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Identification of the entire chromosome

complement of bread wheat by two-colour
FISH. Genome
ARTICLE in GENOME NOVEMBER 1997
Impact Factor: 1.56 DOI: 10.1139/g97-077 Source: PubMed

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Carsten Pedersen

Peter Langridge

University of Copenhagen

University of Adelaide

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Identification of the entire chromosome

complement of bread wheat by
two-colour FISH

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C. Pedersen and P. Langridge

Abstract: Using the Aegilops tauschii clone pAsl together with the barley clone pHvG38 for two-colour fluorescence in situ
hybridization (FISH) the entire chromosome complement of hexaploid wheat was identified. The combination of the two probes
allowed easy discrimination of the three genomes of wheat. The banding pattern obtained with the pHvG38 probe containing the
GAA-satellite sequence was identical to the N-banding pattern of wheat. A detailed idiogram was constructed, including
73 GAA bands and 48 pAsl bands. Identification of the wheat chromosomes by FISH will be particularly useful in connection
with the physical mapping of other DNA sequences to chromosomes, or for chromosome identification in general, as an
alternative to C-banding.
Key words: Triticum aestivum, chromosome identification, fluorescence in situ hybridization, repetitive DNA sequences.

RCsumC : En employant comme sondes les clones pAsl de 1'Aegilops tauschii et pHvG38 de l'orge pour rCaliser une hybridation
in situ ii fluorescence (FISH) bicolore, il a CtC possible d'identifier tous les chromosomes du blC hexaploi'de. La combinaison de
ces deux sondes permet de distinguer aisCment les trois gCnomes du blC. Le motif de bandes obtenu avec la sonde pHvG38,
laquelle contient la sCquence satellite GAA, est identique au motif des bandes N chez le blC. Un idiogramme dCtaillC a CtC produit
incluant les 73 bandes GAA et les 48 bandes pAsl. L'identification des chromosomes du blC par hybridation FISH sera
particuli5rement utile pour rCaliser cargtographie physique de siquences ou encore pour l'identification chromosomique en
gCnCra1, comme alternative ii la coloration des bandes C.
Mots clels : Triticum aestivum, identification chromosomique, hybridization in situ ii fluorescence, sCquences d' ADN rCpCtCes.

[Traduit par la RCdaction]

Introduction
Chromosome identification by C-banding is widely used in
wheat cytogenetic research, and a standard karyotype has been
described (Gill et al. 1991). The in situ hybridization technique
provides a method to localize genes or DNA sequences
directly on chromosomes and has been used intensively for
physical mapping of repetitive DNA sequences and multicopy
and low-copy gene families in plants (Jiang and Gill 1994).
While C-banding has the advantage that all constitutive
heterochromatin is detected, in situ hybridization offers the
opportunity to relate repetitive DNA sequences to distinct
chromosomal sites. Further, two-colour or multicolour fluo-

Corresponding Editor: J.P. Gustafson.

Received December 10, 1996. Accepted April 10, 1997.

C. Pedersen. Environmental Science and Technology

Department, Plant Genetics, Rise National Laboratory,
P.O. Box 49, DK-4000 Roskilde, Denmark.
P. ~angridge.'ARC Special Research Centre for Basic and
Applied Plant Molecular Biology, Department of Plant Science,
Waite Campus, University of Adelaide, Adelaide, South Australia
5064, Australia.
Author to whom all correspondence should be addressed
(e-mail: plangridge @ waite.ade1aide.edu.a~).
Genome, 40: 589-593 (1997)

rescence in situ hybridization (FISH) enables physical mapping of a sequence of interest and chromosome identification
using one or more repetitive sequences simultaneously. The
chromosomal distribution of several repetitive DNA families
has been studied in wheat. The DNA sequences providing the
best banding patterns by in situ hybridization to wheat chromosomes include the Aegilops tauschii clone pAs 1, which
permits identification of the D-genome chromosomes
(Rayburn and Gill 1986, 1987), the rye clone pSc 119.2, which
especially hybridizes to B-genome chromosomes (McIntyre et
al. 1990; Mukai et al. 1993), and the GAA-satellite sequence,
which has multiple hybridization sites on the B-genome chromosomes and some minor sites on A- and D-genome chromosomes (Dennis et al. 1980: Pedersen et al. 1996). However, it
has not been possible to identify the whole chromosome complement by in situ hybridization alone. Mukai et al. (1993)
combined pAs 1 and pSc119.2 for two-colour FISH and were
able to identify 17 of the 21 wheat chromosome pairs. The
hybridization pattern of the GAA-satellite sequence on barley
chromosomes has previously been studied in detail (Pedersen
et al. 1996). It was found that the GAA-satellite sequence
hybridizes to the class of heterochromatin that is stained by
N-banding. In this study we present a detailed wheat molecular karyotype based on both the GAA-satellite sequence and
the pAsl sequence, and show that together ,these two
sequences permit identification of all wheat chromosomes by
two-colour FISH.
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Genome, Vol. 40, 1997

Materials and methods

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Plant material and root-tip and chromosome preparation

Root tips were obtained from seedlings of Triticum aestivum L. cv.
Chinese Spring (CS) and from the ph2a deletion mutant of CS kindly
provided by Prof. Moshe Feldman (Plant Genetics Institute, Israel).
The cell divisions were synchronized with hydroxyurea (Pan et al.
1993). First, the seedlings were germinated on moist filter paper in
plastic boxes at 25C and, when the primary root emerged, were
transferred to filter paper containing 1.25 mM hydroxyurea. After
about 18 h, the seedlings were rinsed in water and incubated on filter
paper with water for another 5.5 h to release the cells from the DNA
block. Subsequently, the roots were pretreated with 4 pM APM (amiprophos-methyl, Miles Ltd.) for 2 h, and with ice water for 20 h,
before fixing in 3: 1 ethanol - glacial acetic acid.
Root tips were digested with 2% cellulase (Sigma), 0.5% cellulase
(Onozuka RlO), and 20% liquid pectinase (Sigma) at 37C for 1 h.
Chromosome preparations were made by the air-drying method
(Olin-Fatih and Heneen 1992). After digestion, the enzyme solution
was carefully removed and replaced with distilled water for 5 min.
The water was then replaced with fresh fixative (absolute ethanol glacial acetic acid 3:l). The meristematic part of the root tip was
transferred to an acid-cleaned slide using a pipette, spread in a drop of
fixative with a pair of tweezers, and air-dried.

DNA probes and hybridization mixture

The barley clone pHvG38 contains the GAA-satellite sequence
(Pedersen et al. 1996), and the clone pAsl contains a 1 kb repetitive
DNA sequence from Aegilops tauschii (Rayburn and Gill 1986). The
inserts of the plasmids were labelled with biotin- 14-dATP or digoxigenin- 11-dUTP by nick translation and mixed to a final concentration
of 1 pg/mL in the hybridization solution, which contained 50% formamide, 10% dextran sulphate, 2x SSC ( l x SSC: 0.15 M Nacl plus
0.015 M sodium citrate), 0.1% SDS, and 50 pg/mL salmon sperm
DNA. The two labelled probes were mixed 1:1 prior to hybridization,
and both combinations of label and clone were tried.

FISH
The slides were incubated in 0.2 pg/mL proteinase K for 10 min at
37"C, washed twice with 2x SSC, and subsequently treated with
100 pg/mL DNase-free RNase in 2x SSC for 1 h at 37"C, washed in
2x SSC, dehydrated in ethanol, and air-dried. Chromosomal DNA
was denatured in 0.2 N HCl at 37C for 10 min, as described previously (Pedersen and Linde-Laursen 1994). The probe mixture was
denatured at 95C for 5 min and chilled on ice. About 50 pL of the
probe mixture was applied per slide, covered with a 24 x 50 mm
cover slip, sealed, and incubated in a moist chamber at 37C overnight. Cover slips were removed and the slides were washed twice in
2x SSC at 37C for 10 min, once in 0 . 2 SSC
~ at 45C for 10 min, and
once in 2x SSC at 37C before transferring to the detection buffer
(4x SSC plus 0.2% Tween 20). The slides were blocked in detection
buffer containing 5% BSA for about 15 min.
For detection of biotin-labelled and digoxigenin-labelled probes
simultaneously, the slides were first incubated with 5 pg/mL
ExtrAvidin-FITC (fluorescein isothiocyanate) (Sigma) in detection
buffer with 5% BSA and 1 pg/mL monoclonal anti-digoxigenin
(Boehringer Mannheim). After washing, the slides were incubated
with 5 pg/mL biotinylated Anti-avidin (Sigma) and 5 pg/mL antimouse Ig digoxigenin (Boehringer Mannheim). The third incubation
was with 5 pg/mL ExtrAvidin-FITC and 5 pg/mL anti-digoxigenin
rhodamine (Boehringer Mannheim). Each incubation was done in a
moist chamber at 37C for 45 min and this was followed by washing
the slides 3 times in detection buffer. After the final washing step, the
slides were dehydrated in 70 and 100% ethanol (3 min each) and airdried. The slides were mounted in an antifade solution (Krenik et al.
1989) containing 0.4 pg/mL 4',6-diamidino-2-phenylindole(DAPI),

or both DAPI and propidium iodide (0.25 pg/mL) when only FITC
detection was used.

Microscopy, photography, and construction of idiograms

The slides were examined with a Zeiss Axiophot microscope
equipped for epifluorescence with the filter combinations for DAPI
and FITC, and a dual band filter for observation of FITC and
rhodamine simultaneously. Photographs were taken on Kodak
Ektachrome P1600 films for colour slides or on Fujicolor 400 Super
HGV films for colour prints.
Chromosomal distances were estimated on the basis of slide projections to a magnification of ca. 20 000 times. The fraction lengths
(FLs) (%) of hybridization sites were calculated as the distance from
the centromere to the hybridization signal relative to the total length
of the chromosome arm. The idiogram of the CS karyotype was constructed using 10 FL measurements of each band. The length of the
individual chromosomes are from Gill et al. (1991).

Results and discussion

For two-colour FISH, the probe pHvG38 was labelled with
biotin and detected with FITC (Fig. 1b) and the pAsl-probe
was labelled with digoxigenin and detected with rhodamine
(Fig. la). The two probes produced hybridization bands on all
wheat chromosomes, with multiple bands on most of the chromosomes. The pHvG38 probe containing the GAA-satellite
sequence hybridized particularly strongly to the B-genome
chromosomes and the pAsl probe hybridized well to the
D-genome chromosomes. Hence, it was easy and fast to
distinguish the three genomes.
The pHvG38 probe alone produced several bands of high
intensity on all B-genome chromosomes, one to a few bands
on the A-genome chromosomes, except IA, and one or two
bands on chromosomes ID, 2D, and 7D (Figs. lb, 2, and 3).
The identification of these chromosomes was based on the
previously described similarity between N-banding patterns
(Endo and Gill 1984) and in situ hybridization with the GAAsatellite sequence (Dennis et al. 1980; Pedersen et al. 1996).
The GAA-banding pattern alone (Fig. 2) enabled identification of 16 chromosome pairs. This study confirmed that the in
situ banding pattern obtained with the GAA-satellite sequence
is similar to the N-banding pattern in wheat, as has been
shown in all other Triticeae species investigated in detail
(Pedersen et al. 1996). The GAA-satellite sequence has previously been used for identifying the chromosomes of barley
(Pedersen and Linde-Laursen 1994). It is present in high-copy
number in Hordeum, Dasypyrum, Aegilops, Elymus species
containing the H genome, and Triticum species containing a
B-genome (Pedersen et al. 1996).
The pAsl probe especially detected bands on D-genome
chromosomes (Figs. 1 and 3), and the pAs 1-banding pattern
described here is in general agreement with Mukai et al.
( 1993), though we detected a few more bands. The hybridization pattern enabled identification of chromosome 1A and all
D-genome chromosomes. The pAsl sequence has previously
been useful for describing and identifying chromosomes of
wheat (Rayburn and Gill 1986; Mukai et al. 1993) and
Aegilops species (Badaeva et al. 1996).
Together these two probes identify all the chromosomes of
CS in 7 metaphases studied in detail and in the ph2a deletion
mutant (Fig. 1) . Only chromosomes 1A and 3A had only one
band, but the A-genome chromosomes were in general quite
easy to identify despite the few bands produced. The few
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Pedersen and Langridge

591

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Fig. 1. FISH to metaphase chromosomes of the ph2a deletion mutant of 'Chinese Spring'. (a)The pAsl probe detected with rhodamine (red)
viewed with a dual filter permitting observation of the yellow FITC signals. (b) The pHvG38 probe detected with FITC (yellow-green). The
strongest pAsl signals (red) are also visible. (c) Chromosomes counterstained with DAPI. Scale bars = 10 pm. Fig. 2. FISH to chromosomes
of 'Chinese Spring' with pHvG38 detected with FITC.The chromosomes are counterstained with propidium iodide. Scale bar = 10 pm.

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Fig. 3. Idiograrn of chromosomes of 'Chinese Spring' wheat showing the locations of pAsl (red bands) and pHvG38 (green bands) on drawn
chromosomes side by side with FISH photographs of the same chromosomes. Chromosomes hybridized with the GAA-satellite sequence
(pHvG38) and counterstained with propidium iodide are on the right of the drawn chromosomes, while two-colour FISH photographs with both
probes are shown pairwise on the left of chromosomes 4A, 5A, 7A, lB, 3B, 6B, 7B, and 2D. The pAsl sites detected with FITC (green) or
rhodarnine (red) are all on the left. Chromosomes are drawn after Gill et al. (1991).

I I:,

NOR

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Pedersen and Langridge

pAsl-bands on some of the A- and B-genome chromosomes
and the GAA bands on chromosomes ID, 2D, and 7D were
often helpful for identifying these chromosomes. Thus, by
combining the two sequences it is possible to identify all the
chromosomes of hexaploid wheat. T o our knowledge, it is the
first report achieving this in wheat by in situ hybridization.
For constructing the idiogram (Fig. 3), 73 GAA bands and
48 pAs1 bands were recorded on chromosomes of CS. Some
of the stronger bands tended to coalesce and were often seen as
one wide band. It was especially pronounced with rhodamine
detection of digoxigenin-labelled probes (Figs. 1 and 3). In
such cases, the measurements were done on chromosomes
with weaker hybridization signals.
Although polymorphisms in C- and N-banding patterns are
common between cultivars of wheat, the overall banding patterns are similar (Endo and Gill 1984). Therefore, the technique used here for chromosome identification in CS will
probably permit identification of all chromosomes in most
wheat cultivars.
Identification of wheat chromosomes by FISH is particularly useful in connection with the physical mapping of other
DNA sequences to chromosomes. The sequence of interest
may be labelled with a third dye for identification by threecolour fluorescence detection (Wiegant et al. 1993). Alternatively, the sequence of interest is first hybridized alone, and
afterwards, the chromosome preparations showing specific
signals can be probed again (Heslop-Harrison et al. 1992;
Pedersen and Linde-Laursen 1994) with the two repetitive
sequences. Other applications include chromosomal characterization of lines with chromosomal abnormalities, such as
aneuploid lines or deletion lines, as indicated here with the
ph2a deletion mutant (manuscript in preparation). In such
cases the two-colour FISH technique serves as an alternative
to C-banding. The idiogram presented here (Fig. 3) will be
useful in this connection.

Acknowledgement
The work was supported by a grant from the Danish Agricultural and Veterinary Research Council to C.P.

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