J. Japan. Soc. Hort. Sci. 78 (3): 279–287. 2009.
Available online at www.jstage.jst.go.jp/browse/jjshs1
JSHS © 2009

Development of a Marker-assisted Selection System for Columnar Growth Habit
in Apple Breeding
Shigeki Moriya1*, Hiroshi Iwanami1, Nobuhiro Kotoda1**, Sae Takahashi1,
Toshiya Yamamoto2 and Kazuyuki Abe1
Apple Research Station, National Institute of Fruit Tree Science, NARO, Shimokuriyagawa, Morioka 020-0123, Japan
National Institute of Fruit Tree Science, NARO, Fujimoto, Tsukuba 305-8605, Japan1
2

To make progress in columnar apple breeding, we constructed genetic linkage maps of the Co genomic region
and identified useful DNA markers that enable correct and efficient selection of columnar-type seedlings in apractical columnar apple breeding program. Linkage maps of three mapping populations were constructed on
the apple linkage group 10, and the Co gene was mapped on two populations, ‘Fuji’ × 8H-9-45 and ‘Fuji’ × 512786. Closely linked marker genotypes of SCAR682, SCAR216, CH03d11, and Hi01a03 were determined on 33
columnar-type cultivars and/or selections, and non-columnar-type cultivars contained 7 ancestral cultivars ofapple breeding in Japan. A 174 bp fragment for primer Hi01a03 was amplified in all columnar-type cultivars,
selections, and ‘McIntosh’, but was not detected in other ancestral apples. Amplified products of 682 bp for
SCAR682 and 177 bp for CH03d11 were detected in almost all columnar genotypes except for one selection, and‘McIntosh’, but the products were not found in other ancestral apples. A 177 bp allele of CH03d11, characteristicof a columnar phenotype, was detected in all 170 columnar-type plants obtained from 18 crosses, suggesting that
it is one of the most tightly linked DNA markers to the Co. These results indicated that CH03d11 was the mostreliable marker to distinguish columnar and non-columnar phenotypes for the MAS system. In our two mostadvanced breeding selections, the Co gene-associated alleles of the original columnar mutant (‘Wijcik’) on
CH03d11 (177 bp) and Hi01a03 (174 bp) remained, indicating that using these two breeding selections as Co
donors in the breeding program, CH03d11 and Hi01a03 enable the selection of seedlings with columnar phenotype.
Key Words: columnar-type apple breeding, linkage map, Malus, simple sequence repeat.

tural types according to the tree growth pattern,
distribution of branches, and fruiting position (Lauri etal., 1995; Lespinasse and Delort, 1986). The genetic
backgrounds of tree forms are complicated and poorly
understood.
The columnar growth habit in apple was initially
discovered in a sport of ‘McIntosh’ (Fisher, 1970). Thismutant, called ‘Wijcik’ or ‘Wijcik McIntosh’, has
peculiar phenotypes, such as reduced branching andincreased spurs on the lateral shoots and a trunk withshort internodes, in contrast to ‘McIntosh’ and othermajor apple cultivars. These characteristics are
considered to be important and advantageous for laborsaving cultivation because of the minimal pruning andtraining required by apple growers; therefore, ‘Wijcik’is an important breeding source to develop columnartype compact apple cultivars. Lapins (1976) revealed
that a single dominant gene, Co, determined the columnar
character; however, one or more modifiers, may be

Introduction
Apples (Malus × domestica Borkh.) belong to thegenus Malus, which is in the subfamily Maloideae ofthe family Rosaceae, and grow readily in all temperate
climatic zones (Luby, 2003; O’Rourke, 2003). Worldwide commercial apple production was about 64 million
tons in 2006 (FAOSTAT, Food and Agriculture
Organization of the United Nations (FAO), http://
faostat.fao.org, October 30, 2008). Tree architecture andshoot morphology traits influence the fruit yield throughpruning or training. Great variability in tree characteristics has been demonstrated in apple cultivars, whichhave been qualitatively classified into several architecReceived; June 30, 2008. Accepted; December 18, 2008.
* Corresponding author (E-mail: moriyas@affrc.go.jp).
** Present address: Okitsu Citrus Research Station, National Instituteof Fruit Tree Science, NARO, Shizuoka 424-0292, Japan.

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S. Moriya, H. Iwanami, N. Kotoda, S. Takahashi, T. Yamamoto and K. Abe

involved in the expression of the character, because thepercentage of plants with a columnar phenotype wasslightly below that expected by the test cross study(Lapins, 1976).
Apple breeding programs for the columnar type were
initiated at the National Institute of Fruit Tree Science(NIFTS, Japan) in 1987. The fruit characteristics of‘Wijcik’ were unsuitable for apple growers andconsumers in Japan because of severe preharvest drop,
low sugar content and high acidity of fruit juice, soft
fruit flesh, and short shelf life; therefore, the breeding
objectives of a dessert columnar-type apple combined acolumnar trait with improved fruit characteristics. To
achieve these objectives, columnar apples have beencrossed with major apple cultivars with good fruit
quality, such as ‘Fuji’. Since it takes many years to
improve the fruit quality of ‘Wijcik’, some columnartype selections introduced from Canada were used as
donors of Co in the apple breeding program at NIFTS.
In order to accelerate columnar apple breeding, early
selection of columnar-type plants in their seedling stage
is quite efficient, because at least 2-year cultivation in
the nursery is required to select columnar-type seedlings
based on their tree form. Long-term cultivation of many
seedlings including the non-columnar type in the nursery
is cost- and labor-intensive for such as fungicide,
insecticide, and associated labor. To reduce these costs,
the establishment of a marker-assisted selection (MAS)system would be valuable for columnar-type apple
breeding.
Several sequence-tagged site (STS) markers linked to
the Co gene have been reported. Hemmat et al. (1997)
developed SSRco from the sequence of random
amplified polymorphic DNA (RAPD) marker OA111000
by bulked segregant analysis (BSA). The SSRco was
mapped on linkage group 10 (LG10) (Maliepaard et al.,1998). A RAPD marker, WB82670, obtained from themapping population of ‘Fuji’ × ‘Tuscan’, was converted
into a sequence characterized amplified region (SCAR)
marker, SCB82670, by amplifying a product of 670 bp(Kim et al., 2003). In another BSA study, a RAPD
marker, S1142682, and an amplified fragment length
polymorphism (AFLP) marker, E-ACT/M-CTA346,
from the mapping population of ‘Spur Fuji’ × ‘Telamon’
were also converted to SCAR markers, designated
SCAR682 (682 bp) and SCAR216 (216 bp), respectively(Tian et al., 2005). Two inter simple sequence repeat(ISSR) markers linked to Co were developed by BSA

from the mapping population of ‘Fuji’ × ‘Telamon’ (Zhu
et al., 2007).
On the other hand, for the last several years, significant
progress in the molecular genetics of apple has been
made, mainly in the development of multi-allelic simple
sequence repeat (SSR) markers and highly informative
genetic linkage maps. The first reference map of apple
was constructed by Maliepaard et al. (1998), including
67 multi-allelic markers (RFLPs, SSRs, and isozyme)
and comprising 17 linkage groups. Liebhard et al. (2002,
2003) and Silfverberg-Dilworth et al. (2006) developed
approximately 300 SSR markers and mapped them ona saturated reference map covering 85% of the genome.
In the report of Silfverberg-Dilworth et al. (2006), 29
SSR markers were mapped on the LG 10; however, it
has been problematic that an integrated genetic linkage
map of these SSR markers and closely linked markers
for important traits, such as Co, has not been available
to date; evaluation and identification of the most efficient
marker in MAS are needed to make progress in columnar
apple breeding.
The objectives of our study were 1) to constructgenetic linkage maps of the Co genomic region forcolumnar apple genotypes important as breeding
materials by using SSR markers and published SCAR
markers and 2) to identify useful DNA markers that will
enable correct and efficient selection of columnar-typeseedlings in a practical columnar apple breeding programin Japan. These DNA markers will be used optically in
the MAS system of columnar-type apple breeding.

Materials and Methods
Plant materials and phenotypic evaluation of growth
characteristics
Thirty-one columnar-type cultivars and/or selections,
10 normal-type cultivars, and 21 F1 populations obtained
from their crosses were used for the phenotypicevaluation of growth characteristics and genetic
mapping, and the evaluation of DNA markers associatedwith the columnar type of apple (Tables 1, 2, and 3).
The growth pattern was visually categorized intocolumnar or non-columnar (normal) in the dormant
season (Fig. 1). Plants possessing a main shoot with little
difference in diameter between the top and base, andsecondary branches with a narrow angle, almost parallelto the main shoot, were scored as columnar according
to the description by Hemmat et al. (1997). In the F1progenies of 187 individuals of three crosses (55 plants

Table 1. Mapping populations for the Co gene and phenotypic evaluation of growth characteristics.

zyMaternal parent

Paternal parent

Tree age at phenotypicevaluation/root system

Number of plants

ColumnarNon-columnar

P value χ2

Fuji

8H-9-45z

5 years/self rooting

55

47

8

0.000 yFuji

5-12786

2 years/JM1

68

30

38

0.332

Underlined cultivars and/or selections were the donors of Co.
Statistically significant difference from the 1 : 1 segregation ratio.

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J. Japan. Soc. Hort. Sci. 78 (3): 279–287. 2009.

Table 2. Marker genotypes of columnar cultivars and/or selections and normal-type cultivars of major ancestors of Japanese apple cultivars andoffspring of ‘McIntosh’ in Japan.
Cultivars and/or
selections

Marker genotype
Phenotype

OriginSCAR682(682 bp)CH03d11

Hi01a03

SCAR216(216 bp)

WijcikColumnar

Bud sport of McIntosh+

126/177y

167/174+

McIntoshNon-columnar

Chance seedling+

126/177

167/174+

TuscanColumnar

Wijcik × Greensleeves+

118/177

174/211+

TrajanColumnar

Golden Delicious × Wijcik+

118/177

174/211+

TelamonColumnar

Wijcik × Golden Delicious+

118/177

174/211+8H-1-13

Columnar

8N-24-57z × 8N-24-49z+

118/177

174/211+8H-2-11

Columnar

8N-24-57z × 8N-24-49z+

118/177

174/211+8H-2-16

Columnar

8N-24-57z × 8N-24-49z+

118/177

174/211+8H-2-26

Columnar

8N-24-57z × 8N-24-49z+

118/177

174/211+8H-4-27

ColumnarUnknown+

118/177

174/211+8H-5-3

Columnar

8N-24-50z × 8N-25-23z+

118/177

174/211+8H-5-29

Columnar

8N-24-50z × 8N-25-23z+

118/177

174/211+8H-6-4

Columnar

8N-24-50z × 8N-25-23z+

118/177

174/211−8H-6-5

Columnar

8N-24-50z × 8N-25-23z+

118/177

174/211+8H-7-15

Columnar

8N-24-50z × 8N-25-23z+

118/177

174/211+8H-9-1

Columnar

Wijcik × Alkmene+

118/177

174/211−8H-9-45

ColumnarUnknown+

118/177

174/211+

8H-12-50

ColumnarUnknown+

118/177

174/211+8C-7-20

Columnar

Wijcik × Empirez+

177/177

174/174+

8C-7-42

Columnar

Wijcik × Empirez+

118/177

174/211+8C-9-111

Columnar

Wijcik × Cox’ Orange Pippin+

177/177

174/211+

8C-14-8

Columnar

8N-24-59z × Wijcik+

177/177

174/174+

8S-60-74

ColumnarUnknown+

118/177

174/211+

5-128

Columnar

8H-1-14 open.

+

122/177

174/174+

5-263

Columnar

8H-6-4 open.

−

118/118

174/174−

5-296

Columnar

8H-6-4 open.

+

118/177

174/187−

5-305

Columnar

8H-6-4 open.

+

122/177

174/211−5-306

Columnar

8H-6-4 open.

+

118/177

174/211−

5-375

Columnar

8H-12-50 open.

+

122/177

174/187+

5-400

Columnar

8H-6-5 open.

+

177/177

174/174+5-8246

Columnar

TSR29T219 × 8H-9-1+

114/177

174/190+5-12786

ColumnarFuji × 8H-2-26+

118/177

174/187+

Ralls Janet

Non-columnar

Chance seedling−

118/118

187/211−

JonathanNon-columnar

Chance seedling−

122/122187/187+

Delicious

Non-columnar

Chance seedling−

118/122187/211−Golden Delicious Non-columnar

Chance seedling−

118/118

211/211−Indo

Non-columnar

Chance seedling−

122/122187/211−

Akane

Non-columnar

Jonathan × Worcester Pearmain−

118/122187/211−

Kitakami

Non-columnar(Worcester Pearmain × McIntosh) × RedGold+

122/177

174/187+

Morioka #61

Non-columnar

Tsugaru × Kitakami−

118/122187/211−

Shinano Red

Non-columnar

Tsugaru × Vista Bella−

118/177

174/211+

8N-24-49 = ‘Golden Delicious’ × ‘Wijcik’; 8N-24-50 = ‘Golden Delicious’ × ‘Wijcik’; 8N-24-57 = ‘Golden Delicious’ × ‘Wijcik’; 8N-24-59 =‘Golden Delicious’ × ‘Wijcik’; 8N-25-23 = ‘Golden Delicious’ × ‘Wijcik’; ‘Empire’ = ‘McIntosh’ × ‘Delicious’.
yUnderlined alleles associated with Co. 8C-14-8 and 5-400 weren’t able to confirm as heterozygous for the columnar locus by marker genotypes
and phenotype segregation data of their offsprings wasn’t available.
zof ‘Fuji’ × 8H-9-45, 68 plants of ‘Fuji’ × 5-12786, and64 plants of ‘Fuji’ × ‘Tuscan’), the growth pattern was
evaluated as shown in Table 1, and used to constructgenetic maps of LG10. Weak selections were performed
twice until investigation of the population of ‘Fuji’ × 8H-

9-45. At the first selection at the end of the first growing
season in 2001, 93 plants with a columnar-type tree form
that grew vigorously were selected from 380 seedlingsin the glass house, and then planted in the nursery. As
a result, the selected plants included both columnar and282

S. Moriya, H. Iwanami, N. Kotoda, S. Takahashi, T. Yamamoto and K. Abe
Table 3. Marker genotypes in the progenies of columnar-type plants.

Cross combination
Fuji × 8H-6-5

zNumber ofcolumnar-type plant

SCAR682

CH03d11+−

Hi01a03177 bpOthers174 bpOthers
0

7

7

0

7

0

7

Sansa × 8H-7-15

11

11

0

11

0

11

0

Jonathan × 8H-7-15

5

5

0

5

0

5

0

Senshu × 8H-7-15

6

6

0

6

0

5

1

Fuji × 8H-7-15

9

8

1

9

0

8

1

Golden Delicious × 8H-9-1

16

16

0

16

0

16

0

Fuji × 8H-9-1

10

10

0

10

0

9

1
1

z

Golden Delicious × 8S-60-74

6

6

0

6

0

5

Senshu × 8S-60-74

17

17

0

17

0

17

0

Ralls Janet × 8S-60-74

2

2

0

2

0

2

0

Jonathan × 8S-60-74

7

6

1

7

0

7

0

Jonathan × 8H-2-26

9

9

0

9

0

9

0

Senshu × 8H-2-26

7

7

0

7

0

7

0

Fuji × 8H-2-26

15

15

0

15

0

15

0

Sansa × 8H-6-4

14

14

0

14

0

14

0

Jonathan × 8H-6-4

5

5

0

5

0

5

0
1

Golden Delicious × 8H-6-4

5

5

0

5

0

4

Senshu × 8H-6-4

19

19

0

19

0

19

0

Total

170

168

2

170

0

165

5

Underlined breeding selections were donors of Co.

DNA isolation
Genomic DNA was extracted from young leaves using
the DNA-isolating system PI-50α (Kurabo Co., Ltd.,
Osaka, Japan). One hundred milligrams of frozen young
leaves were crushed using a machine, SH-48 (Kurabo
Co. Ltd.) and incubated with 1 mL of an isolation buffer(10% PEG#6000, 100 mM Tris-HCl (pH 8.0), 350 mM
sorbitol, and 50 mM EDTA (pH 8.0)) for 30 minutes.
Genomic DNA was then extracted by PI-50α according
to the manufacturer’s instructions.

Fig. 1. Growth pattern of 5-year-old plants of (A) a columnar plantand (B) a non-columnar plant in the progenies of ‘Fuji’ × 8H9-45.

non-columnar plants, because the tree pattern is unstablein one-year-old plants. In the mid-growing season in
2005, 55 plants that grew vigorously were chosen bysecond selection. The other two populations did notundergo any selection. A total of 170 representative F1columnar-type plants of 18 crosses were selected from
intact populations at 4 years old in 1999 and then grafted
onto dwarfing rootstock ‘JM7’ or ‘JM1’, and their
growth pattern was re-evaluated in 2006 to evaluate theutility of DNA markers (Table 3).

SSR analysis
Ten SSR markers located on the LG 10 in the map
constructed by Silfverberg-Dilworth et al. (2006) wereused in this study. These SSR markers were AU223548SSR, CH01f12, CH02a10, CH02c11, CH03d11,
Hi01a03, Hi01b01, Hi03c04p, and Hi22f04, developed
by Liebhard et al. (2002) and Silfverberg-Dilworth etal. (2006). The tenth SSR marker was COL, which was
first developed by Hemmat et al. (1997) as SSRco andwas converted by Gianfranceschi et al. (1998). Since
SSR markers Hi01a03 and Hi01b01 contained an
identical repeat sequence and its flanking region
according to the HiDRAS web site (High-quality Disease
Resistant Apples for a Sustainable Agriculture(HiDRAS), http://users.unimi.it/hidras/, October 30,2008), and their observed PCR products differed onlyin size, Hi01a03 represented these two markers in thisstudy.
SSR analysis was carried out using two methods withslight modifications: the direct fluorescent primer andthe T7-tailed primer methods (Schuelke, 2000; Ziegle

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J. Japan. Soc. Hort. Sci. 78 (3): 279–287. 2009.

et al., 1992). In CH02c11, CH03d11, Hi03c04p, andCOL, the former method was carried out as follows. For
each SSR marker, the forward primer was synthesized
and then conjugated to fluorescent dye, D2 or D3 or D4(Sigma-Aldrich, Tokyo, Japan), at the 5' end. PCR
amplification was performed in a 12 μL solution of
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 eachfluorescent-labeled forward primer and unlabeledreverse primer, 10 ng genomic DNA, and 0.25 units Taq
DNA polymerase (Invitrogen Corp., CA, USA).
Amplification was performed in 35 cycles at 94°C for
1 min (denaturing), 52–55°C for 1 min (annealing), and72°C for 2 min (primer extension). In AU223548-SSR,
CH01f12, CH02a10, Hi01a03, Hi01b01, and Hi22f04,
using the latter method, based on a forward primer foreach SSR primer set, a primer with a 5' tail was designed.
The 5' tail has the partial T7 promoter sequence, 5'AATACGACTCACTATAG-3'. A third T7 primer (5'AATACGACTCACTATAG-3') carries the fluorescentlabel (D2, D3, or D4) at the 5' end that binds to the first
PCR amplicons during the second PCR amplification.
The first PCR amplification was performed in a 12 μL
solution as in the former method, except for the primer
concentration: solutions contained 2 pmol of each of theT7-tailed forward primer and 10 pmol of each unlabeledreverse primer. Amplification was performed in 10
cycles at 94°C for 1 min, 52–55°C for 1 min, and 72°C
for 2 min. After the first amplification, 1 μL of 10 pmol/
μL of the fluorescent-labeled T7 primer was added toeach PCR tube. A second amplification was thenperformed in 35 cycles at 94°C for 1 min, 45°C for 1 min,and 72°C for 2 min. PCR products amplified by bothmethods were separated and detected using the auto
sequencer CEQ 8000 (Beckman-Coulter Inc., Fullerton,
CA, USA). The sizes of the amplicons were determinedon the basis of an internal standard DNA (DNA size
standard kit-400, Beckman-Coulter Inc.) with a CEQGenetic Analyzer (Beckman-Coulter Inc.). The allele
sizes obtained by the T7-tailed primer method contained
about 17–18 bp of tailed sequences.
SCAR marker analysis
Three SCAR markers, designated as SCB82670,
SCAR682, and SCAR216 (Kim et al., 2003; Tian et al.,2005), previously found to be associated with Co, could
generate amplified products for all of the test plants. The
SCAR markers were amplified according to the method
described by Kim et al. (2003) and Tian et al. (2005).
The PCR products were separated on a 1.5% agarose
gel with a 0.5 × TBE buffer, stained with ethidium
bromide, and visualized with ultraviolet light.
The nucleotide sequences of the amplified productsof SCB82670 were determined by direct sequencing using
a DTCS Quick Start Kit for Dye Terminator Cycle
Sequencing (Beckman-Coulter Inc.) and the auto
sequencer CEQ 8000 (Beckman-Coulter Inc.) in

combination with Exo-SAP IT (GE Healthcare UK Ltd.,
Bukinghanshire, England) to purify PCR products
according to the supplier’s protocol.
Linkage analysis
A pseudo-testcross mapping strategy was applied tolinkage analysis (Grattapaglia and Sederoff, 1994).
JoinMap ver. 3.0 (Van Ooijen and Voorrips, 2001) wasused to identify linkage between the columnar trait andDNA markers and to construct a genetic linkage map of
the columnar parent. A logarithm of odds (LOD) scoreof 5.0 was used to define the linkage group. The Kosambi
mapping function was used to convert recombination
units into genetic distances.

Results
Phenotypic evaluation of growth pattern
One-hundred and twenty-three individual progeny ofthe population of ‘Fuji’ × 8H-9-45 and ‘Fuji’ × 5-12786
were clearly categorized into columnar or non-columnar
growth patterns (Table 1). No plants showed theintermediate-type growth habit. The segregation ratio
for the phenotypes fitted the ratio of 1 columnar: 1 noncolumnar for single dominant inheritance in the F1
population of ‘Fuji’ × 5-12786 (χ2 = 0.9, P = 0.33). The
segregation ratio for the phenotypes, however, did not
approximate to 1 columnar: 1 non-columnar in the
population of ‘Fuji’ × 8H-9-45 (χ2 = 27.6, P < 0.01). The
distorted segregation in this population could be due tothe selection of columnar-like plants from the intact
progeny of ‘Fuji’ × 8H-9-45 described in Materials andMethods. The one-year-old population of ‘Fuji’ ×‘Tuscan’ was too young for a phenotypic evaluation to
be made in 2007 and did not show significant differences
regarding the growth pattern. The segregation data forthe growth pattern in the progeny ‘Fuji’ × 5-12786 wasconsistent with the expected ratio, in which the single
dominant gene Co controlled the columnar habit and the
genotype of 5-12786 was heterozygous, Co/co.
Mapping of the Co gene with SCAR and SSR markers
As a result of linkage analysis, linkage maps wereconstructed on LG 10, and the Co gene was mapped on
two populations, ‘Fuji’ × 8H-9-45 and ‘Fuji’ × 5-12786(Fig. 2A, 2B). In ‘Fuji’ × ‘Tuscan’, the map was also
constructed on LG 10, but it did not contain the Co genebecause phenotypic data on the growth pattern were notavailable for this population (Fig. 2C). Six DNA markers,
SCAR682, SCAR216, CH02a10, CH03d11, Hi01a03, andCOL, were successfully mapped on all linkage maps.
The other markers were not mapped on all three linkage
maps since their amplified alleles were homozygous in
specific columnar parent(s) or the amplified productcould not be detected. In the populations of ‘Fuji’ × 8H9-45, the Co gene co-segregated with SCAR682 and the177 bp allele of CH03d11 and, in the population of ‘Fuji’× 5-12786, the Co gene was mapped between SCAR682/

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S. Moriya, H. Iwanami, N. Kotoda, S. Takahashi, T. Yamamoto and K. Abe

CH03d11 and Hi01a03. In the population of ‘Fuji’ ×‘Tuscan’, segregation was detected between SCAR682
and 177 bp of CH03d11, probably due to recombination
between the two markers, and co-segregation between
CH03d11 and Hi01a03 was detected. To confirm theorder between markers and the Co, we drew a graphicalgenotype of all individuals of mapping populations, andno discrepancy was found (data not shown). From allapple cultivars, selections, and F1 plants of the threemapping populations in the study, SCB82670 was
detected only in ‘Tuscan’, TSR29T219, ‘Jonathan’, its
offspring, and ‘Greensleeves’, which was a parent of‘Tuscan’ (data not shown). The nucleotide sequences ofthese amplified products were identical to the sequenceof SCB82670 reported by Kim et al. (2003) in ‘Tuscan’and ‘Greensleeves’, and several single nucleotide
polymorphisms (SNPs) were found in the sequence of‘Jonathan’ (data not shown). In the population of ‘Fuji’× ‘Tuscan’, the recombination frequency and LOD score
between SCB82670 and CH03d11 were 0.484 and 0.01,
respectively. SCB82670 did not show any significant
correlation with the other DNA markers mapped on the
LG 10. The results indicate that SCB82670 was not
located in LG 10; in other words, SCB82670 was not
linked to Co.
Establishing a system for marker-assisted selection using
SCAR682, CH03d11, and Hi01a03
To verify the utility of DNA markers mapped on the
Co region, the marker genotype was determined on 33
columnar-type cultivars and/or selections and 7 noncolumnar-type cultivars, which were ancestral cultivarsof apple breeding in Japan and 3 non-columnar-type
cultivars in Japan, which were offspring of ‘McIntosh’(Table 2). Four markers, SCAR682 and CH03d11 fromthe distal side and Hi01a03 and SCAR216 from the basalside, were chosen from those mapped around the Co
region. A 174 bp fragment of primer Hi01a03 wasamplified in all columnar-type cultivars and selectionsand ‘McIntosh’ but not in ancestral non-columnar apples.
The amplified products of 682 bp for SCAR682 and177 bp for CH03d11 were detected in almost allcolumnar genotypes except for one selection, 5-263, but
the products were not found in ancestral non-columnar
apples. On the other hand, a fragment of primer SCAR216
was not detected in columnar-type selections of 8H-64, 8H-9-1, and their offspring 5-263, 5-296, 5-305, and5-306, although it was found in most of the columnar
apples. The fragment of primer SCAR216 was amplified
even in the non-columnar apple, ‘Jonathan’, in addition
to ‘McIntosh’ and its offspring. These results indicatethat three markers, SCAR682, CH03d11, and Hi01a03,
are candidates for the identification of columnar apple
seedlings in a MAS system in apple breeding. In theoffspring of ‘McIntosh’, ‘Kitakami’, and ‘Shinano Red’showed the same alleles associated with Co for 4 and 3
markers, respectively. On the other hand, ‘Morioka #61’
did not show the same allele associated with Co. In 5263, we confirmed that recombination events should
occur between these markers and the Co gene because
the Co gene was mapped between CH02c11 and COL
in the F1 population of ‘Fuji’ × 5-263 (data not shown);i.e., the Co gene was located on LG 10 of 5-263.
Graphical genotyping of the Co region of several
columnar apples is shown in Figure 3. The Co region
derived from ‘Wijcik’ is the smallest on 5-263 of alltested cultivars/selections.
To investigate the performance of the markers
SCAR682, CH03d11, and Hi01a03 in MAS for columnar
trait, 170 columnar-type plants obtained from 18 crosses
were tested for their marker genotypes. A 177 bp alleleof CH03d11, characteristic of the columnar phenotype,Fig. 2. Linkage maps of the regions around the Co gene for columnar-type cultivar or selections. The genetic maps of (A) 8H-9-45, (B) 5-12786,
and (C) ‘Tuscan’ were aligned using microsatellite and SCAR markers successfully mapped on all maps. Map distances are shown on theleft (cM). Co loci are listed in bold on the right. Observed fragment lengths are shown with SSR names as bp. Segregation distortion isindicated by significance level P of the χ2 test: *: 0.05, **: 0.01, ***: 0.005, ******: 0.0001

J. Japan. Soc. Hort. Sci. 78 (3): 279–287. 2009.

Fig. 3. Graphical genotyping around the Co gene on linkage group10 in several columnar breeding materials. Underlined cultivars
and/or selections showed columnar growth habit. It was notknown whether 8N-24-49, 8N-24-50, 8N-24-57, and 8N-25-23
showed a columnar growth habit. Alleles of each SSR are shownin bp. The genetic distances were according to Figure 2B (512786).

was detected in all 170 plants (Table 3), suggesting that
it is one of the most tightly linked DNA markers to Co.
Fragments of SCAR682 and 174 bp of Hi01a03 were
amplified in 168 and 165 plants, respectively;
recombination occurred between the markers and the Co
gene in 2 plants for SCAR682 and in 5 plants for Hi01a03.
These 7 plants were independent of each other. The
result showed that CH03d11 was the most reliable
marker to distinguish columnar and non-columnar
individuals for the MAS system in apple breeding.
SCAR682 and Hi01a03 could also ensure successful
selection for columnar individuals when these markers
were applied to MAS, although a small degree of error
would be expected when distinguishing columnar plants.

Discussion
Relationship between the Co gene and DNA markers
In previous studies, phenotypic evaluation todetermine whether the tree growth pattern was columnar
was conducted using self-rooting plants that were more

285

than two years old (Hemmat et al., 1997; Kim et al.,
2003; Tian et al., 2005; Zhu et al., 2007); however, Kenis
and Keulemans (2007) conducted a study in which they
replicated two types of plants on the same genotype,which was self-rooted and grafted onto the ‘M.9’
rootstock. They revealed that grafted plants showed more
distinct differences on the main axis traits than selfrooted plants and concluded that the root systemsignificantly affects all the traits determining treearchitecture. To evaluate the phenotype correctly, we
conducted an evaluation using plants grafted onto thedwarfing rootstock of ‘JM1’ in the population of ‘Fuji’× 5-12786 and well-established five-year-old rooting
plants in the population ‘Fuji’ × 8H-9-45; we did notconduct a phenotypic evaluation of the one-year-old
infant population ‘Fuji’ × ‘Tuscan’. Thus, we consideredthat our phenotypic evaluation retained a high level ofconfidence for mapping the Co gene.
In the population of ‘Fuji’ × 8H-9-45 and ‘Fuji’ × 512786, we could not find the intermediate-type plants
described by Hemmat et al. (1997) and Kim et al. (2003).
On the other hand, in ‘Fuji’ × ‘Tuscan’, because all plants
were young and small, unclear tree architecture, such asthat of the intermediate type, was found. In this context,
an intermediate phenotype could be due to its fragile
growth.
To confirm the linkage of published DNA markers to
the Co gene, we constructed three linkage maps of apple
LG 10 (Fig. 2). The Co gene was successfully mapped
between the investigated markers. In the report of Tianet al. (2005), SCAR682 was mapped in the vicinity ofCo than CH03d11; the distance was 2.9 and 3.9 cM,
respectively. The inconsistency in the marker order
between our map and the ‘Telamon’ map could be due
to several reasons, such as the accuracy of phenotypicevaluation, unintentional mistake in marker genotyping,
multiple recombinations occurred in adjacent regions,and differences in the software used in the linkage
analysis.
SCB82670, reported as the DNA marker most tightly
linked to the Co gene (Kim et al., 2003), was not linked
to the Co gene in our results; furthermore, it was not
located on LG 10. Kim et al. (2003) developed SCB82670
by bulked segregant analysis on the F1 population of‘Fuji’ × ‘Tuscan’. We also produced the F1 populationof ‘Fuji’ × ‘Tuscan’ in this study and tried to confirm
the linkage of SCB82670 to the Co gene but failed to do
so. In previous reports, SCB82670 was detected only in‘Tuscan’ (Fernandez-Fernandez et al., 2008; Kenis andKeulemans, 2007; Tian et al., 2005). Since the parentage
of ‘Tuscan’ is ‘Wijcik’ as the maternal columnar-typeparent and ‘Greensleeves’ as the paternal non-columnar
parent, we tried to detect SCB82670 on these cultivars.
As a result, SCB82670 was detected in ‘Greensleeves’and ‘Tuscan’ but not in ‘Wijcik’ (data not shown), andtheir nucleotide sequences were identical to the original
sequence of SCB82670, suggesting that the locus

286

S. Moriya, H. Iwanami, N. Kotoda, S. Takahashi, T. Yamamoto and K. Abe

SCB82670 was inherited by ‘Greensleeves’.
We confirmed that the order of SSRs on each LG 10
was reasonable by drawing graphical genotypes of all
individuals. The order of SSRs on each LG 10 was
consistent with the apple reference map constructed by
Silfverberg-Dilworth et al. (2006) and N’Diaye et al.
(2008). The SSR marker Hi01a03, which was developedby Silfverberg-Dilworth et al. (2006), has not been
investigated to determine whether it links to the Co gene.
In our results, 177 bp of CH03d11 and SCAR682 wereco-segregated in the populations of ‘Fuji’ × 8H-9-45 and‘Fuji’ × 5-12786, and their map distances from the Co
gene were 0 cM and 1.4 cM, respectively. On the other
hand, in the population of ‘Fuji’ × ‘Tuscan’, 177 bp ofCH03d11 was co-segregated with 174 bp of Hi01a03
mapped on the opposite side of the Co gene in the othertwo populations. Furthermore, marker genotypes in theprogenies of columnar-type plants (possessing Co)
showed co-segregation of 177 bp of CH03d11, andrecombination between Co occurred for 2 and 5
independent plants in SCAR682 and 174 bp of Hi01a03,
suggesting that SCAR682 and Hi01a03 are located on
different sides of Co (Table 3). These results indicatedthat 177 bp of CH03d11 was linked to the Co gene more
tightly than SCAR682 and that Hi01a03 was the mosttightly linked to Co on the other side. The reliability ofthese results will be increased by mapping Co on the
population of ‘Fuji’ × ‘Tuscan’; for this objective, further
investigation is required. Fernandez-Fernandez et al.
(2008) mapped Co between CH03d11 and CH02c11.
This might be an artifact because of low marker density
in this region, and accuracy in the marker genotyping
and the phenotypic evaluation. Thus, the relationshipbetween the Co and molecular markers was slightly
different in the respective reports, causing confusion
about the precise location of Co, which retards progressin advanced studies of Co, such as map-based cloning.
To reveal the precise location of Co, it is important todevelop unified criteria for phenotypic evaluation,
increasing marker density in this region, and using alarge number of plants.
MAS in columnar-type apple breeding
With regard to LG 10, some quantitative trait loci
(QTL) have been identified in previous studies. QTL
concerning fruit vitamin C content, harvest date,diameter, weight, firmness, and sugar content weredetected in the vicinity of the region surrounding
CH03d11, i.e., close to the locus of the columnar habit,in the population of ‘Telamon’ × ‘Braeburn’ (Davey etal., 2006; Kenis et al., 2008). In another study, QTL
affecting fruit weight was also identified on LG 10 in
the population of ‘Fiesta’ × ‘Discovery’ (Liebhard et al.,2003). On the other hand, the columnar-type apple,
especially ‘Wijcik’, had various unsuitable characteristics, such as low sugar content and soft fruit flesh. These
results suggested that LG 10 was concerned with diverse

traits of apple, including tree architecture and fruit
quality, and the chromosome region associated with the
Co gene could be linked to such poor characteristics;
therefore, to develop a columnar-type apple with highfruit quality, the replacement of the Co gene-associatedchromosome region with that of cultivars having
excellent fruit quality is considered to be a possible
approach.
When we were trying to use DNA markers for MAS
in a practical breeding program, it was an important step
to develop markers not only linked tightly to usefulgenes, but marker alleles associated with useful traits
are rare in breeding materials (Tanaka, 2006). The
columnar-type-associatedallelesof
SCAR682,
CH03d11, and Hi01a03 were “+” (682 bp), 177 bp, and174 bp, respectively. On the other hand, ‘Delicious’,‘Golden Delicious’, ‘Indo’, ‘Jonathan’, ‘Ralls Janet’, and‘Akane’, which were the ancestral parental cultivars used
reproducibly as parents in apple breeding in Japan(Kitahara et al., 2005), did not have the same alleles(Table 2). In 170 columnar-type seedlings from 18
crosses, 177 bp of CH03d11 showed perfect cosegregation to the columnar trait, while recombination
seemed to occur between SCAR682 and the Co gene in
two individuals (Table 3). These results suggest that
CH03d11 is the most tightly linked DNA marker to the
Co gene on the distal side and the best marker to employ
for MAS in the columnar-type apple breeding programof Japan. On the basal side of the Co gene region,
Hi01a03 is the most tightly linked DNA marker;
however, based on the mapped position and results ofthe detection of Hi01a03 in 170 columnar-type seedlings
obtained from 18 crosses, Hi01a03 seems to be slightly
inferior to CH03d11 in the detection of the columnar
trait in the MAS system of apple breeding.
When offspring of ‘McIntosh’ (non-columnar type)are used to cross with a Co donor, we should pay attention
to their marker genotype in MAS system. ‘Kitakami’,e.g., had the same alleles associated with Co on theinvestigated markers (Table 2); however, the moreadvanced breeding selection ‘Morioka #61’ did not havesuch an allele, so we could apply ‘Morioka #61’ in MAS
easily. ‘Shinano Red’ had the same alleles associatedwith Co on CH03d11, Hi01a03, and SCAR216, but did
not have SCAR682, indicating that SCAR682 enables theuse of ‘Shinano Red’ in the MAS system. It wasconsidered that the lack of SCAR682 in ‘Shinano Red’
was the result of recombination between SCAR682 andCH03d11 on the chromosome of ‘McIntosh’ in thebreeding process.
In Figure 3, the chromosome structures of our mostadvanced breeding selections, 5-8246 and 5-12786, areshown. 5-8246, which is scab-resistant and has high fruit
quality, replaced the chromosome on the basal side of
the Co gene. 5-12786, which has a high sugar content,
retained the ‘Wijcik’ chromosome around the Co region.
In these two selections, Co-associated alleles of ‘Wijcik’J. Japan. Soc. Hort. Sci. 78 (3): 279–287. 2009.

on CH03d11 and Hi01a03 remained, indicating that, if
5-8246 or 5-12786 may be used as Co donors in thebreeding program, the detection of CH03d11 and
Hi01a03 will allow us to select seedlings expressing acolumnar phenotype. On the other hand, a smaller
chromosome region around Co was found in 5-263. In
this material, homozygous alleles on tested SSR markers
or no amplified fragments on SCAR markers wereobserved, indicating the requirement of closer genetic
markers when using this material in a MAS system.
In addition, multiplex PCR of CH03d11 and Hi01a03
is an economical method that will achieve the precise
selection of columnar seedlings, thus saving both timeand money. The identification of the Co gene (i.e., themutation point of ‘McIntosh’) and development of a
DNA marker including that point will ultimately lead toan accurate selection method in MAS of columnar-typeapple breeding. Map-based cloning is one way to identify
the Co gene. In further studies, CH03d11, SCAR682, andHi01a03 could be DNA markers at the starting point of
the positional cloning of Co.

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