Download A new FISH protocol with increased sensitivity for

Journal of Experimental Botany, Vol. 51, No. 346, pp. 965–970, May 2000
A new FISH protocol with increased sensitivity for
physical mapping with short probes in plants
Flavia Guzzo, Evelyn Campagnari and Marisa Levi1
University of Verona, Dipartimento Scientifico e Tecnologico, Strada le Grazie 15, Cà Vignal 1, 37134 Verona,
Italy
Received 25 October 1999; Accepted 21 January 2000
Abstract
Fluorescence in situ hybridization (FISH) is a wellestablished technique used for the detection of
specific DNA regions, that has been applied to
interphase nuclei, pachytene and metaphase chromosomes as well as to extended DNA fibres. This technique allows the physical mapping of specific DNA
sequences both on individual chromosomes and
extended fibres. A new FISH protocol is described here
that enhances the sensitivity of the method. Probes
for small unique DNA sequences of less than 2 kb give
high signal-to-noise ratio with this method, and can
be visualized easily by means of conventional fluorescence microscopy.
Key words: FISH, Asparagus officinalis, physical mapping.
Introduction
Fluorescence in situ hybridization ( FISH ) is a wellestablished technique to detect specific DNA regions,
which can be used for many purposes. Large probes, such
as those derived from BAC and YAC libraries, have been
successfully used to mark chromosomes in species such
as barley (Lapitan et al., 1997) and rice (Ohmido et al.,
1998). Unfortunately, the use of large probes on plants
with a large and complex genome (11–40 pg of DNA)
resulted in a very high background, probably because of
the presence, in the plant genome, of dispersed repetitive
sequences (Fuchs et al., 1996).
FISH has also been used extensively in plants to analyse
interphase nuclei, pachytene and metaphase chromosomes
(Morais-Cecilio et al., 1996; Heslop-Harrison et al., 1993;
Shen et al., 1987; Zhong et al., 1996), in order to
investigate the disposition of chromatin in interphase
nuclei, and to map DNA sequences on chromosomes.
Mapping resolution is higher on pachytene chromosomes
than on metaphase chromosomes (Shen et al., 1987;
Zhong et al., 1996), due to their less contracted structure;
it is even higher in interphase nuclei (Lawrence et al.,
1988, 1990; Trask et al., 1989, 1991). In the latter case,
however, the possibility of identifying individual chromosomes is lost. The use of FISH on extended DNA fibres
further enhances the physical mapping resolution (de
Jong et al., 1999), due to the high linear stretching degrees
of chromatin, evaluated to be around 3.27 kb mm−1,
according to Fransz et al. (Fransz et al., 1996a).
The sensitivity of the FISH techniques so far developed
has allowed the identification of single DNA sequences
as small as 0.25 kb on human chromosomes (Richards
et al., 1994). However, FISH on plants with small probes
( less than 10 kb) seems to be more difficult to perform.
The reports on FISH with probes for small single copy
DNA targets (1–2 kb) are limited in number. The best
results have been obtained by Fransz and coworkers
( Fransz et al., 1996b), who were able to recognize a
1.4 kb DNA fragment on Petunia metaphase chromosomes; by Ten Hoopen and coworkers ( Ten Hoopen
et al., 1996), who recognized a 1.18 kb unique sequence
(a flavonol synthase gene) on Petunia chromosomes and
by Ohmido and coworkers (Ohmido et al., 1998), who
mapped a 1.29 kb RFLP marker on rice chromosomes.
In these works, images were mainly captured with a
cooled CCD camera and often digitally imaged to enhance
the faint signal intensity; direct microscopy images showed
low signal-to-noise ratio.
Within a project devised to study sex determinants in
Asparagus officinalis L., a dioecious species in which males
are heterogametics and sex determinants are located on
the L5 long homomorphic chromosome pair (Loptien,
1979; Biffi et al., 1995), a FISH method was set up which
1 To whom correspondence should be addressed. Fax: +39 45 8027929. E-mail: [email protected]
© Oxford University Press 2000
966 Guzzo et al.
led to the detection of probes shorter than 2 kb, directed
to single copy DNA sequences, on chromosomes and
interphase nuclei, without the help of special detection
systems (such as CCD camera and digital imaging).
Three probes were used, named h23 (1.2 kb), d47
(1.4 kb) and s9 (1.7 kb), generated as RFLP fragment;
the latter ones (d47 and s9) are supposed to be linked to
the sex locus (Spada et al., 1998).
The method described in this paper enabled strong,
specific and reproducible signals, to be obtained with very
low background, which led to a preliminary physical
mapping of short nucleotidic sequences related to traits
of great agronomic and biological importance on
Asparagus officinalis.
Materials and methods
Plant material
Eight-day-old seedlings from two diploid cultivars of Asparagus
officinalis L. ( EROS, all male seeds, XY, and SIRIO, 50% male
seeds, XY, and 50% female seeds, XX ) and from one tetraploid
cultivar ( VIOLETTO DI ALBENGA, 50% male seeds, XYXY,
and 50% female seeds, XXXX ) were used. Seeds were kindly
provided by Dr Agostino Falavigna.
Nuclei preparation and prehybridization
2 mm long root tips were excised and fixed in 4% methanolfree formaldehyde ( Polyscience) in PBS on ice for 15 min and
rinsed three times with the same buffer. Nuclei were extracted
as previously described (Levi et al., 1994); they were spread on
slides and air-dried, post-fixed for 10 min with 4% formaldehyde
in PBS, rinsed three times in PBS and twice in 2× SSC. Slides
were then treated with 200 mg ml−1 RNase-A (Fluka) in 2×
SSC at 37 °C for 1.5 h, washed in the same buffer, then treated
for 10 min with 1 mg ml−1 Proteinase K (Boehringer Mannheim)
in PBS at 37 °C, washed again in PBS, and finally dehydrated
in ethanol series up to ethanol 100% and air-dried.
Probes
Three pUC19 plasmids were used, containing three different
DNA fragments, d47 (1.4 kb), s9 (1.7 kb) and h23 (1.2 kb),
isolated as RFLP fragments, the former two supposed to be
linked to the sex locus. The DNA probes were labelled with
digoxigenin-11-dUTP (Boehringer Mannheim) by Random
Priming (DIG DNA Labelling Kit, Boehringer Mannheim) and
by PCR.
In situ hybridization
Denaturation of nuclear DNA was performed by soaking slides
in 70% deionized formamide in 2× SSC at 72 °C for 5 min,
followed by dehydration with chilled ethanol series (70%, 90%,
100%), 5 min each, and air-drying. Slides were afterward
incubated with 15 ml of previously denatured (3 min at 80 °C )
hybridization mix (2 ng ml−1 Dig-DNA probe, 100 ng ml−1
herring DNA, 100 ng ml−1 yeast RNA, 10% (w/v) dextran
sulphate, 50% deionized formamide in 2× SSC ), for 16 h at
42 °C, in a moist chamber. After hybridization, slides were
washed three times for 5 min in 50% formamide in 4× SSC at
45 °C, and three times for 5 min in 2× SSC at room
temperature.
The hybridization signal was revealed by the ‘Fluorescent
Antibody Enhancer Set for Dig detection’ (Boehringer
Mannheim), according to the instructions of the supplier.
Subsequently, slides were air-dried and mounted with
Vectashield
antifade
containing
5 mg ml−1
DAPI
(4∞,6-diamidino-2-phenylindole) or 1 mg ml−1 propidium iodide
for counterstaining of DNA.
Analysis of the hybridization signal
Hybridization signals were observed with a Leica DM RB
fluorescence microscope, which had been equipped with filter
blocks for DAPI (excitation filter: 340–380 nm; beam splitter:
400 nm; barrier filter: 430 nm), for fluorescein (excitation filter:
470–490 nm; beam splitter: 510 nm; barrier filter: 520 nm) and
for simultaneous detection of fluorescein and propidium iodide
(excitation filter 1: 490/20 nm; beam splitter 1: 505 nm; barrier
filter 1: 525/20 nm; excitation filter 2: 575/30 nm; beam splitter
2: 600 nm; barrier filter 2: 635/40 nm). Photographs were taken
with Scotch 650-T-ASA colour slide film. When DAPI counterstaining was used, two photographs of the same field were
taken with the two different filters for DAPI and for fluorescein.
Flow cytometry
Nuclei extracted from root tips as described above were stained
in suspension with 0.5 mg ml−1 DAPI and analysed for their
relative DNA content with a Bryte HS flow cytometer. The
following filter blocks were used: excitation block: excitation
365 nm, beam splitter 400 nm, emission >420 nm; separator
block (BR): emission 1 450–490 nm, beam splitter 560 nm,
emission 2 >590 nm.
Fluoresbryte fluorescent beads (Polyscience) with a 4.5 mm
diameter were used as the inner standard.
Results
Setting up the FISH technique
After some initial attempts with traditional cell squashes
which gave unsatisfactory results (high background and
poor signal ), nuclei isolated from root tips and smeared
on slides (Levi et al., 1986) were used in order to reduce
the penetrability problems of probes and antibodies and
their non-specific binding. Fixation in formaldehyde
instead of ethanol/acetic acid was necessary for good
isolation of nuclei.
Dig-11-dUTP-labelled probes were initially produced
by Random DNA Priming, but great improvement of the
labelled probe/template ratio was obtained when labelled
probes were produced by PCR (1000 ng of labelled probe
from 0.01 ng of template, compared with 20 ng of labelled
probe from 20 ng of template obtained with Random
DNA Priming).
The hybridization buffer, the timing and temperature
of the probe and target denaturation were optimized,
and, particularly crucial for signal enhancement was the
strict control of the duration and temperature of target
DNA denaturation (slides had to be treated for at least
5 min at a temperature not lower then 72 °C, followed by
an ethanol dehydration to prevent DNA target renaturation). Different signal detection systems were tested:
FISH with increased sensitivity 967
neither anti-Dig Rhodamin or anti-Dig alkaline phosphatase followed by the HNPP Fluorescent Detection set
(Boehringer Mannheim) gave visible signals.
When HNPP was used after a cascade of antibodies it
gave a very high background.
The best results were obtained with the ‘Fluorescent
Antibody Enhancer Set for Dig detection’ (Boehringer
Mannheim), which uses a cascade amplification signal by
three different antibodies, the third one conjugated with
fluorescein.
Detection of the single copy sequences s9, d47 and h23
with FISH
The hybridization experiments were set up with nuclei
from seedling root tips of two different diploid and one
tetraploid Asparagus cultivars. Two different probes were
used, s9 (1.7 kb) and d47 (1.4 kb). s9 and d47 are two
DNA fragments present in single-copy in the Asparagus
genome (A Spada, personal communication). The labelled
nuclei showed a variable number of signals, visible as
brilliant spots; they could be documented by regular
photomicroscopy (Figs 1, 2). The background was very
low; negative controls, in which the probe was omitted,
showed very few spots or none at all ( Fig. 2; Table 2).
Mitotic chromosomes were occasionally present among
the nuclei; some of the long ones were labelled, the spots
being located towards the distal ends of the chromosomes
( Fig. 3). Hybridization with the h23 probe (1.2 kb) also
gave strong signals (not shown).
Determination of specificity of hybridization
To determine the hybridization efficiency and specificity,
the number of spots/nucleus was determined on nuclei
from both diploid and tetraploid cultivars hybridized with
d47 and s9 ( Tables 1, 2). Such determination was
Fig. 1. FISH of s9 (a) and d47 (b) on interphase diploid (a) and
tetraploid (b) nuclei of Asparagus. Fluorescein hybridization signals
are clearly evident over the red propidium iodide counterstained nuclei.
These micrographs, with strong and brilliant signals and a lack of any
background outside the nuclei, were obtained with a regular photocamera. Bar=12 mm.
Fig. 3. FISH of d47 (a) and s9 (b, c, d ) on metaphase chromosomes.
Both d47 and s9 give signals to the end of a long chromosome. All the
micrographs are captured with a photocamera; (a) is the direct image
of the red propidium iodide counterstained chromosome with the
fluorescein signal. (b, c, d) These are the result of a digital overlay of
two photographs showing the fluorescein signals and the blue DAPI
counterstained chromosomes. Bar=15 mm.
Table 1. Efficiency of hybridization in the diploid ‘Eros’ and
tetraploid ‘Violetto di Albenga’ cultivars, expressed as a percentage of labelled nuclei on the total number of scored nuclei
Material
Fig. 2. (a, b) Diploid nuclei hybridized with d47, showing no signals
( lack of hybridization), two signals (the expected amount) and three
signals, of which at least one could be spurious. (c) Diploid nucleus
hybridized with s9, showing two signals. Negative controls (d, e) do
not show any signal. Bar=15 mm.
Diploid plants
d47
s9
Negative control
Tetraploid plants
d47
s9
Negative control
% of Labelled nuclei
Total no. of nuclei
51.7
46.3
0.01
319
518
122
62.3
71.1
0.022
522
881
153
968 Guzzo et al.
Table 2. Specificity of the hybridization on diploid ‘Eros’ and tetraploid ‘Violetto di Albenga’ cultivars, determined counting the number
of spots/nucleus
Nuclei with a different number of spots are expressed as a percentage of labelled nuclei. Since 2 spots are expected on the diploid nuclei and 4 on
the tetraploid nuclei, the percentage of nuclei showing a number of spots within the expected range (from 1 to 2 in the diploid, from 1 to 4 in the
tetraploid) provides an estimation of the specificity of the method.
Material
Diploid plants
d47
s9
Negative control
Tetraploid plants
d47
s9
Negative control
Percentage of nuclei with different number of spots
3 spots
4 spots
>4 spots
% of nuclei with
number of spots
within the
expected range
% of nuclei with
number of spots
exceeding the
expected range
1 spot
2 spots
64.8
53.3
0.01
23.7
30.4
0.0
9.7
8.8
0.0
0.6
5.0
0.0
1.2
2.5
0.0
88.5
83.7
99.99
11.5
16.3
0.01
33.0
18.7
0.01
28.0
37.2
0.0
19.8
12.2
0.006
12.3
17.8
0.006
6.9
14.1
0.0
93.1
85.9
99.978
6.9
14.1
0.022
achieved by excluding the minor areas on the slide where
the hybridization failed entirely (no signal at all ), and
the minor areas in which too many signals were present
both on and out of the nuclei (Morais-Cecilio et al.,
1997). Table 1, which shows the percentage of labelled
nuclei, gives an estimation of the hybridization efficiency,
that is between 45% and 70% depending on the probe
and on the material.
Table 2 shows the distribution of the number of spots
per nucleus and the percentage of nuclei showing the
number of signals within the expected range (two in
nuclei from diploid, four in nuclei from tetraploid plants).
The data suggest that a number of nuclei, between 7%
and 16% depending on probe and on plant material,
could show non-specific spots. Negative controls, where
the probe was omitted, failed to show any hybridization,
suggesting that a non-specific signal within the labelled
nuclei occurred because of non-specific hybridization
rather than non-specific antibody attachment.
Flow cytometry
Since DNA endoreduplication phenomena are quite
common in plants and could be responsible for additional
spots, flow cytometry analysis was performed on nuclei
from diploid and tetraploid plants used for in situ experiments. Cytograms of nuclei extracted and labelled with
DAPI, with fluorescent beads as the internal standard,
are shown in Fig. 4. In nuclei from diploid material about
8% of nuclei had a DNA content higher than 4C, and
about 4% an 8C DNA content, while in tetraploid material about 2% of the nuclei had a DNA content higher
than 8C.
Discussion
Hybridization conditions
FISH detection sensitivity, which can be defined as the
smallest DNA sequence to be detected unambiguously
(de Jong et al., 1999), can be enhanced by increasing the
power of detection tools, such as CCD cameras and
digital imaging, and/or by increasing the signal intensity.
This study worked on enhancing signal intensity, and
within this parameter on optimizing target accessibility,
labelling and hybridization conditions. The optimization
of the technique was made possible because, under nonoptimal conditions, few spots were detectable and their
number and intensity increased slightly when the individual steps were improved. This allowed better experimental conditions to be chosen one by one, but only
when all the optimal conditions were used simultaneously
was it possible to observe a dramatic increase in the
number and intensity of the signals. For this reason it
was not possible to assign a relative importance to any
single step of the procedure. What was definitively clear
is that some steps (denaturation of target DNA, use of
probes produced by PCR, use of an antibody cascade
signal amplification) were particularly crucial for enhancing the intensity and number of signals, whereas other
steps (use of nuclei isolated from root tips instead of cell
squashes, the kind of antibody and of detection system
used in the cascade signal amplification) proved to be of
particular importance in decreasing the background. This
new FISH protocol gave a very high signal-to-noise ratio.
Most of the positive results reported so far with probes
shorter than 2 kb ( Fransz et al., 1996b; Ten Hoopen
et al., 1996; Ohmido et al., 1998) were obtained by means
of capturing images with a cooled CCD camera which
enhanced signal intensity and, occasionally, by resorting
to digital imaging which enhanced the contrast. Fransz
et al. also reported images taken with conventional photomicroscopy, but their signal-to-noise ratio was much
lower than the one revealed in these experiments (Fransz
et al., 1996b). Ohmido et al. emphasized the fact that
effective detection equipment, such as a cooled CCD
camera and digital imaging, is essential in capturing the
faint signal given by short probes (Ohmido et al., 1998).
PCR-produced probes proved to be better than probes
FISH with increased sensitivity 969
Priming labelled probes. The use of isolated nuclei instead
of squashed cells as starting material enabled the problems
of penetrability of such longer probes to be overcome.
This probably also decreased background problems, since
it is common knowledge that cytoplasm displays a sticky
and trapping property in any in situ reaction.
Furthermore, as some authors have discovered, in some
conditions the presence of the cytoplasm not only
increases background signal, but also prevents hybridization or signal detection (Fuchs et al., 1996). The strict
control of target denaturation conditions was also critical
for signal detection. The temperature as well as the
duration of the treatment had to be strictly controlled.
The immersion of the slides in the denaturation solution
caused a transient decrease of the temperature that had
to be accurately checked by an efficient temperature probe
directly dipped in the solution; the 5 min of treatment
were calculated when 72 °C were re-achieved. A shorter
period at 72 °C caused a dramatic decrease in the number
of spots. Ethanol dehydration was performed to prevent
the target DNA renaturation.
FISH efficiency and specificity, and s9 and d47 mapping
Fig. 4. Biparametric cytograms of ‘Eros’ diploid nuclei (a) and ‘Violetto
di Albenga’ tetraploid nuclei (b), stained with DAPI and analysed by
flow cytometry. In (a) nucleus populations with 2C, 4C and 8C DNA
content are shown by their fluorescence emission (FL1) and forward
light scattering (FS ). The population (b) represents the inner standard
(fluorescent beads having a diameter of 4.5 mm). In (b) the tetraploid
4C and 8C DNA content nuclei are evident, while only few of them
have a DNA content higher than 8C.
produced by Random Priming. An efficient hybridization
involving a short single-copy DNA should probably
require that both DNA strands are capable of hybridizing
for the entire length with the labelled probe. This is
probably better achieved with the PCR labelled probes,
since they have a very high labelled probe/template DNA
ratio, and a constant length, rather than with Random
The efficiency of the method devised, calculated as the
percentage of labelled nuclei, was roughly between 50%
and 70%. Unfortunately, these data cannot be compared
with those of other protocols, since their efficiency is not
normally reported. The lack of visible signals on part of
the nuclei could depend on various factors, such as: lack
of hybridization on both DNA target strands; hybridization on one single filament, that could result in a too
faint signal; excess of counterstaining fluorescence of the
nuclei that could mask signal fluorescence.
A number of spots lower than expected could depend
as well on a reduced hybridization efficiency; in the
interphase nuclei a single spot could also depend on
signal overlay.
The specificity of the FISH signals was suggested by
the absence of spots on negative controls and by the
number of spots/nucleus in the labelled nuclei. Since
FISH, in interphase nuclei, does not allow discrimination
between individual chromatids, and hence between G1
and G2 nuclei (Morais-Cecilio et al., 1997), in principle,
two spots were expected in nuclei from the diploid cultivar
and four in nuclei from the tetraploid cultivar. The
percentage of labelled nuclei with the number of spots
within the expected range was always higher than 80%.
The specificity could be even higher since a certain
percentage of nuclei with additional spots might be due
to nuclei which had undergone endoreduplication events
as shown by flow cytometry.
This high specificity was confirmed by the pattern of
hybridization which occurred on metaphase chromosomes. Both with d47 and s9 signals were only found
970 Guzzo et al.
towards the end of the long chromosomes: in the integrated genetic map of Asparagus officinalis published
recently (Spada et al., 1998) the two markers are located
close together (1.6 cM ) and close to the putative sex
locus, which maps toward one end of the linkage group
1, corresponding to the sexual chromosome 5, which
belongs to the group of the long chromosomes.
The use of this method for the detection of unique
short sequences in material enriched in metaphase plates
will allow association between markers of different linkage
groups and their chromosomes. This will also allow
the study of the correlation between genetic recombination frequencies and physical distances within the
chromosomes.
Acknowledgements
This research was supported by MIPA (Ministero per le
Politiche Agricole). The authors are grateful to Dr A Falavigna
(Istituto Sperimentale per l’Orticoltura, Montanaso Lombardo),
Professor G Marziani and Dr A Spada (Dipartimento di
Biologia, Università di Milano), for kindly providing seeds and
probes, and to Dr P Portaluppi for helpful discussions and
revision of the manuscript.
References
Biffi R, Restivo FM, Tassi F, Caporali E, Carboni A, Marziani
GP, Spada A, Falavigna A. 1995. A restriction fragment
length polymorphism probe for early diagnosis of gender in
Asparagus officinalis L. Horticultural Science 30, 1463–1464.
de Jong JH, Fransz P, Zabel P. 1999. High resolution FISH in
plants–techniques and applications. Trends in Plant Science
4, 258–263.
Fransz PF, Alonso-Blanco C, Liharska TB, Peeters AJM,
Zabel P, de Jong HJ. 1996a. High resolution physical
mapping in Arabidopsis thaliana and tomato by fluorescence
in situ hybridization to extended DNA fibres. The Plant
Journal 9, 421–430.
Fransz PF, Stam M, Montijn B, Ten Hoopen R, Wiegant J,
Kooter JM, Oud O, Nanninga N. 1996b. Detection of singlecopy genes and chromosome rearrangements in Petunia
hybrida by fluorescence in situ hybridization. The Plant
Journal 9, 767–774.
Fuchs J, Houben A, Brandes A, Schubert I. 1996. Chromosome
‘painting’ in plants—a feasible technique? Chromosoma
104, 315–320.
Heslop-Harrison JS, Leitch AR, Schwarzacher T. 1993. The
physical organization of interphase nuclei. In: HeslopHarrison JS, Flavell RB, eds. The chromosome. Oxford: BIOS
Publishers, 221–232.
Lapitan NLV, Brown SE, Kennard W, Stephens JL, Knudson
DL. 1997. FISH physical mapping with barley BAC clones.
The Plant Journal 11, 149–156.
Lawrence JB, Singer RH, McNeil JA. 1990. Interphase and
metaphase resolution of different distances within the human
dystrophin gene. Science 249, 928–932.
Lawrence JB, Villnave CA, Singer RH. 1988. Sensitive highresolution chromatin and chromosome mapping in situ:
presence and orientation of two closely-integrated copies of
EBV in a lymphoma line. Cell 52, 51–61.
Levi M, Sparvoli E, Corbetta N. 1994. An antibody against a
sequence of human topoisomerase II gives different immunofluorescence patterns in quiescent and proliferating nuclei of
Pisum sativum L. Journal of Experimental Botany 45,
1157–1162.
Levi M, Tarquini F, Sgorbati S, Sparvoli E. 1986. Determination
of DNA content by static cytofluorimetry in nuclei released
from fixed plant tissue. Protoplasma 132, 64–68.
Loptien H. 1979. Identification of the sex chromosome pair
in asparagus (Asparagus officinalis L.). Zeitschrift für
Pflanzenzuchtung 82, 162–173.
Morais-Cecı́lio L, Delgado M, Jones RN, Viegas W. 1996.
Painting rye B chromosomes in wheat: interphase chromatin
organization, nuclear disposition and association in plants
with two, three or four Bs. Chromosome Research 4, 195–200.
Morais-Cecı́lio L, Delgado M, Jones RN, Viegas W. 1997.
Interphase arrangement of rye B chromosomes in rye and
wheat. Chromosome Research 5, 177–181.
Ohmido N, Akiyama Y, Fukui K. 1998. Physical mapping of
unique nucleotide sequences on identified rice chromosomes.
Plant Molecular Biology 38, 1043–1052.
Richards F, Vogt N, Muleris M, Malfoy B, Dutrillaux B. 1994.
Increased FISH efficiency using APC probes generated by
direct incorporation of labelled nucleotides by PCR.
Cytogenetics and Cell Genetics 65, 169–171.
Shen DL, Wan ZF, Wu M. 1987. Gene mapping on maize
pachytene chromosomes by in situ hybridization. Chromosoma
95, 311–314.
Spada A, Caporali E, Marziani G, Portaluppi P, Restivo FM,
Tassi F, Falavigna A. 1998. A genetic map of Asparagus
officinalis based on integrated RFLP, RAPD and AFLP
molecular markers. Theoretical and Applied Genetics 97,
1083–1089.
Ten Hoopen R, Robbins TP, Fransz PF, Montijn BM, Oud O,
Gerats AGM, Nanninga N. 1996. Localization of T-DNA
insertions in Petunia by fluorescence in situ hybridization:
physical evidence for suppression of recombination. The
Plant Cell 8, 823–830.
Trask B, Massa H, Kenwrick S, Gitschier J. 1991. Mapping of
human chromosome Xq28 by two-color fluorescence in situ
hybridization of DNA sequences to interphase cell nuclei.
American Journal of Human Genetics 48, 1–15.
Trask B, Pinkel D, van den Engh G. 1989. The proximity of
DNA sequences in interphase cell nuclei correlated to genomic
distance and permits ordering of cosmids spanning 250
kilobase pairs. Genomics 5, 710–717.
Zhong X, de Jong JH, Zabel P. 1996. Preparation of tomato
meiotic pachytene and mitotic metaphase chromosomes
suitable for fluorescence in situ hybridization (FISH ).
Chromosome Research 4, 24–28.