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Botanical Studies (2007) 48: 255-261.

Corresponding author: E-mail: whwei@oilcrops.cn; Tel:

+86-27-86722567; Fax: +86-27-86722567.

INTRODUCTION

The Multinational Brassica Genome Project, using

diploid Brassica rapa L. (AA, 2n=20) and Brassica ol-

eracea L. (CC, 2n=18) as two model species, is advancing

rapidly in several laboratories (Rana et al., 2004; Park et

al., 2005; Yang et al., 2005; Ayele et al., 2005; Katari et

al., 2005). It has become another plant genome research

hotspot. The genus Brassica includes many important oil

and vegetable crops, and they play an increasing role in

improving the lives of human beings. For B. oleracea,

including a group of the most important vegetable crops,

such as cauliflower, cabbage, calabrese and Brussels

sprouts, the scientific community is anxious to perform cy -

togenetics research from an evolutionary or breeding point

of view. However, karyotyping B. oleracea and exactly

recognizing all individual chromosomes within a mitotic

metaphase spread is very difficult. This is primarily due to

the small size of the chromosomes as well as the similarity

of chromosome lengths and/or arm ratios for some of the

complement.

At present, distinguishing chromosomes from each

other and the karyotyping of B. oleracea are mainly based

on the number and position of the 45S (or 25S) and 5S

rDNAs on the chromosome (Snowdon et al., 1997; Hast-

erok et al., 2001). FISH signals of 45S rDNA could be

detected on two pairs of chromosomes of B. oleracea.

The copy number of rDNA on the end of the short arm of

chromosome 7 exceeds that on the end of the short arm

of chromosome 4 (Cheng et al., 1995; Fukui et al., 1998).

Sometimes, signals with a very low intensity are detected

on the short arm end of chromosome 2 (Armstrong et

al., 1998). For 5S rDNA, FISH signals with a very low

intensity can detected on the long arms of chromosome

2 (Armstrong et al., 1998; Hasterok et al., 2001). Thus,

rDNA sites are found in only three of nine chromosome

pairs for B. oleracea. In B. rapa, another diploid species of

Brassica, rDNA sites are found in six of ten chromosome

pairs. Compared to B. oleracea, a more exact karyotyping

was performed based on rDNA sites in B. rapa (Koo et

al., 2004; Lim et al., 2005). Five chromosome pairs in B.

oleracea were identified with cDNA and rDNA probes by

Kamisugi et al. (1998), or with three repetitive sequences

by Armstrong et al. (1998). How to exactly identify nine

chromosome pairs of B. oleracea is a pending question,

new markers are obviously needed for karyotyping.

The Cot-1 DNA is enriched with highly and moderately

repetitive DNA sequences. In most eukaryote plant spe-

cies, repetitive sequences comprise a large proportion of

the genome (Flavell et al., 1974; Hake and Walbot, 1980;

MCCouch and Tanksley, 1991). Class I transposable

Karyotyping of Brassica oleracea L. based on Cot-1 and

ribosomal DNAs

Wen-Hui WEI

1,2,

*, Su-Feng ZHANG

, Li-Jun WANG

, Bo CHEN

, Xiao-Ming WU

, and Yun-Chun

SONG

College of Life Sciences, Xinyang Normal University, Xinyang Henan 464000, Peopleˇs Republic of China

Institute of Oil Crops, Chinese Academy of Agricultural Sciences, Wuhan Hubei 430062, Peopleˇs Republic of China

The Key Laboratory of MOE for Plant Developmental Biology, Wuhan University, Wuhan Hubei 430072, Peopleˇs Re-

public of China

(Received June 29, 2006; Accepted April 16, 2007)

ABSTRACT

. To explore a simple, reliable, and effective method of karyotyping Brassica oleracea L., Cot-1

DNA was isolated from its genome, labeled as probe with a Biotin-Nick Translation Mix kit, and in situ hy-

bridized to mitotic spreads. Specific fluorescent bands appeared on each chromosome pair. 25S and 5S rDNAs

were labeled as probes with a DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit, respectively,

and in situ hybridized to mitotic preparations. Signals could be detected on two chromosome pairs for 25S

rDNA, and on only one for 5S rDNA. Cot-1 DNA contains rDNA. The site identity of Cot-1 DNA and 25S

rDNA on the chromosome was determined by dual-colour fluorescence in situ hybridization (FISH). It showed

that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome markers is a

superior alternative. A more exact karyotype of B. oleracea has been developed based on rDNA locations and

Cot-1 DNA fluorescent bands.

Keywords: Brassica oleracea L.; Cot-1 DNA; Karyotyping; Ribosomal DNA.

mOLeCULAR BIOLOgy

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256

Botanical Studies, Vol. 48, 2007

elements, or retrotransposons, represent a major fraction

of all plant genomes (Kumar and Bennetzen, 1999; Alix

et al., 2005) and are important factors in the generation of

diversity because they can transpose from one genomic site

to another. There is evidence that individual retroelement

families have typical patterns of chromosomal localization

(Presting et al., 1998). In situ hybridization has shown that

copia group retrotransposons in B. oleracea are abundant

and distributed along all chromosomes with higher density

in some chromosomal regions (Brandes et al., 1997).

Hence, it is conceivable to band individual chromosomes

of B. oleracea with Cot-1 DNA. The karyotyping results

presented in the present study are those obtained by the

combination of a morphometric study and FISH with

rDNA and Cot-1 DNA probes to B. oleracea chromo-

somes. The applied technique allowed more effective

karyotype construction for this diploid Brassica species.

mATeRIALS AND meTHODS

Plant materials

Brassica oleracea var. acephala developed by the In-

stitute of Oil Crops, Chinese Academy of Agricultural Sci-

ences, was used in the present study. The first-born flower

buds adopted from the field were used for chromosome

preparation, and young plant leaves were used for genomic

DNA extraction.

Preparation of rDNAs

The 25S and 5S rDNAs lodging bacteria supplied by

Dr. Robert Hasterok (Department of Plant Anatomy and

Cytology, The University of Silesia, Poland) were plated

on solid LB medium, a single colony was selected for

cloning in liquid medium, and plasmid DNA was iso-

lated using Qiagen Mini Kit (Cat. No. 12125, supplied by

Wuhan Boyer Bioengineering Co., Ltd; No. 40, Xudong

Road, Wuhan City, China).

Chromosome preparation

The chromosome preparation method was developed

using the technique described by Wei et al.

(2001; 2005)

with some modifications. Briefly, first-born full flower

buds during mitosis were fixed in the mixture (95% etha-

nol : acetic acid glacial=3 : 1) at 4C overnight after being

treated in 4C water for approximately 24 h. Flower buds

were washed three to five times with distilled water, then

digested in 1% (w/v) cellulase "Onozuka" R-10 (Yakult

Honsha, Co., LTD, Japan) and 1% (w/v) pectolyase Y-23

(Yakult Honsha) dissolved in distilled water at 28C for

2.5-3 h. Full flower buds, including tapetum and the wall

of anther, were subjected to a hypotonic treatment in dis-

tilled water for 30 min before being squashed, and the

preparations were dried with flame.

genomic DNA extraction

The extraction of B. oleracea genomic DNA was per-

formed according to the procedure described by Doyle

and Doyle (1990). In brief, 5 g of young leaf tissue was

homogenized in liquid nitrogen and mixed in 20 ml of

preheated (65C) DNA extraction buffer (0.1 M Tris-Cl,

20 mM Na

ethylenediaminetetraacetic acid (EDTA), 1.4

M NaCl and 20% [w/v] hexadecyltrimethylammonium

bromide [CTAB] and 0.2% [v/v] -mercaptoethanol [pH

8.0]) in sterile polypropylene centrifuge tubes and

incubated at 65C in a water bath for 1 h with occasional

gentle swirling. The samples were mixed with chloroform-

isoamylalcohol (24:1). The contents were centrifuged at

9000 g for 15 min at room temperature, and the aqueous

phase was transferred to fresh sterile centrifuge tubes

and mixed well with 0.67 vol (13.4 ml) of isopropanol

by gently inverting the tubes 5-6 times. This mixture was

centrifuged at 9000 g for 10 min to pellet the DNA. The

pellet was washed with 70% (v/v) ethanol and air-dried.

The DNA pellet was dissolved in Tris-EDTA (TE) buffer

(pH 8.0).

genomic DNA shearing and Cot-1 DNA isolation

Genomic DNA shearing and Cot-1 DNA isolation were

performed as described by Zwick et al. (1997) and Wei

et al. (2005). Briefly, the genomic DNA was diluted to a

concentration of 300 ng/L in 0.3 mol/L NaCl, samples

were aliquot and autoclaved (121C, 1.034 ⊙ 10

Pa) for

10 min to make about 100-1000 bp DNA fragments. The

sample tube was then cooled on ice. The DNA was dena-

tured by placing the tube in a 95C water bath for 10 min.

The tube was removed and cooled by swirling in ice water

for 10 s before being incubated in a 65C water bath for a

re-annealing. The time needed for the reannealing reaction

was calculated using the formula C

t = DNA conc (Mole/

L) ⊙ re-naturation time in sec (Ts), C

t =1, Ts = 1/ DNA

conc. C

= (0.300 g/L) / (339 g/mol, an average molecular

weight for a deoxynucleotide monophosphate) = 8.85 ⊙

-4

mol/L, so T = 1 / (8.85⊙10

-4

) = 1130 sec (Zwick et al.,

1997).

After the time allotted for re-annealing had elapsed, the

tube was removed from the 65C water bath. An appropri-

ate amount of 10 ⊙ S1 buffer (Promega, Cat. No. M5761)

was added firstly, and the sample was mixed thoroughly.

The S1 nuclease was then added, and the solution was

mixed thoroughly, but gently. Immediately, the tube was

placed in a 37C water bath for 8 min. The reaction was

stopped by immediate phenol extraction using equal vol-

umes of Tris-equilibrated phenol, and the subsequent steps

in the genomic DNA extraction method were performed

until the Cot-1 DNA was resuspended in 20 L TE. Sam-

ples were stored at -20C after quantitative analysis.

Labeling of probes, FISH, and detection of the

signals

The Cot-1 DNA was labeled with Biotin-Nick Trans-

lation Mix Kit (catalogue no. 11745824910; Roche,

Germany. Supplied by Wuhan Boyer Bioengineering Co.,

Ltd, China), 25S rDNA was labeled with DIG-Nick Trans-

lation Mix Kit (Cat. No. 11745816910; Roche), and 5S

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WEI et al.  Karyotyping of

Brassica oleracea

257

rDNA was directly labeled with biotin-11-dUTP by PCR

using forward primer 5ˇ-GGATGGGTGACCTCCCGG

GAAGTC-3ˇ and reverse primer 5ˇ-CGCTTAACTGCG

GAGTTCTGATGGG-3ˇ (Yang et al., 1998). DNA was

amplified for 35 cycles of 1 min at 94C, 45 s at 55C, 1

min at 72C, and a final period of 5 min at 72C. FISH

and hybridization signal detection were performed accord-

ing to the procedures described by Wei et al. (2002; 2003;

2005). Briefly, chromosome preparations were pretreated

with 100 g/mL RNase (in 2 ⊙ SSC, 0.3 mol/L sodium

chloride plus 0.03 mol/L sodium citrate) at 37C for 1 h

and then rinsed briefly in 2 ⊙ SSC. Chromosomal DNA

was then denatured by immersing the slide in 70% deion -

ized formamide in 2 ⊙ SSC at 70C for 3 min. After de-

hydration of the preparation in an ice-cold 70%, 95%, and

100% ethanol series and air drying, 40 L denatured probe

cocktail (5 ng/L labeled probe DNA, 0.5 g/L sheared

salmon sperm DNA, 10% dextran sulphate, 50% deionized

formamide, 0.1% sodium dodecyl sulfate (SDS), and 2 ⊙

SSC) was added to the slide and hybridization was per-

formed at 37C overnight. Post-hybridization washes in-

cluded a stringent wash in 20% formamide and a wash in 2

⊙ SSC at 42C for 10 min to remove weakly bound probe;

signals were detected with streptavidin-Cy3 (catalogue no.

PA43001; Amersham Biosciences UK Limited, England)

for the biotin labeled probe and with anti-digoxigenin-

fluorescein (Roche) for the digoxigenin labeled probe;

washing in phosphate-buffered saline (0.13 M NaCl, 0.007

M Na

HPO

12H

O, 0.003M NaH

2H

O) was done

for 10 min after each probe detection step. Slides were

counterstained with 2 g/mL 4ˇ, 6ˇ-diamidino-2-phenylin-

dole (DAPI) and examined under a Leica DM IRB fluores-

cence microscope equipped with a DFC300 CCD camera.

ReSULTS

Preparation and shearing of genomic DNA and

isolation of Cot-1 DNA

The preparation and shearing of genomic DNA and the

isolation of Cot-1 DNA results are shown in Figure 1. The

size of genomic DNA is above 20 kb in size, and it became

about 100-2000 bp, mostly under 1000 bp, after being au-

toclaved for 10 min. The isolated Cot-1 DNA was under

1000 bp in size.

FISH

25S rDNA was detected on chromosome pairs 4 and 7,

a fact affirmed by published results (Howell et al., 2002).

Chromosome 7 presented especially bright signals, and

detecting them on these two chromosome pairs was easy

(Figure 2A). 5S rDNA was detected on one chromosome

pair, but the signal was weak (Figure 2B) and detection

was difficult. Individual metaphase chromosomes

showed bright Cot-1 DNA fluorescence bands (Figure

2C, D). The sites of 25S rDNA are in accord with those

of Cot-1 DNA, identified by dual-colour FISH (Figure

3). The Cot-1 DNA fluorescence bands combined with

rDNA loci sites and chromosome morphology should be

powerful for karyotyping because recognition markers

of the chromosomes contain not only specific banding

patterns of Cot-1 DNA but also the rDNA signals and the

chromosome morphology.

Karyotyping

Karyotyping of B. oleracea was performed according

to the method published by Armstrong et al. (1998) and

Howell et al. (2002). The B. oleracea karyotype obtained

in the present study on the basis of Cot-1 DNA hybridiza-

tion bands combined with 25S rDNA sites and morpho-

metric analysis is shown in Figure 4. The green 25S rDNA

hybridization signals were located on chromosome pairs 4

and 7. The red or pinkish white Cot-1 bands are shown on

the long arms of chromosome pairs 6 and 8, the short arms

of chromosome pairs 2, 4, and 7, and the pericentromeric

sites of chromosome pairs 1, 3, 5 and 9. Different chromo-

some pairs could be recognized reliably based on Cot-1

DNA banding patterns and rDNA locations.

DISCUSSION

In the present study, 25S rDNA was detected steadily

on two chromosome pairs, and 5S rDNA accidentally on

one chromosome pair. The numbers of chromosomes with

detectable rDNAs were coincident with former reports

(Cheng et al., 1995; Fukui et al., 1998). However, the

number of chromosome pairs with detectable 25S rDNA

was one less than the result reported by Howell et al.

(2002), and the detection frequency of the 5S rDNA site

Figure 1. Preparation of B. oleracea Cot-1 DNA. M: DNA/

EcoR.+Hind. marker; 1: Total genomic DNA of B. oleracea; 2:

Genomic DNA after autoclaved for 10 min; 3: Cot-1 DNA.

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Botanical Studies, Vol. 48, 2007

was very low. The reasons may be that the copy numbers

of 5S rDNA repetitive sequences are very low at these

sites and more difficult to detect, or the rDNAs sites vary

by cultivars (Weiss and Maluszynska, 2000).

The Cot-1 DNA is enriched with highly and moderately

repetitive DNA sequences. rDNA, a moderately repetitive

DNA sequence, is included in Cot-1 DNA. Hence, the

Cot-1 DNA isolated from a species should contain rDNA.

Theoretically speaking, the sites that present Cot-1 DNA

signals should include the sites of rDNAs loci in FISH,

which has been proved partially by dual-color FISH of

25S rDNA and Cot-1 DNA. This is also the reason that

25S rDNA and Cot-1 DNA were used as chromosome co-

markers. In this study, individual metaphase chromosome

pairs of B. oleracea could be stably banded by Cot-1 DNA.

Based on steady 25S rDNA and Cot-1 DNA chromosome

markers, individual chromosome pairs of B. oleracea

could be identified reliably by dual-color FISH. These two

co-markers exceed any one used alone in chromosome

identification. This technique is a simple, fast, and credible

means of identifying the chromosome set of a species.

The results reported in the present study demonstrate

that Cot-1 DNA FISH can successfully show bands on in-

dividual chromosome pairs in B. oleracea. Moreover, the

Cot-1 DNA FISH banding patterns possessed some impor-

tant features. For example, non-homologous chromosomes

are different, and two members of each chromosome pair

are analogous in the banding patterns. Therefore, based on

Figure 2. 25S rDNA, 5S rDNA and Cot-1 DNA FISH to the metaphases of B. oleracea. A, 25S rDNA showed green fluorescence,

labeled with Digoxigenin-11-dUTP and detected with anti-digoxigenin-fluorescein; B, 5S rDNA showed red fluorescence, labeled with

Biotin-11-dUTP and detected with streptavidin-Cy3; C and D, Cot-1 DNA FISH. Bar, 5 m.

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WEI et al.  Karyotyping of

Brassica oleracea

259

the features of the banding patterns, pairing between two

members of each chromosome pair and recognition among

non-homologs over the entire genome could be performed

easily and effectively. The Cot-1 DNA banding has been

used previously in humans (Wang et al., 1995) and in

Brassica napus (Wei et al., 2005). However, combining

it with rDNA markers in karyotyping has never been at-

tempted. We have successfully completed the karyotyping

of B. oleracea by rDNA locating and Cot-1 DNA banding

for the first time. Obviously, Cot-1 DNA banding could be

further applied to those plants in which karyotyping has

been performed based solely on rDNA locating.

Although Cot-1 bands were confirmed on the long arms

of chromosome pairs 6 and 8, the short arms of chromo-

Figure 3. Dual-colour FISH of 25S rDNA and Cot-1 DNA to the mitotic spreads of B. oleracea. A, 25S rDNA labeled with

Digoxigenin-11-dUTP and detected with anti-digoxigenin-fluorescein showed green fluorescence; B, Cot-1 DNA labeled with Biotin-

11-dUTP and detected with streptavidin-Cy3 showed red fluorescence; C, An overlapping of green fluorescence for 25 rDNA and red

fluorescence for Cot-1 DNA; D, Metaphase of DAPI dyeing corresponding to the spread in C. Bar, 5 m.

Figure 4. Ideogram of Brassica oleracea somatic chromosomes

based on the metaphase plate shown in Figure 3C. Numbers

s how the gene ra l num bering of c hromo som es , which a re

arranged in descending order according to total length taking no

account of the length of chromosome 7 satellite.

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260

Botanical Studies, Vol. 48, 2007

some pairs 2, 4 and 7, and the pericentromeric sites of

chromosome pairs 1, 3, 5 and 9; they are all located in a

domain near the centromere. Sometimes it is difficult to

affirm their sites on long arms, short arms or centromeres.

This question will be probably solved with additional

FISH of the centromere markers. Even now, the specific

Cot-1 band patterns of each chromosome pair provide a

fine maker for chromosome pair identification as rDNA.

Acknowledgements. The authors thank Dr. Robert Hast-

erok (Department of Plant Anatomy and Cytology, The

University of Silesia, Katowice, Poland) for supplying the

25S and 5S rDNA probes and Dr. H. James Price (Depart-

ment of Soil and Crop Science, Texas A & M University,

USA) for supplying his paper on Cot-1 DNA isolation

from plants. This work was supported by the Presidential

Foundation of the Institute of Oil Crops, Chinese Acad-

emy of Agricultural Sciences (200301), the Topping Youth

Foundation of Hubei Province (2005ABB028), and the

Chenguang Program of Wuhan City (20045006071-37).

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