Botanical Studies (2008) 49: 93-99.

*

Corresponding author: E-mail: songwq@nankai.edu.cn;

Tel: +86-22-23508241; Fax: +86-22-23497010.

MOLECULAR BIOLOGY

A genetic linkage map based on AFLP and NBS markers

in cauliflower (Brassica oleracea var. botrytis)

Yu GU

1,2

, Qian-Chen ZHAO

1

, De-Ling SUN

1

, and Wen-Qin SONG

2,

*

1

Tianjin Kernel Vegetable Research Institute, Tianjin 300382, P. R. China

2

Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China

(Received March 26, 2007; Accepted October 4, 2007)

ABSTRACT.

A genetic linkage map of cauliflower (Brassica oleracea var. botrytis) has been constructed

based on AFLP and nucleotide binding site (NBS) markers, in order to identify potential molecular markers

linked to important agronomic traits that could be useful for developing and improving the species. NBS

profiling was first used to map resistance gene analogues (RGAs) in cauliflower (Brassica oleracea var.

botrytis), which simultaneously allowed the amplification and mapping of genetic markers anchored in the

conserved NBS encoding domain of plant disease resistance genes. At the same time, the AFLP method was

also performed in this paper to construct an intervarietal genetic map of cauliflower. A total of 234 AFLP

markers and 21 NBS markers were mapped in an F

2

population derived by self-pollinating a single F

1

plant

(a hybrid between "AD White Flower" and "C-8") based on seventeen AFLP primer combinations and two

degenerate primer/enzyme combinations. The markers were mapped into nine major linkage groups, spanning

668.4 cM, with an average distance of 2.9 cM between adjacent mapped markers. Each of the linkage groups

contained from 12 to 47 loci, and the distance between two consecutive loci ranged from 0 to 14.9 cM. The

AFLP markers were well distributed throughout the nine linkage groups, and eight linkage groups for the NBS

markers. Most NBS markers mapped in this study were organized in clusters, indicating that most of them

could be real RGAs. The maps we have generated provide a firm basis for mapping agriculturally relevant

traits, which will then open the way for application of a marker-assisted selection breeding strategy in this

species.

Keywords: AFLP; Cauliflower (Brassica oleracea var. botrytis); Genetic linkage map; NBS profiling.

Abbreviations: NBS-LRR, nucleotide binding site-leucine-rich repeat; RGAs, resistance gene analogues;

PCs, primer combinations; LGs, linkage groups; cM, centiMorgans; R gene, resistance gene.

INTRODUCTION

Over the past two decades, several genetic maps of

Brassica oleracea have been constructed. The first map

was based on the segregation of 258 restriction fragment

length polymorphism (RFLP) loci in a broccoli ¡Ñ cabbage

F

2

population. The genetic markers defined nine linkage

groups, covering 820 recombination units (Slocum et

al., 1990). Subsequently, at least ten genetic linkage

maps were developed (Landry et al., 1992; Kianian et

al., 1992; Bohuon et al., 1996; Camargo et al., 1997;

Voorrips et al., 1997; Cheung et al., 1997; Hu et al., 1998;

Moriguchi et al., 1999; Sebastian et al., 2000; Chen et

al., 2002; Howell et al., 2002; Wang et al., 2005; Wang

et al., 2007). These maps were based on intraspecific

or intersubspecific populations. An intervarietal genetic

map of cauliflower (Brassica oleracea var. botrytis) was

missing. The high level of genetic variability within an

intraspecific population or the level of DNA polymorphism

in the parents may explain this. Nevertheless, having an

intervarietal linkage map is the most direct and efficient

approach for cauliflower breeding in the future since the

genetic information is derived from cauliflower.

Most studies have dissociated the isolation (cloning)

of resistance gene analogos (RGAs) from their genetic

mapping in segregating progenies. Most often, cloned

RGAs are mapped using RFLPs, which is often time-

consuming (Calenge et al., 2005). Modified amplified

fragment length polymorphisms (AFLP) and nucleotide

binding site (NBS) profiling were proposed as new

strategies by Hayes and Saghai-Maroof (2000) and by

Van der Linden et al. (2004) to generate, simultaneously,

polymorphism and specifically amplify highly conserved

motifs. Both methods are based on the simultaneous use

of an adapter primer matching a restriction enzyme site

94

Botanical Studies, Vol. 49, 2008

and of a degenerate primer targeting the NBS encoding

region. Genetic variation is sampled in the gene region

flanking the primer-binding site. Nevertheless, NBS

profiling has some advantages that improve specificity.

NBS profiling involves a two-step PCR procedure. The

first step is a linear (asymmetric) PCR with a limited

amount of the NBS specific primer. The asymmetric PCR

is then followed by an exponential PCR with an NBS

primer and an adapter primer. An amino linker effectively

blocks extension of the 3¡¦ end of the short arm in the

subsequent PCR reaction. The adapter primer can thus

only participate in the PCR reaction after extension of

the selective NBS primer. Amplification depends highly

on the selectivity and specificity of this NBS primer. To

further increase the specificity of the PCR procedure, the

actual amplification (exponential PCR) is preceded by

linear (asymmetric) PCR with only the NBS primer. In the

modified AFLP method, in the initial digestion and ligation

steps, approximately 87.5% of the soybean genome is

eliminated because Mse/Mse fragments are not detected

in the preceding steps. Nonetheless, NBS profiling can be

identified in all genome. NBS profiling was shown to be

highly effective in generating polymorphic markers with

high sequence homology for RGAs in several species (Van

der Linden et al., 2004).

The purposes of this paper were: 1) To construct an

intervarietal linkage map of cauliflower. This map could be

used to identify more markers, which would eventually be

linked to genes controlling important agronomic characters

and provide a powerful tool to be used in marker-assisted

breeding of cauliflower. 2) To demonstrate the reliability

and efficiency of the NBS profiling approach for mapping

homologous nucleotide binding site-leucine rich repeat

(NBS-LRR) genes in cauliflower, and 3) to determine

the distribution and characterization of NBS-LRR-like

sequences in the cauliflower genome.

MATERIAL AND METHODS

Plant materials and DNA isolation

An F

2

population of 100 progenies was generated by

self-pollination of a single F

1

plant from an intervarietal

cross between "AD White Flower" as pollen parent

and "C-8" as seed parent. The mapping population was

supplied by the Tianjin Kernel Vegetable Research

Institute. "AD White Flower" is an inbred line, developed

from a general Chinese variety through several inbreeding

generations. "C-8" is a self-incompatible line derived from

an individual plant mutation of an exotic line.

Total genomic DNA of mapping progenies was isolated

following the CTAB method (Murray and Thompson,

1980) with some modifications. Fresh leaves (0.2 g) of

both lines were ground in liquid nitrogen. The frozen

powder was directly added to 2 ml lysis buffer (100

mM Tris-HCl, pH 8.0, 50 mM EDTA, 1.4 M NaCl,

0.2% £]-mercaptoethanol, 2% PVP and 1¡ÑCTAB). After

incubation at 56¢XC for 30 min, 2 ml phenol: chloroform:

isoamyl alcohol (25:24:1) was added. The supernatant

was obtained by centrifugation at 10,000 g for 10 min

and extracted once with an equal volume of chloroform:

isoamyl alcohol (24:1). After centrifugation at 11,000 g

for 15 min, a two-thirds volume of cold isopropanol was

added to the supernatant. The mixture was incubated for

30 min at -20¢XC. DNA was pelleted by centrifugation at

11,000 g for 30 min at 4¢XC and dissolved in 100 £gl TE (pH

8.0) buffer. RNA was removed from the DNA by treatment

with 200 £gg £gl-1 RNase A for 1 h at 37¢XC.

AFLP molecular marker analysis

AFLP analysis was performed according to Vos et

al. (1995) with minor modifications. Briefly, 100-150 ng

of DNA was digested with 1.5 U of both EcoR I and Mse

I (Shanghai Sangon, Shanghai China). Aft er ligation and

pre-amplification, selective amplification was conducted

by combining 30 ng of an EcoR I primer containing two

selective nucleotides and an Mse I primer containing

three selective nucleotides. Thermocycling (Mastercycler

Gradient 5331, Eppendorf Germany) was done with

35 cycles and included a 12-cycle touchdown (annealing

temperature was reduced from 65¢XC to 56¢XC a t 0.7¢XC pe r

step for 12 cycles, and subsequently maintained at 56¢XC

for 23 c yc les). Amplification products were separated in 6%

(w/v) denaturing polyacrylamide gels at 65 W for 2.5 h in 1

¡ÑTBE buffer. After silver staining (Bassam et al., 1991), the

gels were dried at room temperature and photographed.

NBS profiling protocol

NBS profiling of the progeny was performed essentially

as described by Van der Linden et al. (2004). In brief,

DNA was digested for 4 h with Mse I, using 400 ng DNA

per individual. An adapter was ligated to the restriction

fragments. Adapter sequences and the adapter primer

were:

Adapter long arm: 5¡¦-ACTCGATTCTCAACCCGAAA

GTATAGATCCCA-3¡¦

Adapter short arm: 5¡¦-TGGGATCTATACTT-3¡¦ (with 3¡¦

amino group)

Adapter primer: 5¡¦-ACTCGATTCTCAACCCGAAAG-

3¡¦

Two different degenerate primers, NBS2 (5¡¦-GTWG

TYTTICCYRAICCISSCAT-3¡¦) and NBS5 (5¡¦-YYTKR

THGTMITKGATGAYGTITGG-3¡¦), were then used for

a two-step PCR procedure. These primers were designed

from part of the conserved P-loop and Kinase-2 motif,

respectively, belonging to the NBS encoding region of

several plant disease resistance genes (Van der Linden

et al., 2004). The first PCR was linear, and only the

degenerate primer (either NBS2 or NBS5) was used. The

second PCR was exponential and was performed with both

the degenerate primer and the adapter primer. Both PCRs

were performed with an annealing temperature of 60¢XC

on a Mastercycler Gradient 5331 (Mastercycler Gradient

5331, Eppendorf Germany), using the following cycling

GU et al. ¡X Cauliflower genetic linkage map

95

program: 30 cycles of 30 s at 95¢XC, 1 min 40 s at 60¢XC and

2 min at 72¢XC. The PCR products were separated in 6%

(w/v) polyacrylamide gels for 2.5 h at 65 W, then dried at

room temperature and photographed.

Genetic linkage map construction

The AFLP fragments were scored as dominant,

presence versus absence of bands, and therefore for the

parents, F

1

and F

2

generation markers were assigned to

either parental allele for map construction. AFLP markers

were designated by the PCs used, followed by a number

reflecting the fragment length on the gel (e.g., M-AAGe-

AC400 = Mse I +AAG/EcoR I +AC, band length is

400 bp). NBS markers were tested against the predicted

3:1 ratio, representing [homozygote + heterozygote]:

[homozygotes], by the presence: absence of the marker

band, respectively. NBS markers were named using the

same protocol/logic as AFLP (e.g., NBS2M60 = NBS2/

Mse I, band length is 60 bp).

Markers originating from each parent were scored

according to the standard coding system using A, B,

C, D and H of JoinMap, Version 3.0 (Van Ooijen and

Voorrips, 2001). Ambiguous genotypes were resolved

by assigning a blank score (-) to the individual locus for

map construction. Chi-square (£q

2

) tests of goodness-of-fit

were performed on segregation data for all markers, with a

0.5% threshold level for significance. To determine marker

order within a linkage group, the following JoinMap

parameter settings were used: Rec = 0.40, LOD = 1.0,

Jump = 5. Markers were assigned to linkage groups (LGs)

by increasing the LOD score for grouping with steps of

one LOD unit. No order was forced during the linkage

analysis. Recombination frequencies were converted to

map distances in centimorgans (cM) using the Kosambi

mapping function (Kosambi, 1943).

RESULTS

Polymorphism analysis using AFLP markers

Initially, sixty-four AFLP PCs were screened for

polymorphism by using two parents. Seventeen most

informative EcoR I+ 2 / Mse I+3 PCs (Table 1) were

selected for mapping according to the number and

reliability of the polymorphic marker. Approximate 1,700

bands were produced by the seventeen PCs in the mapping

population with an average of 100 bands per PC. In

total, 339 polymorphic markers were scored as dominant

markers (Figure 1). The number of polymorphic AFLP

markers per PC ranged from 13 to 26, with a mean of 20

markers per PC (Table 1). Chi-square analysis revealed

234 of the 339 markers (69.9%) fitted a 3:1 ratio. The

remaining 105 (30.1%) showed segregation distortion

within the population.

NBS profiling marker development in

cauliflower

Two degenerate primer/enzyme combinations were

tested in the progeny: NBS2/Mse I and NBS5/ Mse I.

An average of 60-90 monomorphic bands per PC was

obtained. Thirty-one polymorphic bands were identified

with the two combinations and scored as dominant

markers (Figure 1). Ten markers (32.3%) had a distorted

segregation in the progeny as assessed by chi-square tests

and were discarded from data. 21 polymorphic markers

(68%) were reliably mapped on the linkage map (Figure 2).

NBS markers were mapped to 8 of the 9 linkage groups of

Table 1. Primer combinations for AFLP and NBS markers are shown with the number of polymorphic markers generated

for each of them.

Primer combination

Number of markers

Primer combination

Number of markers

M50/E11

18

M48/E13

25

M60/E12

19

M60/E13

13

M59/E13

21

M49/E12

26

M62/E13

20

M60/E11

21

M47/E12

20

M49/E11

23

M47/E11

24

M61/E11

20

M48/E11

25

M48/E12

15

M61/E12

23

NBS2/MseI

13

M62/E12

19

NBS5/MseI

8

M61/E13

17

Total

370

96

Botanical Studies, Vol. 49, 2008

Figure 1. AFLP and NBS markers in a F

2

mapping population. Markers used for this study are indicated by arrow. Primer combinations

for AFLP and NBS profiling were M-CTT/E-AG and NBS5/MseI, respectively.

the genetic maps (Figure 2). Most markers were organized

in more or less wide clusters of 0-18 cM.

Linkage analysis and map construction

The 234 AFLP markers and 21 NBS markers were used

to construct a genetic map. Altogether, 255 of the 370

polymorphic markers were assigned to nine linkage groups

(LGs) (Figure 2) ranging in size from 12 to 60 markers

each. Map distances ranged from 42.6 to 113.0 cM per

LGs (Table 2). The 255 markers (229 loci) gave a total

map length of 668.4 cM and an average genetic distance

between adjacent mapped loci as 2.9 cM (Figure 2).

Linkage relationships of the 255 segregating markers were

established at a 3.0 . LOD . 10.0 and a recombination

fraction smaller than 0.5 Map distances were converted to

cM using the Kosambi mapping function (Kosambi, 1943).

According to Figure 2, we can see the names of markers

are shown at the right and their map position (cM) at the

left. All the markers appear to be distributed randomly

along all LGs, except LGs 3, 4 and 5, which show rather

few markers.

DISCUSSION

Several genetic linkage maps of B. oleracea have been

produced in the past using intraspecific or intersubspecific

mating systems. It is generally believed that the degree

of polymorphism is lower in an intervarietal population

than in an intraspecific or intersubspecific population. The

high level of genetic variability within an intraspecific

population or the level of heterozygosity of the parents

may explain this. However, Hu et al. (1998) compared

three previous independent B. oleracea linkage maps

and pointed out that the distances between markers

often varied from map to map. This demonstrated

that sequence rearrangement is a distinct feature of

this genome. Slocum et al. (1990) reached the same

conclusion. Their study suggested that a fairly high degree

of genetic rearrangement has occurred in the evolution

of B. oleracea. With respect to cauliflower breeding, we

believe that intervarietal crosses should be better than

intersubspecific. Results of this study suggest that an

intervarietal cross allows better map resolution and even

distribution in LGs.

Some published AFLP linkage maps show clustering

of these markers in centromeric regions, due to an excess

of repeats in this area and suppressed recombination

shrinking the genetic map relative to the DNA content

(Jeuken et al., 2001). In this study, do not find any mark on

centromeric regions of any LG (Figure 2). Chromosome

centromeric regions are usually conserved and may not be

polymorphic when self-pollinated. This is in contrast to the

clusters of markers mapped on an interspecific segregating

population (Pearl et al., 2004; Lan et al., 2008).

It was previously demonstrated that the NBS profiling

method allows the generation of polymorphic markers

among different cultivars of potato, tomato, barley,

and lettuce (Van der Linden et al., 2004). The present

investigation is the first to test this new method in

cauliflower and the first to demonstrate its utility for

GU et al. ¡X Cauliflower genetic linkage map

97

Figure 2. Genetic linkage map of cauliflower (Brassica oleracea var. botrytis). Linkage groups are numbered LG1 through to LG9.

Markers are indicated at the right of each linkage group. Recombination distances are in Kosambi¡¦s cM and are indicated at the left of

each linkage group.

Table 2. Marker number and map distance per linkage group.

Linkage group Marker Num. (loci) Marker Num AFLP/NBS (loci) Length (cM) Mean length (cM) LOD

LG1

42(38)

41(37)/1(1)

56.7

1.5

10.0

LG2

24(23)

22(21)/2(2)

46.5

2.0

10.0

LG3

16(16)

14(14)/2(2)

42.6

2.7

10.0

LG4

13(13)

13(13)/0

56.1

4.3

7.0

LG5

12(12)

11(11)/1(1)

65.5

5.5

6.0

LG6

60(47)

56(44)/4(3)

113.0

2.4

6.0

LG7

33(29)

29(25)/4(4)

86.7

3.0

5.0

LG8

34(31)

30(27)/4(4)

90.5

2.9

4.0

LG9

21(20)

18(17)/3(3)

110.8

5.4

3.0

Total

255(229)

234(209)/21(20)

668.4

2.9

98

Botanical Studies, Vol. 49, 2008

mapping studies using a segregating progeny. Thirty-one

polymorphic markers were observed in the intravarietal

progenies across the two-primer/enzyme combinations

tested, of which 21 markers could be mapped. Ten

unmapped markers had a distorted segregation in the

progeny, which could explain why they could not be

reliably mapped. Fewer NBS profiling markers were

obtained than AFLP markers. This is an inherent property

of such markers, as each in principle corresponds to a

member of the NBS-LRR RGA gene family that is present

in far lower numbers in the plant genome than restriction

sites (AFLP) (Syed et al., 2006). The usefulness of NBS

markers is their close linkage to potentially important

resistance genes. The NBS profiling molecular marker

methods have been validated for cauliflower.

In our map, most markers were assembled in clusters.

The best example is the cluster on linkage group LG6,

which includes four markers (Figure 2). Therefore these

markers correspond either to the same gene locus or to

very tightly linked loci. The clustering of RGAs of the

NBS-LRR type has previously been observed in numerous

studies. Most NBS-LRR and other R gene-like sequences

reside in large, extended arrays (Young, 2000). This

clustering of RGAs is not surprising, considering that plant

R genes often, though not always, belong to gene families

with evolutionarily related tandemly repeated genes or to

allelic series (Hulbert et al., 2001). The clustering of the

markers mapped in this study adds to the evidence that

most of them could be real RGAs.

Acknowledgements. The authors thank Ms Lili Liu from

the Tianjin Kernel Vegetable Research Institute of China

for planting and tending the mapping population. Financial

support for this study was provided by the National

Science Foundation of Tianjin (No.06YFJMJC10000).

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