Tree Genetics & Genomes (2010) 6:195–203
DOI 10.1007/s11295-009-0240-y

ORIGINAL PAPER

Genetic mapping of the crown gall resistance geneof the wild apple Malus sieboldii
Shigeki Moriya & Hiroshi Iwanami & Sae Takahashi &Nobuhiro Kotoda & Kouichi Suzaki &Toshiya Yamamoto & Kazuyuki Abe

Received: 16 March 2009 / Revised: 2 September 2009 / Accepted: 8 September 2009 / Published online: 18 September 2009
# Springer-Verlag 2009

Abstract Crown gall, caused by Agrobacterium tumefaciens,
causes severe damage to apple saplings resulting in weakgrowth and loss of commercial value. Developing molecularmarkers linked to crown gall resistance genes, and establishing a marker-assisted selection (MAS) for such a trait would
be an effective way to improve rootstock breeding for crowngall resistance. The wild apple Malus sieboldii Sanashi 63
carries the crown gall resistance gene Cg effective against the
A. tumefaciens strain Peach CG8331 (biovar 2). Applying theCommunicated by E. Dirlewanger
S. Moriya (*) : H. Iwanami : S. Takahashi : N. Kotoda :K. Suzaki : K. AbeApple Research Station, National Institute of Fruit Tree Science,
National Agriculture and Food Research Organization (NARO),
Shimokuriyagawa, Morioka,
Iwate 020-0123, Japane-mail: moriyas@affrc.go.jp
T. Yamamoto
National Institute of Fruit Tree Science,
National Agriculture and Food Research Organization (NARO),
Fujimoto, Tsukuba,
Ibaraki 305-8605, Japan
Present Address:
N. Kotoda
Okitsu Citrus Research Station,
National Institute of Fruit Tree Science,
National Agriculture and Food Research Organization (NARO),
Shimizuokitsunaka-cho, Shizuoka,
Shizuoka 424-0292, Japan
Present Address:
K. Suzaki
Grape and Persimmon Research Station,
National Institute of Fruit Tree Science,
National Agriculture and Food Research Organization (NARO),
Akitsu, Higashi-Hiroshima,
Hiroshima 739-2494, Japan

genome scanning approach on the mapping population JM7(cgcg) × Malus sieboldii Sanashi 63 (Cgcg), Cg was mappedon the linkage group (LG) 2. The constructed linkage map of
LG 2 of Sanashi 63 spans 59.8 cM and has an average marker
density of 3.5 cM per marker. The 191 bp allele of the simple
sequence repeat (SSR) NZmsEB119405 co-segregated perfectly with Cg in a segregating population of 119 individuals.
Quantitative trait loci, accounting for 75.3% to 84.3% of
phenotypic variation were detected in the same position.
Testing eight additional rootstocks with the NZmsEB119405
SSR marker revealed that the 191 bp allele is also present incrown gall-susceptible rootstock accessions. Only the markers
CH03b01 and NZmsPal92 mapping at 0.9 and 4.3 cM fromCg, respectively, showed “private” alleles associated to Cg.
Keywords Crown gall disease . Disease resistance . Malus .
Marker-assisted selection

Introduction
Crown gall, caused by Agrobacterium tumefaciens, occurs
worldwide in apple and pear trees and in numerous speciesof other dicotyledonous plants (Stover and Walsh 1998).
The damages on apple trees results in weak growth and lossof commercial value. This disease is of primary importancefor nurseries and results in discarding of 1–10% of trees
with peaks up to of 80% or more (Moore 1990). Comparedwith M.9 and M.26, the percentage of affected JM1 andJM7 saplings is higher, ranging from 48.1% to 70.9%(Nekoduka et al. 2001). The breeding of resistant rootstock
is an effective strategy to control this disease. Thisapproach has been studied in woody plants such as
grapevine, peach, and aspen (Beneddra et al. 1996; Blisset al. 1999; Mahmoodzadeh et al. 2004; Szegedi et al.

1961984). In a field experiment with grapevine, the use ofcrown gall-resistant rootstocks reduced the disease; 79%of susceptible rootstocks had crown gall, whereas only
9% of resistant rootstocks showed symptoms (Sule andBurr 1998).
In apple, such a strategy just starts to be adopted (Viss etal. 2003). Recently, Moriya et al. (2008) identified a crowngall-resistant wild apple, Malus sieboldii Sanashi 63.
Sanashi 63 is considered to carry a major gene at theheterozygous state for resistance to crown gall. The resultof other testcrosses allowed us to hypothesized that
resistance gene is dominant. However, this resistance isstrain specific (Moriya et al. 2008). In fact, one of the tested
strains (ARAT-001) is able to form galls on Sanashi 63,
indicating that this strain is able to overcome this resistance.
Nevertheless, the strategy focusing on Cg in the resistant
breeding program is valuable, because Peach CG8331 is infact virulent for major rootstock accessions. The supposed
resistance source to ARAT-001 is M. sieboldii Mo-15,
because Mo-15 did not exhibit gall by both Peach CG8331
and ARAT-001 (Moriya et al. 2008).
Crown gall-resistant rootstock breeding has certain
drawbacks, such as the long time it takes and the difficultyin assessing the resistance level of numerous progeny
plants. Therefore, developing molecular markers linked tothe crown gall-resistant gene and applying a markerassisted selection (MAS) system in the selection of crowngall-resistant plants would be an effective way to promoterootstock breeding for crown gall resistance.
Molecular markers are a powerful tool for breeding
disease-resistant apples. In apple, disease resistance breeding has mainly focused on apple scab caused by Venturia
inaquealis. Molecular markers linked to Vb, Vbj, Va, Vm,
Vf, and so on, have been developed (Erdin et al. 2006;
Gygax et al. 2004; Hemmat et al. 2003; Patocchi et al.
2005; Tartarini et al. 1999). For other diseases and insect
damages, molecular markers and/or quantitative trait loci
(QTL) linked to powdery mildew, fire bright, rosy leafcurling aphid, and wooly apple aphid, have been studied(Calenge and Durel 2006; Calenge et al. 2005a; Khan et al.
2006; Cevik and King 2002; Bus et al. 2008). To readily
map major resistance genes and identify molecular markers
linked to them, the genome scanning approach (GSA) was
proposed by Patocchi and Gessler (2003). GSA is based on
the detection of distortion of the expected 1:1 segregation
ratio of the alleles of the resistant parent for a simple
sequence repeat (SSR) marker within a small subset ofprogeny plants. The GSA was successfully applied to
identify the map position of Vb (Erdin et al. 2006), Vbj(Gygax et al. 2004), and Vm (Patocchi et al. 2005). In
Japanese pear, the map position of genes for susceptibility
to black spot disease was also determined by the GSA(Terakami et al. 2007).

Tree Genetics & Genomes (2010) 6:195–203

In this study, we identified a SSR marker closely linked
to crown gall resistance by GSA and mapped the resistancegene Cg of Sanashi 63 against Agrobacterium tumefaciens.
To our knowledge, this is the first time that a crown gall
resistance gene is mapped in apple.

Materials and methods
Plant material
One hundred and twenty progenies of JM7 (M.9 × M.
prunifolia Morioka Seishi) × M. sieboldii Sanashi 63 wereused for this study. Three replications of each plant wereprepared by grafting dormant shoots on JM7 rootstockpotted in fumigated soil. Each plant had three shoots.
DNA extraction
One hundred milligrams of young leaves were first crushed
using an SH-48 machine (Kurabo, Osaka, Japan) using
liquid nitrogen and the obtained fine leaf powder was
incubated for 30 min at 37°C with 1 mL of isolation buffer(10% PEG#6000, 100 mM Tris-HCl (pH 8.0), 350 mM
sorbitol, and 50 mM EDTA (pH 8.0)) as described by
Moriya et al. (2009). Then, genomic DNA was extractedusing the automated DNA extracting machine PI-50α(Kurabo) according to the manufacturer’s instructions.
Extracted DNA was eluted in Tris-EDTA (TE) buffer andquantified by agarose gel electrophoresis as described by
Yamamoto et al. (2006). Finally, quantified DNA was
diluted to 10 ng/μL with 1/10 TE buffer.
Inoculation and evaluation of crown gall resistance
The A. tumefaciens strain Peach CG8331 (biovar 2),
isolated from peach at Yamagata, Japan, was used for
inoculation. The bacterial inoculum was cultured in a YP
broth at 28°C for 2 days and adjusted to a 109 colonyforming unit (cfu)/mL. The inoculation was conductedwhen almost all the shoots were 30 cm long. The
inoculation was performed as described by Moriya et al.
(2008); the bacterial suspensions were taken up into
sterilized disposable syringes (5 mL, Terumo, Tokyo,
Japan) with a sterilized needle (0.55 × 25 mm, Terumo),
the needle was inserted into the internode of each growing
shoot, and then one drop of bacterial suspension was
injected. Needles were replaced after each injection. Sincenine sites per potted plant were inoculated, in total, 27 sites
per genotype were inoculated. The inoculation test was
repeated twice over 2 years.
Six months after inoculation, each inoculation site wasvisually assessed to determine whether a crown gall had

Tree Genetics & Genomes (2010) 6:195–203

formed and the number of galls at the inoculated sites wascounted for each progeny (Fig. 1). The frequency of galloccurrence was then determined for each progeny. Since the
distribution of individual phenotypic values indicated theputative presence of a major gene, these values weretransformed into binary data for genetic mapping. Transformation was performed according to the scale of Moriya
et al. (2008) slightly modified: resistant (0) and moderately
resistant (>0 to ≤0.3) were classified as resistant in thisstudy. The progenies showing a frequency of >0.3 wereclassified as susceptible.
Genome scanning approach
To identify the linkage group (LG) carrying the crown gall
resistance Cg and then precisely map the gene, GSA wasapplied after Patocchi et al. (2005). For the first GSA
round, 22 resistant plants and the two parents were used.
SSRs used for GSA round 1 were chosen on the basis of areference map published by Silfverberg-Dilworth et al.
(2006). For each linkage group, SSRs were selected anda197

tested until two to three informative (i.e., Sanashi 63
segregating alleles) SSRs per linkage group were identified(Table 1).
Polymerase chain reaction (PCR) was performed using
two methods: the direct fluorescent primer method (Ziegle
et al. 1992) and the M13-tailed primer method (Schuelke
2000) with the following modification: the M13-tailed
sequence of the forward primer was substituted with the
T7 promoter sequence. PCR products amplified by bothmethods were separated and detected using the autosequencer CEQ 8000 (Beckman-Coulter Inc., CA, USA).
The sizes of the amplicons were determined on the basis of
an internal standard DNA (DNA Size Standard Kit-400,
Beckman-Coulter) with a CEQ Genetic Analyzer(Beckman-Coulter). The allele size obtained by the T7tailed primer method contained 17–18 bp of tailed sequencelength. Once segregation distortion (distortion above 16:6;
p<0.05) was detected, the distorted SSR were tested on thewhole progeny (GSA round 2). Upon identification of thelinkage group carrying Cg, SSRs previously mapped on
this LG of apple and pear (Celton et al. 2009; Fernandez-

c

d

b

e

Fig. 1 Degree of resistance for gall formation. a and b no formation of crown gall (resistant phenotypes), c–e formation of crown gall at theinoculation sites (susceptible phenotypes). Gall formation at 90 after inoculation

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Table 1 Informative simple sequence repeats (SSR) and segregationof the alleles of Malus sieboldii Sanashi 63 in the 22 crown gallresistant progenies of JM7 × Sanashi 63 cross
Linkage
group

SSRa

Segregation
ratio alleles
Sanashi 63ab

Table 1 (continued)
Linkage
group

SSRa

χ2 valueb

Segregation
ratio alleles
Sanashi 63
a

1
1
2
2
3
3

CH05g08
Hi02c07
CH03d10CH05e03
CH03g07
MS14h03

14
16
4
6
14
13

8
6
18
16
8
9

1.64
4.55*
8.91**
4.55*
1.64
0.73

4
4
5
5
6
6
7
7
8
8
8
9
9
9
10
10
11
11
11

CH02h11a
CH02c02b
CH05e06
CH04e03
CH03d07
CH05a05
Hi03a10CH04e05
CH01c06
CH02g09
CH01h10NH029aCH05c07
CH01h02
CH01f12CH03d11CH02d08
CH02d12
Hi06b06

11
12
11
11
10
11
20
5
11
10
10
11
10
8
7
16
12
11
13

11
10
11
11
12
11
2
17
11
12
12
11
12
14
15
6
10
11
9

0
0.18
0
0
0.18
0
14.73***
6.55*
0
0.18
0.18
0
0.18
1.64
2.91
4.55*
0.18
0
0.73

12
12
12
13
13
14
14
14

CH05d04
CH04g04
CH03c02
Hi04g05
CH03a08
CH01g05
CH01e01
CH05g11

9
10
12
15
12
14
8
7

13
12
10
7
10
8
14
15

0.73
0.18
0.18
2.91
0.18
1.64
1.64
2.91

15
15
15
16

CH03b06
CH02d11CH03b10CH05c06

14
17
16
9

8
5
6
13

0.20
6.55*
4.55*
0.73

16
17

CH05a04
CH05g03

12
7

10
15

0.18
2.91

χ2 valuebb

17

CH01h01

7

15

2.91

17

Hi07h02

12

10

0.67

*0.05; **0.01; ***0.001
a

The following SSRs (LG) have been tested, but the alleles of Sanashi
63 were not polymorphic or identical to those of JM7: CH03g12 (1),KA4b (1), CH-Vf1 (1), CH02f06 (2), Hi15b02 (3), Hi01e10p (4),CH01d03 (4), Hi04a08p (5), CH03a09 (5), Hi04d02p (5), CH03a04(5), Hi04d10p (6), CH01f03 (9), Hi05e07p (9), CH02b07 (10),CH02a08 (10), AF057134-SSR (10), CH02a10 (10), CH02c11 (10),Col (10), CH02g12 (12), CH01d09 (12), CH05h05 (13), AU223486SSR (13), CH03h03 (13), CH05f04 (13), CH01d08 (15), CH02c09(15), CH04f10 (16), Hi02f12 (17)bSegregation distortion is indicated by means of significance level
P of the χ2 test

Fernandez et al. 2008; Liebhard et al. 2003; SilfverbergDilworth et al. 2006; Yamamoto et al. 2007) were used togenerate a genetic map of the LG of Sanashi 63 (Table 2).
Cleaved amplified polymorphic sequence analysis

Second locus
unknown

Second locus
unknown

The expressed sequence tag CN493139 was transformed ina cleaved amplified polymorphic sequence (CAPS) marker.
Primers (F: 5′- AAA CTG GTA CAT ACC GCT GGA -3′
and R: 5′- GCA GGATTT CTATAATAT CGG AAA AG -3′)
were designed using the Primer3 software (Rozen and
Skaletsky 2000). PCR was performed in a 20 μL solutionof 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2,
0.01% gelatin, 0.2 mM each dNTP, 10 pmol of each forward
and reverse primer, 20 ng of genomic DNA, and 0.5 units of
Taq DNA polymerase (Invitrogen Corp., CA, USA). The
amplification was performed in 35 cycles at 94°C for 30 s(denaturing), 60°C for 30 s (annealing), and 72°C for 1 min(primer extension). PCR products were sequenced and apolymorphism was found in an intron. Amplicons weredigested with Tsp 509I (New England BioLabs Inc., MA,
USA) and separated on 1.5% agarose gels. The gels were
stained with ethidium bromide and visualized with ultraviolet
light.
Mapping and QTL analysis
JoinMap ver. 3.0 (Van Ooijen and Voorrips 2001) was used
to calculate the genetic linkage maps. The Kosambi
mapping function was used to convert recombination units
into genetic distances. The map of Sanashi 63 wasgenerated using MapChart 2.2 (Voorrips 2002). QTL were

Tree Genetics & Genomes (2010) 6:195–203
Table 2 Simple sequence repeat (SSR) markers used for saturation of
linkage group 2
SSR

Reference

CH02c02a
CH02c06
CH02b10
CH02f06
CH03b01
CH03d01
CH03d10CH04e12b
CH05e03
AJ251116-SSR
AT000400-SSR
CN493139-SSR
CN581493-SSR

Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Liebhard et al. (2002)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)

Hi02a07
Hi05g12Hi08g12Hi22d06
Hi07d12NZmsPal13
NZmsPal92
NZmsEB149808
NZmsEB153909
NZmsEB106592
NZmsEB119405
NZmsEB107305
NH033b

Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Silfverberg-Dilworth et al. (2006)
Celton et al. (2009)
Celton et al. (2009)
Celton et al. (2009)
Celton et al. (2009)
Celton et al. (2009)
Celton et al. (2009)
Celton et al. (2009)
Yamamoto et al. (2002)

detected using the interval mapping procedure of MapQTL
ver. 4.0 (Van Ooijen et al. 2002). QTL with a maximum
logarithm of odds (LOD) score greater than or equal to 3.0
were declared significant. For each significant QTL,
confidence intervals corresponding to a LOD score dropoff of 2 on both sides of the likelihood peak were
calculated. The confidence interval is supposed to containthe QTL with a probability over 95% (Van Ooijen 1992).

Results

199

outlier and excluded from subsequent analysis. In total, 61
plants were scored as resistant to crown gall and 58 plants
were scored as susceptible (Fig. 2). This segregation ratio
does not significantly differ from the expected 1:1 ratio(χ2 =0.076) and is indication of monogenic inheritance.
Genome scanning approach
In the first round of GSA, out of the 71 SSRs tested, 41
markers were informative for Sanashi 63 (Table 1). Two or
three informative SSRs were obtained on every LG.
Significant distortion from 1:1 of the alleles of Sanashi 63
was found for eight SSRs of five LGs: LG 1, 2, 7, 10, and
15. These SSRs were considered as putatively linked to Cg;
therefore, the GSA round 2 was carried out with allprogeny plants and the eight distorted SSRs. As a result,significant linkage to Cg was detected only on CH03d10
and CH05e03 of LG 2 with recombination values 0.202(LOD 9.83) and 0.252 (LOD 6.63), respectively, while nosignificant linkage was found with the other SSRs.
Recombination values between Cg and Hi02c07p (LG 1),
Hi03a10 (LG7), CH04e05 (LG7), CH02d11 (LG 15), andCH03b10 (LG 15) were all higher than 0.445 (LOD ≤0.30).
Fine mapping of Cg on LG 2
Publicly available SSRs located on LG2 were tested tobetter define the position of Cg on Sanashi 63 LG. Ten out
of 18 tested CH or Hi SSRs (Liebhard et al. 2002;
Silfverberg-Dilworth et al. 2006), four out of seven NZ
SSRs (Celton et al. 2009), and one NH SSR (Yamamoto etal. 2002) were successfully mapped (Table 3, Fig. 3).
Because the SSR CN493139 was mapped between Hi02a07
and AT000400-SSR by Silfverberg-Dilworth et al. (2006),
this SSR should be located close to Cg. However, the SSR
CN493139 showed an indistinguishable fragment patternon tested plants. It was converted into a CAPS marker andmapped distinct position than in the previous publication. A
SNP, made visible by a restriction digestion with the
enzyme Tsp 509I, was identified. JM7 is A/A while Sanashi
63 is A/G, with the A allele of Sanashi 63 being in couplingwith Cg. The constructed linkage map of LG 2 of Sanashi
63 spanned 59.8 cM and had an average marker density of
3.5 cM per marker (Fig. 3). The SSR NZmsEB119405
perfectly co-segregated with Cg (LOD 35.8).

Segregation of crown gall resistance
QTL analysis
As a positive correlation (R2 =0.68; data not shown)
between the phenotyping data of the 2 years, has been
observed, the average value of crown gall frequency overthe 2 years was calculated and used to discriminate between
resistant and susceptible plants for the GSA and the genetic
mapping. A single progeny was clearly classified as an

Since the gall occurrence frequency was a quantitative trait,a QTL analysis was also performed for LG 2. A QTL witha high LOD score was detected on the same position as in
the previous mapping using the phenotype data of both
years (Fig. 4). Their effect on the phenotypic variation was

200

Tree Genetics & Genomes (2010) 6:195–203

Sanashi 63
0a Sanashi 63 LG 2

b LG 2 (apple disease resistance)

Percentage of hybrids (%)

30

JM7
0.67

25

0.0

0

CH02c02a
Vr2

5

Vh4

43

CH02b1047

CH05e0363

CH03d01

CN581493-SSR

20

Powdery
mildew
QTL

15
18.6
22.1

10

25.5

5

46.2
47.6
51.6
59.8

NZmsEB107305

0Frequency of gall occurrence

Fig. 2 Distribution of the frequency of gall occurrence within the F1progeny derived from JM7 and Malus sieboldii Sanashi 63 inoculatedwith Agrobacterium tumefaciens strain Peach CG8331. The frequencyof each individual was calculated averaging the frequencies observedin 2 years (2005 and 2006). Close and open arrows indicate thefrequency of Sanashi 63 and JM7, respectively. Reproduced bypermission of Japanese Society for Horticultural Science

noteworthy strong; R2 =84.3 and 75.3% in 2005 and 2006,
respectively. The marker closest to the peak with thehighest LOD is NZmsEB119405. The confidence intervalsof two years overlapped and distribute around
NZmsEB119405. Significantly skewed markers on LG 1, 7,
10, and 15 in the first round of GSA were also analyzed using
interval mapping procedure. The LOD score of these markers
ranged from 0.00 to 0.55, indicating no presence of QTL.
Verification of the allele coupling with Cg
Perfectly co-segregating SSR NZmsEB119405 and closely
linked SSRs Hi02a07, AT000400-SSR, CH03b01, andNZmsPal92 were tested on additional 8 rootstock accessions (Table 4). All these accessions has been using as
parents in rootstock breeding program, and considered to becrown gall susceptible by the inoculation test and/or fieldobservations. For NZmsEB119405, three alleles wereobserved among 10 accessions. The 191 bp allele ofCg

NZmsEB119405
AT000400-SSR
CH03b01
NZmsPal92
CN493139-CAPS
Hi07d12NH033bNZmsPal13
CH02c06
NZmsEB149808
CH03d10CH02b10CH05e03

26.4

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

CH02c02a
Hi02a07

29.8
32.5
38.6
40.4
44.7

Vh2
Vh8
Vbj
VT57
Scab QTL

Fig. 3 a Genetic map of the genomic region around the crown gall
resistance locus Cg of Malus sieboldii Sanashi 63. b Representation of
the distribution of apple scab resistance genes on LG2. Genetic
distances of apple scab resistance genes are represented as in Bus et al.
(2005b). Map distances are indicated in centimorgans

NZmsEB119405, which is associated with Cg in Sanashi
63, was also observed in crown gall susceptible accessions ofM. prunifolia Mo-84a, M. prunifolia Morioka Seishi, JM1,
and JM5 (the latter 2 accessions are descendants of Morioka
Seishi).The first SSRs most closely associated to Cg,
showing “private” alleles among the tested rootstocks wereCH03b01 (allele 193 bp), and NZmsPal92 (null allele),
mapped at 0.9 cM and 4.3 cM from Cg, respectively,.

Discussion
In this study, we identified the map position of the crowngall resistance gene, Cg, derived from Malus sieboldii

Table 3 Segregation of the markers closely linked to the crown gall resistance gene Cg in the progenies of JM7 × Sanashi 63
MarkerAllele of Sanashi 63a

Resistantb

Susceptibleb

Total

Recombination frequency (LOD)

Hi02a07257 bpNull
191 bp200 bp232 bp/158 bp
244 bp/193 bp
264 bp/G
Null/A

60
1
61
0
0
61
3
58

3
55
0
58
57
1
56
2

63
56
61
58
57
62
59
60

0.034

(28.4)

0(35.8)

0.008(33.6)

0.042(26.9)

NZmsEB119405
AT000400-SSR/CH03b01
NZmsPal92/
CN493139-CAPSa

The alleles in bold coupling with CgbMean of the frequency of gall occurrence over 2 years

Tree Genetics & Genomes (2010) 6:195–203

201

50
2005
2006
Confidence
interval

LOD

40
30
20
10

40

60
NZmsEB107305

20
Hi02a07
NZmsEB119405
CH03b01
AT000400-SSR

0CN581493

0

2005
2006

Distance (cM)

Fig. 4 LOD plot for the crown gall resistance QTL identified on thelinkage group 2 using the phenotype data of 2005 and 2006. LOD-2
confidence intervals are indicated with boxes

Sanashi 63. Testing as little as 22 resistant plants with 71
SSRs was sufficient to identify 5 LGs candidate to carry theCg gene. After the second GSA round, only SSR on LG 2
of Sanashi 63 showed a significant linkage to Cg,
indicating that the crown gall resistance gene was locatedon LG 2 of Sanashi 63. An SSR marker, NZmsEB119405,
co-segregates in a population of 119 progeny plants has
also been identified (Table 3). The same locus was
identified also performing a QTL mapping. A strong QTL
accounting for about 80% of the phenotypic variation was
identified in the same region. The high phenotypic variation

explained by the QTL is consistent with our hypothesis thata major gene is responsible for the resistance. To ourknowledge, this is the first report of the mapping ofresistance gene to crown gall disease in apple.
Genomic regions with distorted segregation were foundon LG 1, 7, 10, and 15 of Sanashi 63. During othermapping experiments, regions with variable degree ofmarker segregation distortion were found on various
linkage groups (Patocchi et al. 2005). Gao and van deWeg (2006) reported sub-lethal genes linked to Vf mappedon LG 1. The distortion detected on LG 7 and 15 wasconsistent with the reports of Igarashi et al. (2008). The
segregation distortion on LG 10 was frequently detected(Kenis and Keulemans 2005; Liebhard et al. 2003;
Maliepaard et al. 1998) and these distortions wereexplained by the presence of alleles of genes conferring
low viability (Liebhard et al. 2003).
SSR markers tightly linked to crown gall resistance canbe directly used in MAS. Therefore, the frequency of thealleles in coupling of the most closely linked SSRs wasverified in 10 rootstock accessions (Table 4). The alleles
191 bp, 257 bp and 244 bp in coupling with Cg of
NZmsEB119405, Hi02a07 and AT000400-SSR were also
found in crown gall susceptible accessions of M. prunifolia
Mo-84a, M. prunifolia Morioka Seishi, and Morioka Seishi
descendants JM1 and JM5. Only CH03b01 that cosegregated with AT000400-SSR (at 0.9 cM from Cg) and
NZmsPal92 (at 4.3 cM from, Cg) presented unique alleles
among the investigated accessions. These results indicatethat for MAS CH03b01 and NZmsPal92 may be better
suited than NZmsEB119405, Hi02a07, and AT000400SSR. However, CH03b01 and NZmsPal92 have both aninconvenience. At our conditions, CH03b01 amplified

Table 4 Allele compositions of selected rootstocks tested with the five SSRs most tightly linked to Cg

Distance in centimorgans from Cg
Sanashi 63
JM1
JM5
JM7
Mo-84aMorioka Seishi
M.9
M.26
M.27
G.65
Number of allelesab

Crown gall-resistant

Hi02a07

NZmsEB119405

AT000400-SSR

CH03b01a

NZmsPal92

Yes
No
No
No
No
No
No
No

3.4
257b/null
257/299257/299274/299257/274
257/274
299/299272/2990
191/200
191/206
191/206
206/206
191/206
191/206
206/206
206/2060.9
232/244232/244232/244232/232232/244232/244232/232232/2440.9
159/193
171/185
171/185
183/185
171/183
171/183
185/185
185/185

4.3
264/null
245/277245/277
277/279245/283245/279
271/277
269/277

265/299299/2996

200/206
206/2063

228/232/249
228/232/249
4

181/185
181/1855271/277
255/277
9No
No

Minor bands, presumed second loci, were present in the 190–215 bp range

Alleles in bold associated with Cg. All SSRs were detected by a T7-tail primer method (see Materials and methods). Underlined are the alleles ofthe same size of those associated to Cg but identified in susceptible rootstocks

202

some weak bands of about the same size of the allele incoupling with Cg. These bands may complicate the scoringof CH03b01. The problem of NZmsPal92 is that the allele
associated with the Cg is a null allele making impossible todistinguish amplification failure form resistant progenies. In
MAS, therefore, using several SSRs to generate haplotypes
is considered to be reasonable selection method; e.g.,
NZmsEB119405, CH03b01, and NZmsPal92.
LG 2 appears to be deeply involved in disease resistance inapple. In fact, on apple LG 2, six scab resistance major genes
have been mapped, including Vh2, Vh4, Vh8, VT57, Vbj, andVr2 (Bus et al. 2005a, b; Gygax et al. 2004; Patocchi et al.
2004; Fig. 3). Major QTL for apple scab resistance (Calenge
et al. 2004) and powdery mildew (Calenge and Durel 2006)
were also identified on LG 2 using Discovery × TN108 progeny (Fig. 3). Moreover, several resistance gene analogsof the NBS-type have been mapped on the LG 2 andparticularly on the top of the LG (Baldi et al. 2004; Calenge
et al. 2005b; Celton et al. 2009; Naik et al. 2006). Comparing
the map position of the crown gall resistance Cg and apple
scab resistance genes and QTL mapped on this LG, it can beobserved that Cg maps about in the same position as Vr2,
Vh4, and powdery mildew QTL. Therefore, on the top of LG
2, not only resistance genes against fungal disease are present
but also resistance genes against a bacterial disease.
Sanashi 63 exhibits no gall at all with strain Peach
CG8331. On the other hand, numerous moderately resistantprogeny plants exhibited some crown gall (freqency >0 and≤0.3). The genetic background of Sanashi 63 probably
consisted of Cg major gene and putative polygenic factors
having moderate/minor but complementary effects. The
lack of these polygenic factors (modifiers) could decrease
the level of resistance of some progenies carrying Cg.
Modifiers have been proposed for the apple scab resistancegene Vf (Gessler 1989). Genome wide QTL study will
reveal such minor resistance factors.
One of our main goals is the development of durable crowngall-resistant and dwarfing rootstock suitable for the Japaneseclimate. Rootstocks adapted to the Japanese climate can beobtained by crossing Sanashi 63 with rootstocks that are well
adapted to the Japanese climate such as JM1, JM5, and JM7.
Recently, the map position of Dw1, a gene conferring dwarf
growth to the scion derived from M.9 rootstock, has been
determined and molecular markers closely linked to the gene
have been identified (Pilcher et al. 2008). Therefore,
combining these markers and our Cg-linked markers will
enable apple breeders to save time and labor in the selectionof dwarfing rootstocks resistant to some strains of crown gall.
Acknowledgement We would like to thank Dr. Y. Takikawa of
Shizuoka University for providing the bacterial strain and Dr. S.
Terakami of National Institute of Fruit Tree Science for valuable
discussions and computer analysis.

Tree Genetics & Genomes (2010) 6:195–203

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