Indian Journal of Biotechnology

Vol.4, July 2005, pp. 307-315

FISH and GISH: Modern cytogenetic techniques

J Devi1*, J M Ko2 and B B Seo3
1
Department of Plant Breeding and Genetics, Assam Agricultural University, Jorhat 785 013, India
2
National Yeongnam Agricultural Experiment Station, Milyang 627 130, Korea
3
Department of Biology, Kyungpook National University, Taegu 702 701, Korea
Received 19 March 2004; revised 23 July 2004; accepted 5 August 2004

In recent years, advances in the molecular cytogenetic technique of fluorescence in situ hybridization (FISH), which
enables the direct chromosomal localization of labelled DNA probes and genomic in situ hybridization (GISH), which
determines the inter-species distribution of repeated sequences have enabled a resurgence of cytogenetic analysis in plant
genome research and molecular breeding. Practical applications of these techniques in chromosome mapping, genome
analysis, determination of phylogenetic relationship, detection of chromosomal aberrations and alien chromatin in plant
breeding programmes, study of chromosome organization at interphase nuclei and analysis of somaclonal variations in tissue
culture have been presented.

Keywords : FISH, GISH, in situ hybridization, rDNA, physical map, probe labelling
IPC Code: Int. Cl.7 A01H1/00

Introduction location of genes or DNA can be visualized on

The age of classical cytogenetics has, however,
been largely superseded by the implementation of
DNA techniques during the past few decades. In situ
hybridization (ISH) is now recognized as an important
technique in many areas of molecular biological
research and its associated clinical studies. The
technique is used to locate the physical position of a
known DNA sequence on a chromosome. In this
technique, treating the cells that have been squashed
on a slide denatures DNA within the cell. The
squashed cells can then be incubated in a solution of
labeled DNA, whose position on a chromosome, we
are interested in knowing. Repeated or unique DNA
sequences, isolated from an organism or artificially
synthesized, can be utilized as radioactively labelled
or biotinylated probes for a study of the location of
these sequences on the chromosomes.
FISH and GISH Techniques
In a modification of in situ hybridization technique,
a fluorescent molecule is deposited at the site of in
situ hybridization. The sites located will exhibit
fluorescence and can be photographed with a
fluorescent microscope. Thus, precise physical
_________________
*Author for correspondence:
Tel: 91-376-2340098; Fax: 91-376-2340101
E-mail: jdevi@aau.ac.in
chromosomes. The technique is popularly described
as FISH (Fluorescence in situ hybridization). The
advantages of FISH over ISH are faster detection,
higher resolution, sensitivity and speed. There has
been considerable refinement in the technique since
its development in the area of human molecular
cytogenetics about a decade ago. A variety of probe-
labelling schemes are now available for simultaneous
detection of two or more sequences in the same
nucleus.
There are two methods for multicolour FISH. The
indirect method uses biotin, digoxigenin and
dinitrophenol (DNP) as reporter molecules. They are
detected by fluorochrome-conjugated avidin or
antibodies. Fluorochrome-labelled nucleotides are
used for probe labeling in the direct method. The
direct coupling of reporter molecules like
fluorochromes to probes eliminates the need for
immunocytochemical detection. Thus, the direct
method has two advantages over indirect method, i.e.,
better resolution and speed. Nederlof et al1 first
described a method of detecting more than three
target. DNA sequences using only three fluorescent
dyes by labelling a probe with more than one hapten
and detecting with more than one fluorochrome.
Using three haptens (single, double, and triple
labelling) and three fluorochromes, in principle, a
total of seven probes should be resolvable. Using
308 INDIAN J BIOTECHNOL, JULY 2005
variable ratios of each hapten could further increase
the total number of probes, which could be detected.
When total genomic DNA (consisting of the entire
nuclear DNA of a plant species) is used as a probe in
hybridization experiments to chromosomal DNA in
situ, the technique becomes known as GISH
(Genomic in situ hybridization). Repeated sequences,
which comprise 40-95% of the genomic DNA in
higher plants reanneal more rapidly than the unique
sequences of the genome. Genomic hybridization
method examines the inter-species distribution and
organization of these sequences. It involves extraction
of genomic DNA from one of the species of interest,
for use as a probe by either Southern hybridization to
DNA digests or in situ hybridization to chromosome
preparations from the species or hybrids being
studied. Many of the DNA sequences within the two
or more genomes under investigation may be
sufficiently different so that genomic probing
discriminates them.
Applications of FISH and GISH
Some very useful studies have been conducted
utilizing these techniques both in animals and plants.
Initially, studies made involved repeated DNA
including sat-DNA from Drosophila and mouse. The
application of in situ hybridization techniques in
plants has lagged behind compared to its use in
mammalian cytogenetics. However, they are now
finding increasing application in plants, especially in
the breeding programmes. Although the first
published work was within Tritiaceae, the method has
been successful in other families of both
monocotyledons and dicotyledons. Some of the
applications of these techniques are cited below.
Chromosome Mapping
The utilization of in situ hybridization technology
is of particular interest to those engaged in
chromosome walking or genome mapping projects.
FISH has been utilized in many plants to identify
chromosome accurately, using species-specific
repetitive sequences, ribosomal genes and even
unique sequences. Because of their universal
occurrence and redundancy, the ribosomal genes are
of great value for karyotype analysis and comparative
studies of genome organizations. FISH techniques
using florochrome allows the visualization of
multigenic families, such as 5S and 18S-5.8S-26S
ribosomal RNA genes for their location on
chromosomes. Physical localization of multicopy
gene families, such as 5S and 18S-26 rRNA genes
have been reported in wheat2, tomato3, barley4, garlic5
and in Aegilopes umbellulata6. In cotton, multicopy
genes were mapped on specific chromosomes in
meiosis7. Recently, FISH been used for the physical
mapping of ribosomal genes, microsatellite and
transposable DNA sequences on sugar beet
chromosome8, 9.
Lee and Seo10 constructed an accurate physical map
showing the localization of the 5S and the 18S-26S
rRNA genes in Allium wakegi by bicolor FISH. The
signal of 5S rDNA was detected on the intercalary
region of short arm of chromosome 15 (one region)
and 9 (two region) while the signal of 18S-26S rDNA
were detected on terminal region of short arm of
chromosome 6, 10 and 14 including satellite and
secondary constriction regions. 5S rDNA has been
reported in the intercalary region for wheat, and
pea11, 12, while in tomato and sugar beet this site is in
the region proximal to the centromere3. The tendemly
array of 5S rDNA sites on the satellite has been also
reported in Vicia faba and Allium sativum5,13.
Nucloelus organiser region (NOR) that is
cytologically identifiable as a secondary constriction
on satellite chromosome contain expressed
18S-5.8S-26S ribosomal genes. Thus, the 18S-26S
rDNA probe was used to confirm the presence of
NORs2,14. In addition to the secondary constriction,
this probe heavily hybridized to the entire satellite in
tomato3, garlic5 and Aegilopes squarossa 2.
A digoxigenin-labelled 5S rDNA probe containing
the 5S rRNA genes and the adjacent intergeneric
spacer was used for in situ hybridization to metaphase
and interphase chromosomes of a trisomic stock from
sugar beet16. Three chromosomes of primary trisomic
line IV revealed the signals close to the centromere.
Polymorphism of 5S rDNA repeats in a segregating
population was used to map genetically the 5S rRNA
genes within a cluster of markers in linkage group II
of sugar beet. The concentration of genetic markers
around the centromeres presumably reflected the
suppressed recombination frequency in centromeric
region. The correlation of physical and genetic data
allowed the assignment of a linkage group to sugar
beet cc IV according to line of the primary trisomic.
Linc et al17 conducted FISH analysis of genomic
constitution of Aegilopes cylindrica Host
(2n=4X=28, DcDcCcCc). One major 18S-5.8S-26S
rDNA loci was identified in the short arm of
chromosome 1Cc, 5Dc, 5Cc, and 1Dc. Chen et al18
 DEVI et al.: FISH & GISH : MODERN CYTOGENETIC TECHNIQUES
attempted direct mapping of a tendemly repeated
sequences MR 68 by FISH to recognize its
distribution on maize chromosome. The sequence was
found to be located on the subtelomeric region of the
long arm chromosome 3 and 6, as well as on the
satellite of chromosome 6. Snowdon et al19 developed
FISH methods for the accurate localization of
repetitive DNA sequences at chromosomal sub-arm
level in Brassica sp. and thus allowing more reliable
chromosome identification and giving new
information on genome structure and evolution.
Genome Analysis
GISH permits characterization of the genome and
chromosome of hybrid plants, allopolyploid species
and recombinant breeding lines. Thus, the ancestry of
hybrid and polyploid species can be elucidated by
genomic southern and in situ hybridization. In
essence, the analysis involves hybridization of
labelled genomic DNA from suggested ancestors or
relatives to chromosome spreads or southern blots of
DNA from the species under investigation.
Hybridization strength, uniformity, and presence of
positive or negative bands are then assessed to
indicate relationships. Traditionally, genome
relationship was analyzed by study of chromosome
painting but there may be several limitations of
chromosome pairing. The amount of pairing not only
depends on the degree of homology between the
pairing chromosomes but also on genetic and
environmental factors.
Multicolour FISH (mFISH) using total genomic
DNA probes is a promising approach for
simultaneously discriminating each genome in natural
or artificial amphidiploids. It uses various
fluorescence dyes to represent different painting
probes at the same time. Moreover, this technique is
powerful tool for investigating genome homology
between polyploid species and their diploid
progenitors. Using fluorescent probes produced by
shearing the total genomic DNA of a particular
progenitor species, it may be possible to identify all
chromosomes belonging to a particular genome of the
amphidiploids.
Multicolour in situ hybridization has been used to
distinguish three genomes in hexaploid wheat20.
Biotinylated total genomic DNA of the diploid A
genome progenitor Triticum urartu, digoxigenin-
labelled total genomic progenitor Aegilopes squarossa
and non-labelled total genomic DNA of one of the
possible B genome progenitor Ae. speltoides were
309
hybridized in situ to metaphase chromosome spreads
of Triticum aestivum cv. Chinese Spring. For
detection, only two fluorochromes, fluorescein and
rhodamine, were used. The A, B, and D genomes
were simultaneously detected by their yellow, blue
and red fluorescence, respectively. Bennett et al21 by
using genomic in situ hybridization, demonstrated the
allopolyploids origin of Milium montianum (2n=22)
and the homology between eight large chromosomes
of this species and M. vernale (2n=8).
Sometimes it is difficult to identify the parental
origin of chromosomes in the inter-
generic/interspecific hybrid or amphidiploids using
total genomic DNA alone as a probe. However,
addition of a large excess of unlabelled genomic
blocking DNA from the species not used as a probe to
the hybridizing solution, improves differentiation
between species of different genera. The genomes of
Hordeum bulbosum and H. vulgare, two closely
related species were clearly distinguished in the
hybrid, both in situ and on southern transfer only after
addition of an excess of the genomic DNA from the
species not used as a probe22. The major effect of the
blocking DNA may be attributed to the hybridizing of
the blocking DNA to the sequence in common
between the blocking DNA, probe DNA, and
chromosomal DNA in situ or bound to the southern
membrane, thereby mainly leaving species-specific
sequences as sites for labelled probes hybridization.
In an intergeneric hybrid between H. chilence and H
africanum, after FISH, the seven large chromosome
of H. africanum were labelled while the seven
chromosomes of H. chilence origin showed little cross
hybridization and were stained with the DNA counter
stain. At interphase, the two parental genomes were
organized in separate domains23.
Phylogenetic Relationship
GISH offers new opportunities in phylogenetic and
taxonomic studies for determining and testing
genomic relationship of wild and cultivated plant
species. It gives unique information about similarities
between DNA from related species. Furthermore, it
provides data about the physical distribution of
sequences, which are common or differ between the
species being probed and the species used to supply
the probe DNA. Together, the information can be
used to support and develop theories about
phylogenic, hybridization, and diversification of plant
species. Since plant breeding involves genomic
reconstitutions, these informations help to plan
310 INDIAN J BIOTECHNOL, JULY 2005
effective breeding programmes designed to transfer
desired genes or gene clusters from alien species into
otherwise superior cultivars of crop plants.
Genomic divergence in Gibasis spp. has been
investigated using GISH24. Despite the similarity of the
karyotypes and close taxonomic affinity of
G. karwinskyana and G. consobrina, probing with
genomic DNA distinguished the chromosomes, and only
the region proximal to each nucleolus organizer was
strongly conserved between the two chromosome sets.
The 5S ribosomal RNA (rRNA) genes of higher
plants are organized into clusters of tandem repeats
with thousands of copies at one or more positions in
the genome. Each repeat consists of a highly
conserved 5S rRNA coding region of approximately
120 base pairs in length and of NTS regions that vary
in size between 100 and 700 base pairs. Most repeats
appear to be uniform in a species. On the basis of the
high degree of stability during the course of evolution,
comparative studies of the nucleotide sequences of
rRNA genes provide a means for analyzing
phylogenetic relationship over a wide range of
taxonomic levels25. The variation in sizes and
sequences of the NTS of the 5S rRNA gene was
found to be useful for the phylogenetic reconstruction
of species26, and to discover differences between
cultivars in barley and wheat, and between the
breeding lines in maize27.
Lee et al28 classified eleven diploid species of
Allium into five types, A to E, based on the
chromosomal localization and distribution patterns of
the 5S rRNA genes by means of FISH. Data on the
amphidiploids with genomes of type B and C, A.
deltoid-fistulosum is an allotetraploid resulting from a
cross between a B type species and a species of an
unknown type. Do and Seo29 demonstrated the
phylogenetic relationship among Allium subgenus
Rhizirideum species based on the molecular variation
of 5S rRNA genes. The size of this region was 120
base pairs in all the species and the sequences were
highly conserved. The size of non-transcribed spacer
(NTS) regions varied from 194 bp in A. deltoid-
fistulosum to 483 bp in A. sativum. Snowdon et al19
applied GISH for identification and characterization
of parental genome components in oilseed rape
(B. napus) hybrids.
Analysis of Somaclonal Variations
Somaclonal variations arising in tissue culture have
been looked upon as a novel source of genetic
variation for crop improvement. Tissue culture phases
may impose stress, and induce instability
(chromosome breakage and the DNA transposition)
leading to karyotyping changes. Genetic instability
may be associated with the fraction of repeated
sequences of DNA present in the plant genome30.
Analysis of genetic variation in the regenerated plants
is necessary for identification and utilization of the
proper somaclonal variation for crop improvement.
Examination of the chromosomal distribution of 5S
and 18S-26S rRNA is useful in identifying the types
of genomic changes that might occur during in vitro
culture31.
Physical map showing the localization of 5S and
18S-26S rRNA genes was constructed by bi-colour
FISH in amphidiploid Allium wakegi cultivar and an
amphidiploid tissue culture regenerant10. A rhodamine
labelled 5S rDNA and a biotin labelled 18S-26SrDNA
were used as probes. The signals of 5S rDNA were
detected on the intercalary region of short arm in
chromosomes 9 (two region) and 15 (one region). The
signals of 18S-26S rDNA were detected on the
terminal region of short arm of chromosome 10 and at
same regions of chromosomes 6 and 14 including the
satellite. In an amphidiploid regenerant, homologous
chromosomes were identified by chromosomal
localization of rRNA gene families.
Using digoxigenin-labelled 5S rRNA and biotin
labelled 18S-26S rDNA gene probes. Lee et al32
compared the FISH patterns of regenerated
autotetraploid plants with the diploid wild type in
Allium cyaneum. The physical localization of rRNA
genes in tetraploid regenerants corresponded with that
of diploid species. Thus, the results of FISH
suggested that tetraploid regenerants originated from
exact doubling of normal diploids.
In a karyotype analysis of somaclonal variants of
A. tuberosum (2n=4X=32), the chromosomal
positions of rRNA genes were physically mapped33.
Both normal A. tuberosum and aneuploid regenerant
(At 30) exhibited two sets of 5S rDNA sites, one on
the proximal position of the short arm of chromosome
3, and the other on the intercalary region on the long
arm of chromosome 6. There was one 18S-5.8S-26S
rDNA site in the terminal regions on the short arm of
chromosome 8 including secondary constriction and
satellite. However, At 30 showed only 3 labelled
chromosomes 8 indicating that this was one of the lost
chromosomes of At 30. Do et al34 further identified
the lost chromosomes in three aneuploid somaclonal
variants of A. tuberosum. Chromosome compositions
 DEVI et al.: FISH & GISH : MODERN CYTOGENETIC TECHNIQUES
of these variants were confirmed as being fixed lines
during two years of greenhouse cultivation. The 5S
rRNA gene signals in all variants as well as the wild
type were detected as two sets while only one
18S-5.8S-26S rRNA gene site was located. The three
lost chromosomes of At 29 variant (2n=29) were all
chromosome 2, the two for At 30 (2n=30) were
chromosome 7 and 8, and At 31 (2n=31) was missing
one of the chromosome 2.
Detection of Alien Chromatin
Interspecific and intergeneric crosses have been
made in plants with the aim of transferring desirable
traits, such as disease and pest resistance from wild or
related species into cultivated species. Following
hybridization if the donor has at least one genome in
common with the recipient, and then the
recombination between the homologous genome in
common with the recipient, then recombination
between homologous genome can readily take place
and, through several cycles of backcrossing and
selection, the desired trait can be transferred. If the
donor and the recipient genomes are not homologous,
the preferred method of handling such hybrid is to
continue backcrossing and chromosome screening to
produce series of addition or substitution lines of the
genome of the donor parent. Chromosome medicated
alien gene transfers through hybridization have
resulted in the genetic improvement of many crops.
Recently, development of direct gene transfer
methods have further helped to engineer genes of
importance into crops.
In plant breeding programme, alien chromosome,
chromosome segments, and genes can be identified
and characterized by FISH and GISH. They can be
visualized and counted in wide hybrids and
amphidiploids, not only in high quality metaphase
spreads, but also within interphase nuclei.
Subsequently, alien chromosomes can be followed
through backcrosses and recombinant lines. FISH
technology has been used to identify partial
amphidiploids derived from crosses of wheat with
Thinopyrum intermedium and Lophopyrum elongatum
with the resistance to BYDV35 and wheat streak
mosaic virus36.
Highly repetitive sequences such as those in 5S loci
have been used to identify certain addition lines of Ae.
umbellulata6. Sequential C banding and GISH have
been used to identify addition lines of Haynaldia
villosum37 and substitution lines of Lophopyrum
ponticum38, while FISH was used to characterize
311
addition lines of L. elongatum showing resistance to
Cephalosporium graminicum38. Pickering et al39
characterized progeny from H. vulgare X H.
bulbosum crosses by GISH, confirmed the presence of
a monosomic alien substitution plant and established
the presence of a distal H. bulbosum introgression on
chromosome 3 HL.
F1 hybrid of a cross T. aestivum cv. Olmil X S.
cereale cv. Paldanghomil was backcrossed to T.
aestivum cv. Olmil as a male parent and progenies
were advanced to BC1F6 generation40. The presence of
rye chromatin was identified in 32 plants put of 467 in
BC1F5 by GISH technique. The analysis showed that
the mode of rye chromatin in these plants was almost
telocentric. FISH at meiosis of wheat lines in BC1F6
generation also depicted the presence of rye
chromatin. Zhang et al41 used GISH to investigate
meiotic crossing-over in hybrids between H.
bulbosum and H. vulgare and FISH with an
oligonucleotide sequence (CCT) 10 followed by
GISH to map introgressions in selfed progeny from
hybrids. GISH established that pairing is intergenomic
and pairing frequency exceeded recombination.
Detection of Chromosomal Aberration
The identification of structural abnormalities by
routine and high-resolution cytogenetic studies plays
an important role in diagnosis and treatment of
disease. However, this analysis is relatively gross and
only permits the visual diagnosis of aberrations of
single chromosome bands on the order of seven
million or so base pairs. ISH technique has felicitated
the diagnosis and identification of chromosomal
aberrations particularly for human and animal
chromosomes. Using chromosome-specific DNA
libraries, it permits the identification of small
chromosome aberrations, which are not readily
detected by standard high resolution banding
techniques. Therefore, this technique may be used in
prenatal and postnatal cytogenetic studies. For
example, women who have an increased risk of
carrying abnormal fetuses can undergo cytogenetic
analysis of fetal cells to rule out chromosomal
aberrations but it requires time. In that case, FISH can
provide a rapid and accurate identification for the
most common autosomal trisomics and sex
chromosome abnormalities. With mFISH analysis all
24 human chromosomes can be hybridized using
fluorochrome labelled chromosome specific DNA
libraries. The advantage of this staining method is the
demonstration of structural aberrations, which cannot
312 INDIAN J BIOTECHNOL, JULY 2005
be detected by conventional staining techniques. The
STARFISH system is another excellent method for
the identification of single human chromosomes, and
allows the detection of translocations and insertions
on metaphase chromosomes.
Recently developed m Band FISH technique yields
high resolution multi-colour banding based on region-
specific partial chromosome paints. The use of m
Band FISH42, 43 was tested to determine the inter- and
intra-arm chromosome exchanges in human.
Altogether, seven overlapping microdissection DNA
libraries of chromosome 5 were constructed, 2 within
the p-arm and 5 within the q-arm.
These techniques may also be used to identify
marker chromosomes, clarify translocations, define
chromosome duplications, or analyze complex
chromosome rearrangements. Analysis at both
meiotic prophase and metaphase 1 gives maximum
amount of information about genetic relationships
between homologous and homeologous chromosomes
in a hybrid, or species where there may be
duplications by identifying pairing partners at the
early meiotic stages. In many polyploid species, there
are intergenomic translocations, which are clearly
shown by GISH.
Genomic in situ hybridization has detected
translocations in fusion hybrids between Nicotiana
plumbaginifolia and gamma irradiated Petunia
hybrida protoplasts44. A combination of Giemsa
banding and GISH has illuminated the karyotype
change, which have taken place in early wheat
evolution45. There was a 4A-5A-7B cyclic
translocation specific to T. turgidum AABB relative
to the hexaploid T. aestivum AABBDD species, and a
different cyclic translocation in T. timopheevii,
genome AAGG, strongly supporting the diphyletic
origin of tetraploid wheats.
Total genomic DNA has been used as a probe from
the putative diploid progenitors to show the presence
of A and C genome in the tetraploid Avena
maroccana46,47. The results demonstrated that
intergenomic translocations are present in
A. maroccana; four C-A translocations were
observed, of which three were nonreciprocal in
nature. A probable A-C translocation was also
observed. Jellan et al47 used GISH to also identify
A/D-C translocations in allotetraploid and hexaploid
oat species. The amphiploid Nicotiana has two
component genomes, designated S and T. Three
different Nicotiana tabacum genotypes showed up to
nine homozygous translocations between
chromosomes of the S and T genomes48.
Chromosome Organization at Interphase Nuclei
Simultaneous visualization of total genomic and
highly repeated DNA as probes is also useful for
investigating chromosome organization in the
interphase nucleus, orientation of telomeres and
centromeres, spatial location of individual
chromosomes, and the relationship between
chromatin decondensation and gene expression49.
Leitch50 examined the structural organization of
interphase nuclei using a range of examples from the
plants, animals and fungi and showed nuclear
organization to be an important phenomenon in cell
differentiation and development. FISH was used to
simultaneously visualize specific chromosomal
regions and functional nuclear domains and to
elucidate the relationship between specific
chromosome arrangements and nuclear functions
especially the extent to which changes in higher-order
nuclear organization are implicated in the
etiopathogenesis of human disease51, 52. A few studies
have investigated the arrangement of telomeres or
centromeres in interphase cells by in situ
hybridization53, 54. Plant telomeric sequences have
been cloned from Arabidopsis thaliana55 and
tomato56, but no plant centromeric sequences have
been cloned. The translocation line of wheat,
4AS-6RL.4AL has a good centromere marker. A tiny
segment of rye chromatin is inserted near the
centromere of wheat chromosome 4A57.
Centromere-specific multi-colour FISH
(cenM-FISH) is a new technique that allows the
simultaneous characterization of all human
centromeres by using labelled centromeric satellite
DNA as probes. This approach allows the rapid
identification of all human centromeres by their
individual pseudo-colouring in one single step58.
Barley interphase nuclei showed strong polar
arrangement of chromosomes with telomeres and
centromeres located at the opposite nuclear poles
(Rabl-orientation), as shown by two-colour FISH
experiments using the barley subtelomeric 118 bp
repeat HvTO1 and a BAC containing centromere-
specific retroelements and satellite sequences59.
Chromosome Specific Painting in Plants
Determination of karyotype based on chromosome
size, centromeric index and banding patterns is
limited by the similar morphology of chromosomes in
 DEVI et al.: FISH & GISH : MODERN CYTOGENETIC TECHNIQUES
many species. FISH or PRINS (primed in situ
labelling) overcomes these limitations by providing
specific labelling patterns useful for discrimination of
similar chromosomes. Additional cytogenetic
landmarks can be obtained using species- or genus-
specific satellite repeats that are often amplified to
high copy numbers and form discrete bands or spots
on chromosomes. These repeats frequently occur at a
higher number of genomic loci and may therefore
produce signals characteristic for each chromosome
within the karyotype. Successful painting of a specific
plant chromosome wthin its own genome was
reported by Vega et al60. Dissected isochromosomes
for the long arm of chromosome 5 of the wheat B
genome (5BL) were amplified and used as probes.
Hybridization signal data suggested that chromosome
and homoeologous group-specific sequences are more
abundant in 5BL than in genome specific sequences.
FISH had been used61 to analyse the structure of the
rye B chromosome. GISH demonstrated high level of
similarity between A and B chromosomes of rye. The
B-specific repeat families D1100 and E 3900 were
analysed in terms of their physical location and
possible contiguity.
Accurate identification of individual chromosomes
of Secale montanum Guss was achieved using
simultaneous and (or) successive FISH and
C-banding62. FISH identification was performed using
total rye DNA, three highly repetitive rye DNA
sequences (pSc 119.2, pSc74, and pSc34), and the
ribosomal RNA probes pTa71 and pTa794. Three
families of highly repeated sequences from rye and
the rRNA multigenes (NOR and 5S) have been
mapped63 by FISH and C-banding, in chromosomes of
triticale. The pSc 119.2 probe showed intestitial
hybridization in chromosome arms 1RS, 1RL, 4Rl,
5RL, 6RL, 6RS, 6RL, 7RS and 7RL and was very
effective for chromosome identification of rye
chromosomes in triticale. Numbers and positions of
hybridization signals provided cytogenetic landmarks
suitable for unambiguous identification of all
chromosomes, and establishment of the karyotypes in
four Vicia species (V. sativa, V. grandiflora,
V. pannonicica and V. narbonensis)64.
These techniques have been useful for the
simultaneous mapping of different DNA sequences
and genome allocation of genes of interest. The
genome probing methods supplement data from other
methods of genomic analysis, gives complementary
and novel data about genomes and their relationships,
313
including identification of parents or ancestors in
unknown crosses or in polyploid species, information
about genomic regions which have diversified
between species, and enabling clear identification of
pairing partners at meiosis and evolutionary
translocations between genomes in polyploid and
hybrids. Application of FISH to somaclonal variants
is a useful tool for identifying and understanding
chromosomal changes during the tissue culture
process.
However, there are some limitations of these
techniques. Multicolour FISH can only be used
successfully on polyploids with at least one known
progenitor species. Closely related genomes in certain
allopolyploids cannot be discriminated using GISH
technique. Multicolour FISH is less sensitive and
shows a lower degree of detection resolution than
single colour FISH due to multiple exposure
photographs65.
Advances in mammalian genetics involving the use
of techniques outlined above provide promise for
future progress in plant molecular cytogenetic
research. There is no doubt that the applications of
these techniques will multiply in coming years and
enable the investigation of even more difficult
problems of biology.
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