This article was published in the above mentioned Springer issue.The material, including all portions thereof, is protected by copyright;all rights are held exclusively by Springer Science + Business Media.

The material is for personal use only;commercial use is not permitted.

Unauthorized reproduction, transfer and/or usemay be a violation of criminal as well as civil law.

ISSN 0014-2336, Volume 173, Number 3

Molecular mapping of a gene conferring resistanceto Aphanomyces root rot (black root) in sugar beet(Beta vulgaris L.)

Kazunori Taguchi • Kazuyuki Okazaki •

Hiroyuki Takahashi • Tomohiko Kubo •

Tetsuo Mikami

Received: 9 December 2009 / Accepted: 16 February 2010 / Published online: 4 March 2010

� Springer Science+Business Media B.V. 2010

Abstract Caused by Aphanomyces cochlioides

Drechsler, Aphanomyces root rot is a serious disease

of sugar beet (Beta vulgaris L.), for which sources of

resistance are scarce. To identify the segregation

pattern of the rare resistance trait found in Japanese

sugar beet line ‘NK-310mm-O’, F1 and BC1F2 see-

dings, drawn from a cross between ‘NK-310mm-O’

and susceptible line ‘NK-184mm-O’, were inoculated

with zoospores and their survival evaluated in the

greenhouse. Resistance segregation followed was that

of a single dominant gene, which was designated Acr1

(Aphanomyces cochlioides resistance 1). Molecular

markers tightly linked to Acr1 were identified by

bulked segregant analysis of two BC1F2 populations.

Fourteen AFLP markers linked to Acr1 were identified,

the closest located within ±3.3 cM. Three F5 lines and

two BC2F1 lines, selected on the basis of their Acr1-

AFLP markers, were tested for their resistance to

Aphanomyces root rot in a highly infested field.

Results indicated that Acr1 conferred significant

resistance to Aphanomyces root rot at the field level.

Based on its linkage with CAPS marker tk, a repre-

sentative marker for chromosome III, Acr1 was

located on this chromosome. The clear linkage between

tk and Rhizomania resistance trait Rz1, suggests

the clustering of major disease resistance genes on

chromosome III.

Keywords Aphanomyces cochlioides �Sugar beet � AFLP � Marker assisted selection �Resistance � Linkage analysis

Introduction

Aphanomyces cochlioides Drechsler, a soil-borne

phytopathogenic Peronosporomycete, causes root rot

in spinach (Spinacia oleracea L.), sugar beet (Beta

vulgaris L.), and other members of the Chenopodi-

aceae. In sugar beet, A. cochlioides is the causal agent

of both an acute seedling disease (damping-off) and a

chronic rot of the mature root (black root) (Schneider

and Whitney 1986). As because the disease’s prev-

alence and severity in the field is exacerbated by wet

soil conditions and high temperatures, outbreaks of

Aphanomyces root rot have become a serious prob-

lem in many sugar beet growing areas, especially

those of Japan. Aphanomyces root rot, when severe,

can lead to complete plant degeneration death and

drastically reduced sugar yield. While although

seedling damping-off can be controlled by fungicide

application to the seed at planting, chemicals and

improved cultivation methods have not proven an

K. Taguchi (&) � K. Okazaki � H. Takahashi

Memuro Upland Farming Research Station, National

Agricultural Research Center for Hokkaido region,

(NARCH), Hokkaido, Japan

e-mail: ktaguchi@naro.affrc.go.jp

T. Kubo � T. Mikami

Laboratory of Genetic Engineering, Research Faculty

of Agriculture, Hokkaido University, Hokkaido, Japan

123

Euphytica (2010) 173:409–418

DOI 10.1007/s10681-010-0153-8

Author's personal copy

efficient way to control Aphanomyces root rot. An

alternative mode of disease control is to use cultivars

in which resistance has been introduced from resis-

tant germplasm, but this requires the identification of

appropriate germplasm. Such identification would be

enhanced if the mode of inheritance were known and

genetic marker(s) tightly linked to the resistant trait

were identified, making it possible to conduct

marker-assisted selection (MAS), rather than labori-

ous phenotype-based selection.

Little is known about the genetic basis of

resistance to Aphanomyces-caused diseases. Early

research by the USDA suggested that the resistance

trait to behave in a dominant manner, since because

experimental hybrids exhibited resistance (Bochstahler

et al. 1950). However, given that environmental

factors can strongly influence phenotypic evaluation

of resistance in the field, the progress of these genetic

studies in genetic improvement was slow (Doxtator

and Finkner 1954). A recent QTL analysis of

Aphanomyces root rot resistance using a field where

the disease existed at highly and uniformly infectious

levels (Taguchi et al. 2009), showed resistance to be

associated with a major QTL located on chromosome

III. However, such fields are not always available,

and environmental effects cannot be excluded when

field trials are conducted. A greenhouse screening

method for determining seedling resistance to

damping-off by Aphanomyces rot (Schneider 1954;

Coe and Schneider 1966; Schneider 1978), which

contributed to recent advances in Aphanomyces

resistance breeding programs throughout the world

(Panella 2005), was altered to evaluate resistance of

sugar beet root to Aphanomyces rot (Okazaki et al.

2005). Knowing whether Aphanomyces root rot

resistance expressed in the field can also be expressed

in the greenhouse and whether the same gene is

involved, is of interest, for if such were the case, field

and greenhouse screening could complement each

other. This is an important issue for sugar beet

disease resistance breeding programs, because differ-

ing levels of resistance between field and greenhouse

tests has been noted in the case of Rhizomania

resistance (Grimmer et al. 2007).

The public molecular markers on the nine chro-

mosomes of sugar beet are not sufficiently dense to

conduct MAS. Bulked segregant analysis (BSA)

(Michelmore et al. 1991) is an alternative method

to obtain markers tightly linked to the desired genetic

trait, provided that phenotypic evaluation of a plant

strictly correlates with its genotype. To obtain

molecular markers tightly linked to Aphanomyces

root rot resistance, we believe it most effective to

combine BSA with greenhouse tests, which can

discriminate between resistant/non-resistant plants

more accurately than field trials. In the present

report, we show for the first time that the Aphano-

myces root rot resistance derived from Japanese sugar

beet line ‘NK-310mm-O’ behaves as a dominant and

monogenic trait in greenhouse tests. The chromo-

some III-borne AFLP linkage markers obtained by

BSA, suggest that resistance to Aphanomyces root rot

is governed by the same gene in both field and

greenhouse. We also show the possibility that Apha-

nomyces root rot resistance can be introgressed into

sugar beet varieties through MAS, using AFLP

markers.

Materials and methods

Plant materials

Sugar beet crosses completed in this study are

diagrammed in Fig. 1. All sugar beet lines used in

this study were developed at the National Agricul-

tural Research Center for Hokkaido Region

(NARCH) in Japan. A resistant maintainer line

for Owen cytoplasmic male sterility (O-type),

‘NK-310mm-O’, and a susceptible O-type line

‘NK-184mm-O’ are self-incompatible, and self-fertile,

respectively. A single F1 plant was obtained by the

cross between a single ‘NK-310mm-O’ as a female

parent and a single ‘NK-184mm-O’ as pollen donor.

Ten of the F2 plants were used as pollen donors to a

hand-emasculated ‘NK-184mm-O’ plant to generate

56 BC1F1 plants to be used for marker-assisted

selection for the qAcr1 region (Taguchi et al. 2009).

Two of the BC1F1 plants were selfed to obtain BC1F2

populations, which were genotyped using the same

set of the markers. Using marker-assisted selection,

two BC2F1 genomic backgrounds were generated by

backcrossing lines with positive-effect segments to

‘NK-184mm-CMS’. F2 plants were propagated by

controlled self-pollination using the single-seed

descent method to the F5 generation, at which stage

genotyping was undertaken with the same set of

markers as for positive-effect segments.

410 Euphytica (2010) 173:409–418

123

Author's personal copy

Inoculation of the zoospores and evaluation

of the resistance

Inoculation followed the artificial inoculation method

of Okazaki et al. (2005). Aphanomyces cochlioides

strain AP-16, isolated from a field in Hokkaido

prefecture, Japan, was grown on potato dextrose agar

for 7 days at 25�C in a 90 mm Petri dish. Seven

8-mm diameter disks were then aseptically removed

from the margin of the mycelium and placed in a

90 mm Petri dish flooded with 20 ml of sterile

deionized water. After 24 h’ growth in the dark at

25�C, released zoospores were counted with a

hemocytometer.

Each of the parental, F1 and two of the BC1F2

populations were seeded in paper pots (19 mm

diameter and 130 mm height, Nippon Beet Sugar

Mfg. Co., Ltd.) on April 10, 2004, and grown in a

greenhouse for about 40 days. Seedling-bearing pots

were then soaked continuously for a week with a

zoospore suspension (3.0 9 104 zoospores per plant).

After inoculation (mid-May), each seeding was then

transplanted (mid-May) into a Wagner pot (1/5000 a),

allowed to grow in the greenhouse at 15�C for

30 days, and thereafter at [20�C. In addition to

regular watering, plants received extra water

(300 ml pot-1 day-1) for the first 10 days after

transplanting and on day 30 after transplanting, to

promote infection. Roots were harvested on August

21. Visual symptoms of Aphanomyces rot were rated

from 0 (no symptoms) to 5 (fully decayed) (Taguchi

et al. 2009); plants were considered as ‘‘healthy’’

when the rating was in the range of 0–2 and ‘‘rotted’’

when in the range of 3–5.

DNA isolation and genotyping with molecular

markers

Total cellular DNA was extracted from fresh leaves

collected on July 1, according to the procedure of

Roger and Bendich (1988). Amplified Fragment

Length Polymorphism (AFLP) was detected using an

AFLP Analysis System I (Invitrogen). The restriction

endonucleases EcoRI and MseI were used in this

analysis. The adapter-ligated DNA was pre-amplified

with primers having a single selective nucleotide. For

selective amplification, EcoRI-NNN and MseI-NNN

primers were employed. The amplified products were

electrophoresed in a High Efficiency Genome Scan-

ning (HEGS) system (Kawaguchi et al. 2001; Hori

et al. 2003; Kikuchi et al. 2003) using discontinuous

non-denatured acrylamide gel and TBE buffer. The

gels were scanned after staining with Vistra Green I

(GE Healthcare) and photographed under a UV

transilluminator (ATTO). Genomic regions for

Cleaved Amplified Polymorphic Sequence (CAPS)

analysis were amplified by using primer sets (Mohring

et al. 2005). PCR products were digested with one of

NK-310mm-O× NK-184mm-O

F1 (single seed)

F2 population F2-7 × NK-184mm-O

self

SSD

BC1F1

(QTL analysis)

self

BC1F2 (PopulationA&B)

(Linkage analysis)

Single-Seedd (SSD)

F5-1, F5-2, F5-3

F4 (n=80)

F3 (n=80)

SSD

MAS(n=192)

Descen

MAS (n=56)

(QTL analysis)

NK-184mm-CMS(BC5F1) ×

BC2F1-1, BC2F1-2

MAS (n=96, n=94)

BC1F2-1&BC1F2-2F5(n=80)

MAS (n=80)

Fig. 1 Scheme of crosses

used to generate the

populations studied.

Abbreviations: CMScytoplasmic male sterility,

O non-restorer genotype

Euphytica (2010) 173:409–418 411

123

Author's personal copy

the 13 restriction endonucleases: HaeIII, HhaI, TaqI,

HapII, MboI, AfaI, XspI, AluI, AccII (Takara Bio,

Ohtsu, Japan), TspEI (TOYOBO, Osaka, Japan), MseI,

HpyCh4IV, or NlaIII (New England BioLabs). The

resultant fragments were electrophoresed in a 2%

agarose gel.

Bulked segregant analysis and linkage analysis

Bulked segregant analysis (BSA) was performed, as

described by Michelmore et al. (1991). Five individ-

ual plants were bulked according to their resistant

(healthy) or susceptible (rotted) phenotype. DNA

samples were pooled after pre-amplification. A set of

resistant and susceptible bulks were compared on the

basis of the AFLP profiles generated with 2600

primer combinations. Integrity of the polymorphic

bands was tested by confirming the presence/absence

of the bands in individual DNA samples drawn from

the bulks.

Map distance in centi Morgans (cM) were calcu-

lated from recombination frequencies using the

Kosambi function (Kosambi 1944). The derived

AFLP and CAPS markers were grouped at a

logarithm of odds (LOD) threshold of 3.0 and a

maximum distance of 30 cM. Marker order in each of

the linkage groups was verified using MAPMAKER/

EXP ver 3.0 (Lander et al. 1987).

Evaluation of Aphanomyces root rot resistance

in the field

Seedlings were transplanted (May 18, 2005) to an

area of high Aphanomyces oomycetes density chosen

in an Aphanomyces-infested field in Ikeda, Hokka-

ido, Japan, known to have housed epidemic levels of

typical Aphanomyces root rot for over 5 years. Tests

were implemented in a complete randomized block

design with four replications. Each of the parental,

F1, F5 and BC2F1 lines consisting of 104 plants were

evenly distributed into the four plots (i.e. 26 plants/

plot). Sugar beet roots were harvested by hand

(October 6), and disease symptoms at the whole-

plant level assessed according to an index of root rot

severity (Taguchi et al. 2009) ranging from 0 (no

symptoms) to 5 (fully decayed). Data were averaged

across replications.

Results

A single dominant gene is involved

in the resistance to Aphanomyces root rot

Field-level resistance of Japanese sugar beet line

‘NK-310mm-O’ to Aphanomyces root rot appears to

involve the QTL designated as qAcr1 (Taguchi et al.

2009). We wished to establish whether the resistance

trait expressed by ‘NK-310mm-O’ in the field was

also expressed in the greenhouse and, if so, to

identify molecular markers linked to the resistance

gene for further study and breeding purposes.

Whereas roots of Aphanomyces-inoculated ‘NK-

184mm-O’ sugar beet were fully rotted, those of

similarly inoculated ‘NK-310mm-O’ and F1 (‘NK-

310mm-O’ 9 ‘NK-184mm-O’) plants were healthy

(Fig. 2, Table 1), indicating that resistance was also

expressed at the greenhouse level, and was governed

by one or more dominant genes.

The genetic behaviour of the resistance trait was

then investigated in segregating populations. The

above-mentioned F2 plants were used to develop BC1

populations, with ‘NK-184mm-O’ being used as a

recurrent parent; however, number of plants obtained

was insufficient for the genetic analysis. Therefore,

we selfed the BC1 plants to obtain sufficient BC1F2

seeds. Those BC1F1 plants heterozygous for the

interval between AFLP marker e45m23-4 and CAPS

marker tk, which bears the qAcr1 (Taguchi et al.

2009; see Fig. 4), were selected before selfing,

because the chromosomal region containing the

qAcr1 was expected to be involved in greenhouse

level resistance.

Two BC1F2 populations, A and B, consisting of 96

and 94 plants, respectively, were subjected to green-

house tests. In the population A, 73 plants remained

healthy (root rot intensity B 2, mean = 0.4), and 23

plants were rotted, (3 B root rot intensity B 5,

mean = 4.8) (Fig. 3). Comparatively, in population

B, 70 plants remained healthy (root rot intensity B 1,

mean = 0.3), and 24 plants were rotted, (3 B root rot

intensity B 5, mean = 4.6). In both populations,

segregation of healthy and rotted plants fitted the

expected 3:1 ratio for monogenic inheritance

(Table 1), leading us to tentatively designate this

single dominant gene Acr1.

412 Euphytica (2010) 173:409–418

123

Author's personal copy

Fig. 2 Aphanomyces root

rot symptoms observed

under greenhouse

inoculation.

(A: NK-310mm-O,

B: F1, C: NK-184mm-O,

D: Population A)

Table 1 Phenotypic disease ratios for reaction of sugar beet to Aphanomyces root rot under greenhouse assessment conditions

(2004)

Materials No. of plants Observed segregation ratios v2 (ratio) df P-value

Resistant Susceptible Total Resistant Susceptible

NK-310mm-O 10 (0.0) 0 (–) 10 1.00 0.00 0.00 (1:0) 1 P [ 0.99

NK-184mm-O 0 (–) 9 (5.0) 9 0.00 1.00 0.00 (0:1) 1 P [ 0.99

F1 10 (0.0) 0 (–) 10 1.00 0.00 0.00 (1:0) 1 P [ 0.99

Population A 73 (0.4) 23 (4.8) 96 0.76 0.24 0.06 (3:1) 1 0.9 [ P [ 0.7

Population B 70 (0.3) 24 (4.6) 94 0.74 0.26 0.01 (3:1) 1 0.9 [ P [ 0.7

‘(–)’ is mean of resistance scores (0–5 scale: 0 = healthy and 5 = complete root rot)

v2 value and probability compare to the segragation of monogenic resistance model

Population A

n=96

0

10

20

30

40

50

60

Disease indices

Num

ber

of p

lant

s

Population B

n=94

0

10

20

30

40

50

60

0 1 2 3 4 5 0 1 2 3 4 5

Disease indices

Num

ber

of p

lant

s

Fig. 3 Frequency

distribution for

Aphanomyces root rot

indices in B1F2 population

A and B of ‘NK-310mm-

O’ 9 ‘NK-184mm-O’

Euphytica (2010) 173:409–418 413

123

Author's personal copy

Molecular markers linked to the Acr1 gene

To isolate AFLP markers linked to the Acr1, popu-

lation A was used for bulked segregant analysis

(BSA) (Michelmore et al. 1991). Four sets of bulked

DNA were used, each set consisting of DNA samples

from five healthy and five rotted plants. Of the 18700

AFLP fragments amplified with 1440 primer combi-

nations, 19 were polymorphic between the bulked

samples in all four sets. After confirming the

presence/absence of the polymorphic bands in the

healthy/rotted individuals in all the bulks, we finally

identified 13 polymorphic AFLP markers (e19m4-9,

e14m40-7, e14m6-9, e10m47-9, e10m46-7, e19m13-

6, e11m36-8, e18m14-9, e19m6-8, e20m6-7, e20m7-

10, e20m13-6, and e10m37-9). In addition to these 13

markers, we expected that five AFLP markers and

one CAPS marker near qAcr1 (e2m38-82, e2m42-9,

e45m26-10, e45m23-4, e30m44-5 and tk) (Taguchi

et al. 2009) to also be linked to the Acr1 locus. A total

of 18 AFLP markers and one CAPS marker were

subjected to segregation analysis using population A.

Genetic linkage between the Acr1 and each of the 19

markers was assessed (Table 2) and mapped with

Table 2 Linkage analysis for Aphanomyces root rot resistance in sugar beet: association between the Acrl locus and DNA markers

(2004)

Marker(a) Marker(b) No. of plants (a:b) v2 RV ± SE cM ± SE

?? ?- -? -- Total

Population A

e30m45-5 Acrl 65 12 8 11 96 1.39 25.36% ± 5.28% 27.94 ± 5.30 a

e45m23-4 Acrl 66 9 7 14 96 0.50 19.12% ± 4.55% 20.14 ± 4.56 a

e10m37-9 Acrl 71 6 2 17 96 1.39 9.17% ± 3.12% 9.27 ± 3.12 BSA

e20m6-7 Acrl 73 4 0 19 96 1.39 4.46% ± 2.16% 4.47 ± 2.16 a

e20m13-6 Acrl 73 4 0 19 96 1.39 4.46% ± 2.16% 4.47 ± 2.16 BSA

e45m26-10 Acrl 73 2 0 21 96 0.50 2.17% ± 1.51% 2.17 ± 1.51 a

Acrl e2m38-82 71 2 2 21 96 0.06 4.32% ± 2.13% 4.33 ± 2.13 a

Acrl e2m42-9 71 2 2 21 96 0.06 4.32% ± 2.13% 4.33 ± 2.13 a

Acrl tk 70 3 2 21 96 0.06 5.39% ± 2.38% 5.41 ± 2.38 a

Acrl e11m36-8 68 5 5 18 96 0.06 11.22% ± 3.45% 11.41 ± 3.46 BSA

Acrl e18m14-9 68 5 5 18 96 0.06 11.22% ± 3.45% 11.41 ± 3.46 BSA

Acrl e19m6-8 68 5 5 18 96 0.06 11.22% ± 3.45% 11.41 ± 3.46 BSA

Acrl e20m7-10 68 5 5 18 96 0.06 11.22% ± 3.45% 11.41 ± 3.46 BSA

Acrl e19m13-6 68 5 6 17 96 0.06 12.53% ± 3.66% 12.81 ± 3.66 BSA

Acrl e10m46-7 68 5 6 17 96 0.06 12.53% ± 3.66% 12.81 ± 3.66 BSA

Acrl e10m47-9 68 5 6 17 96 0.06 12.53% ± 3.66% 12.81 ± 3.66 BSA

Acrl e14m6-9 67 6 6 17 96 0.06 13.64% ± 3.82% 14.00 ± 3.83 BSA

Acrl e14m40-7 68 5 7 16 96 0.06 13.90% ± 3.86% 14.28 ± 3.86 BSA

Acrl e19m4-9 67 6 10 13 96 0.06 19.53% ± 4.60% 20.63 ± 4.62 BSA

Population B

e30m45-5 Acrl 58 11 12 13 94 0.13 27.85% ± 5.61% 31.42 ± 5.64 a

e45m26-10 Acrl 69 2 1 22 94 0.01 3.24% ± 1.86% 3.25 ± 1.86 a

Acrl e2m38-82 64 6 3 21 94 0.01 9.75% ± 3.25% 9.88 ± 3.25 a

Acrl e2m42-9 65 5 3 21 94 0.01 8.69% ± 3.06% 8.78 ± 3.07 a

Acrl tk 63 6 2 22 93 0.03 8.61% ± 3.07% 8.69 ± 3.07 a

Acrl e11m36-8 62 8 3 21 94 0.01 11.85% ± 3.59% 12.08 ± 3.60 BSA

Acrl e14m40-7 61 9 6 18 94 0.01 16.82% ± 4.30% 17.50 ± 4.31 BSA

RV recombination value, SE standard errora Taguchi et al. 2009, BSA:AFLP marker developped from bulked segregant analysis

414 Euphytica (2010) 173:409–418

123

Author's personal copy

MAPMAKER (Fig. 4). In population A, AFLP

marker e45m26-10 was very closely linked (2.2 cM

distant) to Acr1 (map distance is 2.2 cM), while two

further AFLP markers e2m38-82 and e2m42-9 were

also closely linked (both 4.3 cM distant) to Acr1.

Analysis in population B of the linkage between Acr1

and these three markers (Fig. 5), showed their

arrangement to be identical to that in population A.

The linkage map includes the AFLP marker e11m36-

8 and the CAPS marker tk, whose relative location is

conserved between populations A and B.

Marker-assisted selection is effective for selecting

Aphanomyces root rot resistant beets

To evaluate the effectiveness of selecting for Apha-

nomyces root rot resistance at the field level through

MAS, the three AFLP markers closest to Acr1 were

used to select sugar beet lines bearing the resistance

allele. Three F5 lines and two BC2F1 lines expected to

bear the Acr1 resistant allele based on their marker

genotypes, were field tested for their resistance to

Aphanomyces root rot. Resistance was evaluated

based on whole plant symptoms in mature beets

according to the root rot intensity index (Table 3).

With mean (±standard error) root rot intensity values

of 0.25 (±0.17) and 0.75 (±0.27), respectively,

Acr1-donor line ‘NK-310mm-O’ and F1 (‘NK-

310mm-O’ 9 ‘NK-184mm-O’) plants exhibited Apha-

nomyces root rot resistance in the heavily infested

field. Under the same conditions, susceptible line

‘NK-184mm-O’ roots were almost fully decayed,

with a mean root-rot intensity values of 4.75 (±0.17).

The root rot intensity for the three F5 lines and two

BC2F1 lines ranged from 0.18 to 1.18, which can be

considered as highly resistant. Therefore, MAS

selection for a genomic region bearing Acr1 con-

ferred significant resistance to sugar beets.

Discussion

Based on the results of artificial inoculation of sugar

beets with Aphanomyces zoospores, Schneider

(1954) observed a general concurrence in the resis-

tance at the greenhouse and field levels (Panella

2005). However, the genetic basis as to what gene(s)

governs the two levels of resistance has remained

obscure for over 50 years. Therefore, identification of

resistant gene at field level (i.e. qAcr1, Taguchi et al.

2009) and at greenhouse level (Acr1, this study) is a

major advance for sugar beet breeding.

Under greenhouse conditions sugar beet line

‘NK-310mm-O’ exhibited strong Aphanomyces root

rot resistance, clearly distinguishing it from suscep-

tible lines. Resistance segregated in a dominant and

monogenic manner in its offspring, which prompted

us to conduct a segregation-based genetic study. The

in-greenhouse resistance gene, Acr1, was mapped to

the same region of chromosome III as that found to

bear the in-field resistance site qAcr1, suggesting the

e10m46-7 e10m47-9

1.1 e14m6-91.1 e19m13-6

e11m36-8 e11m36-8 e18m14-9 e19m6-8 e20m7-10

1.1 e2m38-82 e2m38-82 e2m42-9 e2m42-9

e20m6-7 e20m13-6

e30m45-5

Population A Population B

8.8

3.3

2.2

2.3

4.7

e10m37-9

e45m26-1

4.6

5.5

4.4

8.6

8.7

e45m23-4

e45m26-1

Acr1

e19m4-9

e14m40-7

tk/XspI

Acr1

tk/XspI

3.8

2.0

Fig. 4 Linkage map focusing on the chromosomal region

around Acr1 locus on chromosome III, derived from two

BC1F2 population developed from the cross of ‘NK-310mm-O’

and ‘NK-184mm-O’. Marker loci codes are listed on the right

and the map distance on the left (Kosambi, cM)

Euphytica (2010) 173:409–418 415

123

Author's personal copy

two genes may be tightly linked or the same.

Supporting this idea, the involvement of Acr1 in

field-level resistance was also shown by using a MAS

approach: sugar beets bearing Acr1 exhibited resis-

tance in the Aphanomyces-infested field. Therefore,

Acr1 is of great interest for breeding Aphanomyces

root rot resistance in sugar beets: introgression of

Acr1 into sugar beet varieties through, for example,

MAS could provide a solution to the control of

Aphanomyces root rot. However, it should be noted

that, in contrast to the nearly complete penetrance of

Acr1 at the greenhouse level, the genetic behaviour of

the ‘NK-310mm-O’ derived resistance at field level

was as quantitative trait loci, where the explained

variance of qAcr1 was 20% and 65% in the F2

population and F3 lines, respectively (Taguchi et al.

2009). While the major portion of field-level resis-

tance is likely contributed by the Acr1 gene, other

factors including environmental effects and genetic

background cannot be ignored, as they may modulate

the gene’s manifestation. In addition, because the

constitution of bulks were not covered accurately

around Acr1 region but covered the larger qAcr1

region, unfortunately, the markers density around the

Acr1 were quite low compared to the high number of

AFLPs tested during BSA. Molecular cloning of Acr1

will be necessary as a starting point in elucidating the

mechanism of Aphanomyces root rot resistance. Such

information is useful not only for sugar beet, but also

for other crops threatened by Aphanomyces.

That Aphanomyces resistance maps to chromo-

some III is interesting from the viewpoint of sugar

beet disease resistance genomics. A number of

important sugar beet disease resistance genes have

been mapped to chromosome III: Rz1 to Rz5 for

Rhizomania (caused by Beet necrotic yellow vein

virus (BNYVV)) and a major QTL for Cercospora

leaf spot (caused by fungus Cercospora beticola

Sacc.) (Barzen et al. 1999; Scholten et al. 1999;

Fig. 5 Image of AFLP

marker e2m42-9, e2m38-82

and e45m26-10,

respectively. Three resistant

(R) and three susceptible

(S) individuals from

population A were used in

these panels

Table 3 Disease index means for MAS lines under evaluation

in an Aphanomyces infested field (2005)

Line name Intensity index (mean ± SE)

NK-184mm-O 4.75 ± 0.17

NK-310mm-O 0.25 ± 0.17**

F1 0.70 ± 0.37**

F2 1.88 ± 0.73**

F5-1 1.10 ± 0.20**

F5-2 0.18 ± 0.05**

F5-3 0.85 ± 0.29**

BC2F1-1 1.08 ± 0.22**

BC2F1-2 1.18 ± 0.33**

F-test **

LSD (5%) 0.34

LSD (1%) 0.47

Lines were compared to ‘NK-184mm-O’

SE standard error, LSD least significant difference

** Significant difference of means at 1% level

416 Euphytica (2010) 173:409–418

123

Author's personal copy

Gidner et al. 2005; Grimmer et al. 2007; Schafer-

Pregl et al. 1999; Setiawan et al. 2000). Close linkage

was shown between Rz1 (probably allelic to Rz4 and

Rz5) and Rz2 (probably the same as Rz3), although

physical mapping has not yet been to be applied to

determining the precise distance. A similarly close

linkage was shown to exist between the Rz1-marker

OPC15-1800 and CAPS marker MP_tk (Grimmer

et al. 2007), the latter of which was included in our

study as tk, and found to be closely linked to Acr1

(see Fig. 3). This suggests that Acr1, Rz1 and, hence,

Rz2 may be clustered on chromosome III. Showing

organizational similarity to known resistance genes in

other plants, a number of resistance gene analogues

(RGAs) have been cloned from sugar beet, amongst

which some have been mapped to clusters on

chromosome III (Lein et al. 2007). This situation is

reminiscent of the hypothesis proposed by Michel-

more and Meyers (1998), where clustered RGAs

facilitated the evolution of resistance to diverse

pathogens, in this case to Aphanomyces and BNYVV.

However, further study is needed to clarify the

evolution of sugar beet chromosome III and the RGA

cluster. We are now investigating whether there are

any RGAs on chromosome III that are linked to Acr1,

or identical to Acr1 itself.

Acknowledgements The authors wish to thank Chiho

Tanaka, Sachiko Nishimura and Naoko Yamada for technical

assistance. This work was supported in part by the grant from

the Ministry of Agriculture, Forestry and Fisheries, Japan,

(Research and development projects applied in promoting new

Agriculture Forestry and Fisheries policies, No. 18058), and

the Program for Promotion of Basic and Applied Research for

Innovation in Bio-oriented Industry (BRAIN).

References

Barzen E, Stahl R, Fuchs E, Borchardt DC, Salamini F (1999)

Development of coupling-repulsion-phase SCAR markers

diagnostic for the sugar beet Rr1 allele conferring resis-

tance to Rhizomania. Mol Breed 3:231–238

Bochstahler HW, Hogaboam GJ, Schneider CL (1950) Further

studies on the inheritance of black root resistance in sugar

beets. Proc Am Soc Sugar Beet Technol 6:104–107

Coe GE, Schneider CL (1966) Selecting sugar beet seedlings

for resistance to Aphanomyces cochlioides. J Am Soc

Sugar Beet Technol 14:164–167

Doxtator CW, Finkner RE (1954) A summary of results in the

breeding for resistance to Aphanomyces cochlioides(Drechs) by the American Crystal Sugar Company since

1942. Proc Am Soc Sugar Beet Technol 8(2):94–98

Gidner S, Lennesfors B-L, Nilsson N-O, Bensefelt J, Johansson

E, Gyllenspetz U, Kraft T (2005) QTL mapping of

BNYVV resistance from WB41 source in sugar beet.

Genome 48:279–285

Grimmer MK, Trybush S, Hanley S, Francis SA, Karp A,

Asher MJC (2007) An anchored linkage map for sugar

beet based on AFLP, SNP and RAPD markers and QTL

mapping of a new source of resistance to Beet necroticyellow vein virus. Theor Appl Genet 114:1151–1160

Hori K, Kobayashi T, Shimizu A, Sato K, Takeda K, Kawasaki

S (2003) Efficient construction of high-density linkage

map and its application to QTL analysis in barley. Theor

Appl Genet 107:806–813

Kawaguchi M, Motomura T, Imaizumi-Anraku H, Akao S,

Kawasaki S (2001) Providing the basis for genomics in

Lotus japonicus: the accessions Miyakojima and Gifu are

appropriate crossing partners for genetic analyses. Mol

Genet Genomics 266:157–166

Kikuchi S, Taketa S, Ichii M, Kawasaki S (2003) Efficient fine

mapping of the naked caryopsis gene (nud) by HEGS

(High Efficiency Genome Scanning)/AFLP in barley.

Theor Appl Genet 108:73–78

Kosambi DD (1944) The estimation of map distance. Ann

Eugenics 12:505–525

Lander ES, Green P, Abrahamson J, Barlow A, Daly M (1987)

MAPMAKER: an interactive computer package for con-

structing primary linkage maps of experimental and nat-

ural populations. Genomics 1:174–181

Lein CL, Asbach K, Tian Y, Schulte D, Li C, Koch G, Jung C,

Cai D (2007) Resistance gene analogues are clustered on

chromosome 3 of sugar beet and cosegregate with QTL

for Rhizomania resistance. Genome 50:61–71

Michelmore RW, Meyers BC (1998) Clusters of resistance

genes in plants evolve by divergent selection and a birth-

and-death process. Genome Res 8:1113–1130

Michelmore RW, Paren I, Kesseli RV (1991) Identification of

Markers linked to disease-resistance genes by bulked

segregant analysis: a rapid method to detect markers in

specific genomic regions by using a segregation popula-

tion. Proc Natl Acad Sci USA 88:9828–9832

Mohring S, Salamini F, Schneider K (2005) Multiplexed,

linkage group-specific SNP marker sets for rapid genetic

mapping and fingerprinting of sugar beet (Beta vulgarisL.). Mol Breed 14:475–488

Okazaki K, Ogata N, Takahashi H, Taguchi K, Nakatsuka K

(2005) An artificial inoculation method to evaluate sugar

beet breeding line for resistance to Aphanomyces root rot.

Proc Am Soc Sugar Beet Technol 33:151–165

Panella L (2005) Black root. In: Biancardi E, Campbell L, De

Biaggi M, Skaracis GN (eds) Genetics and breeding of

sugar beet. Science Publishers, Enfield (NH), Plymouth,

pp 101–110

Roger SO, Bendich AJ (1988) Extraction of DNA from plant

tissues. Plant Mol Biol Manual A6:1–10

Schafer-Pregl R, Borchardt DC, Barzen E, Glass C, Mechelke

W, Seitzer JF, Salamini F (1999) Localization of QTLs

for tolerance to Cercospora beticola on sugar beet linkage

groups. Theor Appl Genet 99:829–836

Schneider CL (1954) Methods of inoculating sugar beets with

Aphanomyces cochlioides Drechs. Proc Am Soc Sugar

Beet Technol 8:247–251

Euphytica (2010) 173:409–418 417

123

Author's personal copy

Schneider CL (1978) Use of Oospore Inoculum of Aphano-myces cochlioides to initiate black root disease in sugar

beet seedlings. J Am Soc Sugar Beet Technol 20:55–62

Schneider CL, Whitney ED (1986) Black root. Page 17. In:

Whitney ED, Duffus JE (eds) Compendium of beet dis-

eases and insects. American Phytopathological Society,

St. Paul, MN

Scholten OE, De Bock ThSM, Klein-Lankhorst RM, Lange W

(1999) Inheritance of resistance to beet necrotic yellow

vein virus in Beta vulgaris, conferred by a second gene for

resistance. Theor Appl Genet 99:740–746

Setiawan A, Koch G, Barnes SR, Jung C (2000) mapping

quantitative trait loci (QTLs) for resistance to Cercospora

leaf spot disease (Cercospora beticola Sacc.) in sugar beet

(Beta vulgaris L.). Theor Appl Genet 100:1176–1182

Taguchi K, Ogata N, Kubo T, Kawasaki S, Mikami T (2009)

Quantitative trait locus responsible for resistance to

Aphanomyces root rot (black root) caused by Aphano-myces cochlioides Drechs. in sugar beet. Theor Appl

Genet 118:227–234

418 Euphytica (2010) 173:409–418

123

Author's personal copy