Botanical Studies (2008) 49: 33-37.

*

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

cn; lan_ty1982@yahoo.com.cn; Tel: +022-2350-8241; Fax:

+022-2349-7010.

INTRODUCTION

Ginkgo biloba is a dioecious gymnosperm species

with both male and female plants having 2n = 2x = 24

chromosomes, consisting of four metacentrics and twenty

subtelocentrics. Several studies on the sex determination

system and sex chromosomes of ginkgo have appeared

since the 1950s. Newcomer (1954) and Chen et al. (1987)

both reported the ZW-type sex chromosome system might

be present in ginkgo. Chen et al. (1987) found that the size

of the Ag-NOR on chromosome 1 from females is different

while it is the same from the male. So far, however, no

further study on the chromosome 1 has been reported.

The direct strategy for isolating sequences from

chromosomes of interest is to separate them by a flow-

sorting procedure, or by microdissection. A chromosome

microdissection technology was developed in 1981

(Scalenghe et al., 1981).

Subsequently, it has evolved

into an efficient tool for generating chromosome specific

DNA libraries of many species (Ponelies et al., 1997;

Thalhammer et al., 2004).

Chromosome painting refers to the hybridization of

fluorescently-labeled, chromosome specific, composite

probe pools to cytological and structural chromosomal

aberrations with high sensitivity and specificity (Ried

et al., 1998). The concept of chromosome painting was

first introduced in 1988 (Lichter et al., 1988; Pinkel

et al., 1988). It has over the last few years become an

established procedure in laboratories working with

mammalian chromosomes (Antonacci et al., 1995).

In plants, however, chromosome painting is relatively

underdeveloped. In plants, to ensure specific hybridization

to related chromosome segments, repetitive sequences

need to be excluded from the hybridization process by,

for example, blocking with a large excess of unlabelled

total genomic DNA or the Cot-1 fraction of genomic

DNA (Houben et al., 2002). Painting of sex chromosomes

has been performed in Rumex acetosa by Shibata et al.

(1999) and in Silene latifolia by Hobza et al. (2004).

Hobza et al. (2004) used a modified FAST-FISH protocol

based on a short hybridization time combined with a low

concentration of probe and succesfully distinguished the

sex chromosomes by differential labelling patterns.

Here, by applying microdissection and painting the

W chromosome, we found a different labelling region,

meaning a different sequence structure, there on the sex

chromosome.

MATERIALS AND METHODS

Plant materials and chromosome preparation

Root tips and tender buds of 15 male and 12 female

ginkgo plants were used in this study. Slides were prepared

by cell wall degradation hypotonic method according to

Chen et al. (1979) with minor modifications. In brief, root

tips were removed and immersed successively in saturated

Microdissection and painting of the W chromosome in

Ginkgo biloba showed different labelling patterns

T. Y. LAN

1

, R. Y. CHEN

1

, X. L. LI

1

, F. P. DONG

1

, Y. C. QI

2

, and W. Q. SONG

1,

*

1

Laboratory of Chromosome Research, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China

2

College of Life Sciences, Peking University, Peking 100000, P.R.China

(Received April 26, 2007; Accepted August 16, 2007)

ABSTRACT.

The chromosome 1 with bigger satellite, supposed to be

the W chromosome, was

microdissected from the metaphase spreads of female ginkgo root-tip cells with a fine glass needle controlled

by a micromanipulator. The dissected chromosome was amplified in vitro by the Sau3A linker adaptor

mediated PCR (LA-PCR) technique. Southern hybridization analysis indicated that DNA from the single W

chromosome was successfully amplified. FISH analyses with the PCR products have been performed in the

metaphase spreads of female and male ginkgo. FISH signals were observed along the entire W chromosome,

while along about 1/2 length at the end of the long arm of the Z chromosome, the intensity of signals was

lower than at other segments. This suggested that the chromosome 1 of ginkgo might harbor different DNA

sequence structures at the 1/2 length end of the long arm, and this region on the W chromosome might be a

female-specific region which formed during evolution of the sex chromosomes.

Keywords: Chromosome painting; FISH; Ginkgo biloba; Microdissection; Sex chromosome.

CYTOGENETICS

34

Botanical Studies, Vol. 49, 2008

para-dichlorobenzene for 3 h, rinsed in double-distilled

water for 30 min, treated with 2.5% (w/v) cellulose

and pecitinase (Sigma, Germany) for 30 min, rinsed in

double-distilled water for 15 min, and finally fixed in 70%

ethanol for 5 min. The fixed material was put on the sterile

coverslip (22¡Ñ60-mm) with a drop of 70% ethanol, using

a microscope slide as a carrier to stabilize the coverslip.

The material was torn into fine pieces and the debris was

removed. Two drops of 70% ethanol were added on the

slide and then dried with hot air. Metaphase spreads for

FISH were prepared on a common microscope slide.

Microdissection and LA-PCR amplification

Each air-dried chromosome specimen was immediately

used for microdissection. The target chromosome, the

W chromosome, was isolated using an traditional light

microscope (BH-2, Olympus, Japan) equipped with a

micromanipulator (MMO-203, Narishige, Japan) and

transferred by a fine glass needle pulled by an OC-10

puller (Narishige, Japan) into a 0.5 ml tube according to Li

et al. (1998).

The Sau3A linker adaptors, with the sequences

5¡¦-CGGGAATTCTGGCTCTGCGACATG-3¡¦ and 5¡¦-

GATCCATGTC-3¡¦ were prepared as described by Deng

et al. (1992). Isolated chromosomal DNA was treated in

10 £gl of 50 ng/£gl proteinase K (Merck, Germany) solution

at 37¢XC for 2 h. The proteinase K was then inactivated at

65¢XC for 20 min. The chromosomal DNA was digested

by Sau3A (0.002U in 1¡ÑT4 ligase buffer, Takara, Japan)

at 37¢XC for 2 h. The Sau3A was inactivated at 65¢XC for

20 min, and 20 £gM of prepared Sau3A linker adaptors

and 1U of T4 ligase (Takara, Japan) were added. The

ligation between the adaptors and digested chromosomal

DNA was performed at 16¢XC for 16 h. The first round

of PCR was carried out in the same tube by adding 10

£gl of 10¡ÑTaq buffer, 6 £gl of 25 mM MgCl

2

, 2 £gl of 10

mM dNTPs, 6 £gl of 10 £gM 24-mer primer, 2.5 U of Taq

DNA polymerase (Takara, Japan), and distilled water to

100 £gl. PCR amplifications were performed using the

following programme: after denaturation at 94¢XC for 5

min, amplification was performed with 35 cycles of 1 min

at 94¢XC, 1 min at 56¢XC, and 2.5 min at 72¢XC, followed by

a final extension at 72¢XC for 10 min. The second round of

PCR was carried out using 2 £gl of the first round products

as template. The method was the same as described above,

except that 20 cycles of amplification were carried out.

To monitor possible extraneous DNA contamination,

we maintained a negative control (no template DNA)

and a positive control (1 pg genomic DNA as template)

throughout the whole process.

Southern blot hybridization analysis

The ginkgo genomic DNA was isolated from leaf

tissue using the CTAB method according to Murray

and Thompson (1980). DNA molecular weight was

checked for quality and quantity by agarose gel (0.8%)

electrophoresis and fluorometry (ND-1000, NanoDrop,

America). Appropriate amounts of Sau3A digested

genomic DNA of female and male plants, and the second

rounds PCR products from chromosome and controls were

transferred onto nylon membranes (Pall, American) after

0.8% agarose gel electrophosis and hybridization with

DIG-labeled ginkgo genomic DNA at 42¢XC for 16 h. The

membranes were washed with 0.2¡ÑSSC containing 0.1%

SDS at 65¢XC for 30 min. Labeling and detection were

performed following the instruction of the Roche DIG

High Prime DNA Labeling and Detection Starter Kit II

(Roche, Germany).

Fluorescence in situ hybridization (FISH)

FISH was carried out as described by Qi et al. (2002)

with minor modifications. The second round PCR products

were labeled with DIG-dUTP (Roche, Germany) by

randomly-primed DNA synthesis. Hybridization buffer

contained 50% deionized formamide; 2¡ÑSSC; 50 mM

sodium phosphate, pH 7.0; 5% dextran sulfate; 10 ng

£gl

-1

probe and 200 ng £gl

-1

unlabelled total genomic DNA.

The probe and the unlabelled total genomic DNA were

mixed and denatured at 94¢XC for 10 min before being

used. Slides with metaphase spreads were treated with

70% deionised formamide in 2¡ÑSSC at 70¢XC for 2 min.

Denatured hybridization buffer (10 £gl) was then applied to

the slides, which were incubated at 37¢XC for 2 h. Finally,

the slides were washed with 30% deionised formamide in

2¡ÑSSC at 37¢XC for 5 min and twice in 2¡ÑSSC at 37¢XC for

5 min. Metaphase spreads were counterstained with 100 ng

ml

-1

4¡¦, 6-diamidino-2-phenylindole (DAPI). Fluorescence

signal was detected using anti-DIG-fluorescent-conjugate

(Roche). The hybridization signals were visualized and

recorded using Nikon 80i fluorescent microscope and a

cooled CCD camera and were then processed using Adobe

Photoshop.

RESULTS

Individual W chromosome microdissection

Using root tips and tender buds as source material and

cell wall degradation hypotonic method, we succeeded in

preparing good-quality slides of chromosomes in ginkgo.

As shown in Figure 1 A, chromosomes were spread

evenly on the slide with a low background. The somatic

chromosome number was 2n=2x=24, consisting of four

metacentrics and twenty subtelocentrics. Chromosome 1,

which harbors a satellite, was the biggest chromosome. In

male, the satellites of chromosome 1 are homoeomorphic,

while in female they are heteromorphic, and one is

apparently bigger than the other

(Figure 1 A). The

chromosome 1 with the bigger satellite was microdissected

from the metaphase spreads of female ginkgo root-tip cells

with fine glass needle controlled by a micromanipulator

(Figure 1)

and

then

used for two rounds of LA-PCR

amplification. The southern blot analyses of the second

round PCR products confirmed that the products were

amplified from the ginkgo genome (Figure 2).

LAN et al. ¡X Microdissection and painting of W chromosome

35

Chromosome painting of W chromosome

Blocking with twenty-fold excess of unlabelled

genomic DNA, the DIG-labeled second-round LA-PCR

products originating from individual microdissected

chromosome 1 were hybridized to mitotic metaphase

spreads. Signals were mainly observed along the entire W

chromosome while along about 1/2 length at the end of the

long arm of the Z chromosome, the signals were weaker

than at other parts of the Z chromosome. At the same

time, some signals were also observed on the terminal,

centromeric, or other regions of other chromosomes

(Figure 3).

DISCUSSION

In plants, no specific painting of the chromosomes

was obtained although a number of different approaches,

including pre-hybridization with a large excess of

total unlabelled genomic DNA, were tested. Several

experiments with DOP-PCR amplified probes from

microdissected chromosomes or chromosome regions

hybridized to metaphase chromosome complements,

unequivocally revealed dispersed hybridization signals

Figure 1. Isolation of an individual chromosome 1 in ginkgo by micromanipulator. A: Mitotic metaphase spread of ginkgo before

microdissection. A suitable chromosome 1 with big satellite was found under the inverted microscope (arrow); B: The target

chromosome adhering to the tip of a glas s needle; C: The target chromosome was rem oved from the slide. Bar=5, 10, 5 £gm,

respectively.

Figure 2. Southern blot hybridization analysis of second-round

LA-PCR products with DIG-labeled genomic ginkgo DNA.

Lane 1 was the negative control (no DNA template in LA-PCR),

lane 2-3 were the Sau3A digested total genomic ginkgo DNA

of female and male respectively, lane 4-5 were the LA-PCR

products, and lane 6 was the positive control (genomic DNA as

template in LA-PCR).

Figure 3. Chromosome painting patterns on female (A) and male (C) metaphase chromosomes of ginkgo using DIG-labeled second-

round LA-PCR products. chromosome marked as "a" was chromosome 1 with big satellite while "b, c, d" were chromosome 1 with

small satellite. FISH signals on the segments marked with arrows in B were apparently weaker than other segments of chromosome 1.

Bar=5 £gm.

36

Botanical Studies, Vol. 49, 2008

on all chromosomes in the case of Vicia faba, Hordeum

vulgare, Triticum aestivum, Picea abies and Petunia

hybrida (Fuchs et al., 1996). In our study, we modified the

FISH procedure including changing the hybridization time

and the amount of DNA probe or using unlabelled genomic

DNA for blocking (data not shown). The results were still

similar to the cases described above that all chromosomes

harbor signals. However a clear difference emerged

between the intensity of the signal on the chromosome

of probe origin and that of the other chromosomes. Such

uniform hybridization patterns were found irrespective of

the blocking conditions and the concentrations of genomic

DNA used in these experiments. The number of repetitive

sequences in plants is too large for efficient blocking by

conventional prehybridization procedures. In the nuclear

genome of higher plants, especially in the repetitive

sequence, most cytosine are methylated. The unmethylated

loci maily disperse in the low and single copy sequence

regions. Methylation sensitive restriction enzyme cannot

recognize the methylated cytosine loci. If it could, then

low and single copy sequences would be relatively rich

in the products. Many researchers used the methylation

sensitive restriction enzyme such as Sau3A to digeste the

DNA. The LA-PCR method also can concentrate the low

copy sequences, and it can amplify longer fragments than

DOP-PCR.

In plants, chromosome painting is possible only

on specialized chromosomes, such as the B and Y

chromosome or on chromosomal regions that contain

specific highly repetitive sequences. Painting of sex

chromosomes has been successfully performed in Rumex

acetosa by Shibata et al. (1999) and in Silene latifolia by

Hobza et al. (2004). In Rumex acetosa, strong signals were

abserved on the Y

1

andY

2

chromosomes, and weak signals

were also observed on the X chromsome and autosomes.

In Silen latifolia, Hobza et al. (2004) found that the X

chromosome probe revealed a clear signal on the entire X

chromosomes while the signal on the other chromosomes,

including the Y, was of lower intensity. Similar results

were obtained with the Y chromosome probe when the Y

chromosome was strongly labeled. The different labeling

patterns of sex chromosomes showed that the composition

of the DNA sequences in the X and Y chromosome differs.

In our study, different labelling patterns have been shown

on the sex chromosomes of Ginkgo biloba. Along about

1/2 length at the end of the long arm of the Z chromosome,

the signals were weaker than at other parts of chromosome

1. This indicated that the composition of DNA sequences

in the W chromosome and the Z chromosome might

differ, especially in the parts where the FISH signal

intensity differed.

We believed this part on W chromosome

became specialized as a result of the accumulation of

chromosome-specific repetitive sequences in the process

of sex chromosome evolution. It would harbor sex-specific

sequences and might be a non-recombining region.

Further study, such as construction of a W chromosome-

specific genomic library and isolation of female specific

DNA markers would reveal more information about the

evolution and divergence of the sex chromosomes in

Ginkgo biloba.

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