete and that some chromosomes are probably represented by more than one
linkage group. The L. maculans map described by Cozijnsen et al. (2000) was based on 159 markers mapped on 21
linkage groups with 18 loci remaining unlinked and those
of Kuhn et al. (2006) were based on between 177 and 443
markers mapped on 15 major groups. Unfortunately, the
use of a novel set of genetic markers makes a detailed
comparison with other maps of L. maculans impossible.
Overall, SRAP markers provided an inexpensive system
for genetic mapping in L. maculans, that required very little
development. SRAPs are a molecular-marker system with
some advantages over RAPD, RFLP, and AFLP markers
(Li and Quiros 2001). They have been used for genetic
mapping in plants (Li and Quiros 2001; Ferriol et al. 2003;
Sun et al. 2007; Dusabenyagasani and Fernando 2008) and
fungi (Dodds et al. 2004) and also in genetic diversity
studies in a range of organisms (Fernando et al. 2006; Sun
et al. 2006; Zhang et al. 2005). The clustering of SRAP
markers (Fig. 3) was one reason for the marker coverage of
the genome being incomplete despite the large number of
marker-loci assayed. Marker-dense regions of the L. maculans genome were also reported by Kuhn et al. (2006).
Table 3 Interaction phenotypes of the L. maculans parental isolates and of a set of 30 single-spore progenies all tested on the same panel of
B. napus lines, each carrying a defined resistance gene
Brassica lines and the resistance genes they are known to carry
Westar
22-1-1
Rlm3
Falcon
Rlm4
23-1-1
Rlm7
Darmor
Rlm9
ddm-126s-1
LepR1
99-56
V
V
A
A
V
A
87-41
V
V
A
A
V
V
Single-spore isolatesa
0:30b
0:30
30:0
30:0
0:30
15:15
L. maculans isolates
V virulent interaction, A avirulent interaction
a
b
A set of 30 single-spore isolates derived from crossing L. maculans isolates 87-41 and 99-56
The ratio of the number of avirulent isolates to the number of virulent isolate (A:V) in the 30 isolates tested
123
512
AvrLepR1 relative to other avirulence genes. A polymorphic molecular marker specific for AvrLm1 allowed this
gene to be positioned on linkage group 13, well removed
from the AvrLepR1 locus on linkage group 4. New
molecular markers specific for the DNA sequence variants
of the AvrLm4-7 gene resident in the two parental isolates,
87-41 and 99-56, allowed the segregation at the AvrLm4-7
locus to be scored in the mapping population and the
positioning of the AvrLm4-7 locus relative to the AvrLepR1
locus (Fig. 4). The obvious recombination between AvrLepR1 and AvrLm4-7 demonstrated that they are distinct
genes but their close linkage suggests that AvrLepR1 is
probably a new member of the L. maculans AvrLm3-4-7-9
genetic cluster, identified previously by Balesdent et al.
(2005). The reaction phenotypes of isolates 99-56 (that
carries AvrLepR1) and 87-41 (that does not carry AvrLepR1) (Table 3) demonstrated that AvrLepR1 has a distinct specificity from those of AvrLm3, AvrLm4, AvrLm7
and AvrLm9. However, because both isolates were virulent
on host plants carrying Rlm3 or Rlm9 (Table 3) it was not
possible to map any of the corresponding L. maculans
avirulence genes (AvrLm3 or AvrLm9) based of segregation
for the virulence/avirulence phenotypes in the new
L. maculans mapping population. Following the finding the
AvrLm4 and AvrLm7 are two distinct specificities of the
same avirulent gene (Parlange et al. 2009), the possibility
that AvrLm3 and/or AvrLm9 are allelic variants of AvrLepR1 cannot be dismissed.
Nine L. maculans avirulence genes, each conferring
specificity to one or two distinct Brassica resistance genes,
have been analyzed genetically (Balesdent et al. 2005;
Gout et al. 2006; Parlange et al. 2009). Three of these
genes have been cloned; AvrLm1 (Gout et al. 2006),
AvrLm6 (Fudal et al. 2007) and AvrLm4-7 (Parlange et al.
2009). We have now defined the reaction phenotype and
identified the locus encoding a novel avirulence gene,
AvrLepR1, which specifies incompatibility towards the
LepR1 resistance gene carried by B. napus line ddm-12-6s1. The detailed characterization of isolate 87-41 (avrLepR1, AvrLm1) and isolate 99-56 (AvrLepR1, avrLm1)
has established this pair of isolates as ideal for surveying B.
napus germplasm for the presence of the LepR1 and Rlm1
resistance genes. SRAP markers tightly linked to AvrLepR1
have been identified. These results are the first steps
towards cloning AvrLepR1, uncovering its molecular
function and defining its molecular interaction with LepR1.
Acknowledgments The authors thank Dr. Thierry Rouxel and
Dr. Marie- Hélène Balesdent for providing seed of lines 22-1-1 and
23-1-1. We also would like to thank Dr. Julian Thomas, Erica Reidel
and Sasanda Nilmalgoda, at the Cereal Research Center of Agriculture and Agri-Food Canada, and Paula Parks, at the University of
Manitoba, for their technical assistance in this research. This work
was supported by an NSERC Discovery Grant from the Government
123
Theor Appl Genet (2012) 124:505–513
of Canada, awarded to W.G.D. Fernando, and funding from the AgriFood Research & Development Initiative (ARDI), Government of
Manitoba.
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