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Fish and Gish: Modern Cytogenetic Techniques: J Devi, Jmko Andbbseo
Fish and Gish: Modern Cytogenetic Techniques: J Devi, Jmko Andbbseo
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
variable ratios of each hapten could further increase gene families, such as 5S and 18S-26 rRNA genes
the total number of probes, which could be detected. have been reported in wheat2, tomato3, barley4, garlic5
When total genomic DNA (consisting of the entire and in Aegilopes umbellulata6. In cotton, multicopy
nuclear DNA of a plant species) is used as a probe in genes were mapped on specific chromosomes in
hybridization experiments to chromosomal DNA in meiosis7. Recently, FISH been used for the physical
situ, the technique becomes known as GISH mapping of ribosomal genes, microsatellite and
(Genomic in situ hybridization). Repeated sequences, transposable DNA sequences on sugar beet
which comprise 40-95% of the genomic DNA in chromosome8, 9.
higher plants reanneal more rapidly than the unique Lee and Seo10 constructed an accurate physical map
sequences of the genome. Genomic hybridization showing the localization of the 5S and the 18S-26S
method examines the inter-species distribution and rRNA genes in Allium wakegi by bicolor FISH. The
organization of these sequences. It involves extraction signal of 5S rDNA was detected on the intercalary
of genomic DNA from one of the species of interest, region of short arm of chromosome 15 (one region)
for use as a probe by either Southern hybridization to and 9 (two region) while the signal of 18S-26S rDNA
DNA digests or in situ hybridization to chromosome were detected on terminal region of short arm of
preparations from the species or hybrids being chromosome 6, 10 and 14 including satellite and
studied. Many of the DNA sequences within the two secondary constriction regions. 5S rDNA has been
or more genomes under investigation may be reported in the intercalary region for wheat, and
sufficiently different so that genomic probing pea11, 12, while in tomato and sugar beet this site is in
discriminates them. the region proximal to the centromere3. The tendemly
array of 5S rDNA sites on the satellite has been also
Applications of FISH and GISH reported in Vicia faba and Allium sativum5,13.
Some very useful studies have been conducted Nucloelus organiser region (NOR) that is
utilizing these techniques both in animals and plants. cytologically identifiable as a secondary constriction
Initially, studies made involved repeated DNA on satellite chromosome contain expressed
including sat-DNA from Drosophila and mouse. The 18S-5.8S-26S ribosomal genes. Thus, the 18S-26S
application of in situ hybridization techniques in rDNA probe was used to confirm the presence of
plants has lagged behind compared to its use in NORs2,14. In addition to the secondary constriction,
mammalian cytogenetics. However, they are now this probe heavily hybridized to the entire satellite in
finding increasing application in plants, especially in tomato3, garlic5 and Aegilopes squarossa 2.
the breeding programmes. Although the first A digoxigenin-labelled 5S rDNA probe containing
published work was within Tritiaceae, the method has the 5S rRNA genes and the adjacent intergeneric
been successful in other families of both spacer was used for in situ hybridization to metaphase
monocotyledons and dicotyledons. Some of the and interphase chromosomes of a trisomic stock from
applications of these techniques are cited below. sugar beet16. Three chromosomes of primary trisomic
line IV revealed the signals close to the centromere.
Chromosome Mapping
The utilization of in situ hybridization technology Polymorphism of 5S rDNA repeats in a segregating
is of particular interest to those engaged in population was used to map genetically the 5S rRNA
chromosome walking or genome mapping projects. genes within a cluster of markers in linkage group II
FISH has been utilized in many plants to identify of sugar beet. The concentration of genetic markers
chromosome accurately, using species-specific around the centromeres presumably reflected the
repetitive sequences, ribosomal genes and even suppressed recombination frequency in centromeric
unique sequences. Because of their universal region. The correlation of physical and genetic data
occurrence and redundancy, the ribosomal genes are allowed the assignment of a linkage group to sugar
of great value for karyotype analysis and comparative beet cc IV according to line of the primary trisomic.
studies of genome organizations. FISH techniques Linc et al17 conducted FISH analysis of genomic
using florochrome allows the visualization of constitution of Aegilopes cylindrica Host
multigenic families, such as 5S and 18S-5.8S-26S (2n=4X=28, DcDcCcCc). One major 18S-5.8S-26S
ribosomal RNA genes for their location on rDNA loci was identified in the short arm of
chromosomes. Physical localization of multicopy chromosome 1Cc, 5Dc, 5Cc, and 1Dc. Chen et al18
DEVI et al.: FISH & GISH : MODERN CYTOGENETIC TECHNIQUES 309
attempted direct mapping of a tendemly repeated hybridized in situ to metaphase chromosome spreads
sequences MR 68 by FISH to recognize its of Triticum aestivum cv. Chinese Spring. For
distribution on maize chromosome. The sequence was detection, only two fluorochromes, fluorescein and
found to be located on the subtelomeric region of the rhodamine, were used. The A, B, and D genomes
long arm chromosome 3 and 6, as well as on the were simultaneously detected by their yellow, blue
satellite of chromosome 6. Snowdon et al19 developed and red fluorescence, respectively. Bennett et al21 by
FISH methods for the accurate localization of using genomic in situ hybridization, demonstrated the
repetitive DNA sequences at chromosomal sub-arm allopolyploids origin of Milium montianum (2n=22)
level in Brassica sp. and thus allowing more reliable and the homology between eight large chromosomes
chromosome identification and giving new of this species and M. vernale (2n=8).
information on genome structure and evolution. Sometimes it is difficult to identify the parental
origin of chromosomes in the inter-
Genome Analysis generic/interspecific hybrid or amphidiploids using
GISH permits characterization of the genome and total genomic DNA alone as a probe. However,
chromosome of hybrid plants, allopolyploid species addition of a large excess of unlabelled genomic
and recombinant breeding lines. Thus, the ancestry of blocking DNA from the species not used as a probe to
hybrid and polyploid species can be elucidated by the hybridizing solution, improves differentiation
genomic southern and in situ hybridization. In between species of different genera. The genomes of
essence, the analysis involves hybridization of Hordeum bulbosum and H. vulgare, two closely
labelled genomic DNA from suggested ancestors or related species were clearly distinguished in the
relatives to chromosome spreads or southern blots of hybrid, both in situ and on southern transfer only after
DNA from the species under investigation. addition of an excess of the genomic DNA from the
Hybridization strength, uniformity, and presence of species not used as a probe22. The major effect of the
positive or negative bands are then assessed to blocking DNA may be attributed to the hybridizing of
indicate relationships. Traditionally, genome the blocking DNA to the sequence in common
relationship was analyzed by study of chromosome between the blocking DNA, probe DNA, and
painting but there may be several limitations of chromosomal DNA in situ or bound to the southern
chromosome pairing. The amount of pairing not only membrane, thereby mainly leaving species-specific
depends on the degree of homology between the sequences as sites for labelled probes hybridization.
pairing chromosomes but also on genetic and In an intergeneric hybrid between H. chilence and H
environmental factors. africanum, after FISH, the seven large chromosome
Multicolour FISH (mFISH) using total genomic of H. africanum were labelled while the seven
DNA probes is a promising approach for chromosomes of H. chilence origin showed little cross
simultaneously discriminating each genome in natural hybridization and were stained with the DNA counter
or artificial amphidiploids. It uses various stain. At interphase, the two parental genomes were
fluorescence dyes to represent different painting organized in separate domains23.
probes at the same time. Moreover, this technique is
powerful tool for investigating genome homology Phylogenetic Relationship
between polyploid species and their diploid GISH offers new opportunities in phylogenetic and
progenitors. Using fluorescent probes produced by taxonomic studies for determining and testing
shearing the total genomic DNA of a particular genomic relationship of wild and cultivated plant
progenitor species, it may be possible to identify all species. It gives unique information about similarities
chromosomes belonging to a particular genome of the between DNA from related species. Furthermore, it
amphidiploids. provides data about the physical distribution of
Multicolour in situ hybridization has been used to sequences, which are common or differ between the
distinguish three genomes in hexaploid wheat20. species being probed and the species used to supply
Biotinylated total genomic DNA of the diploid A the probe DNA. Together, the information can be
genome progenitor Triticum urartu, digoxigenin- used to support and develop theories about
labelled total genomic progenitor Aegilopes squarossa phylogenic, hybridization, and diversification of plant
and non-labelled total genomic DNA of one of the species. Since plant breeding involves genomic
possible B genome progenitor Ae. speltoides were reconstitutions, these informations help to plan
310 INDIAN J BIOTECHNOL, JULY 2005
effective breeding programmes designed to transfer may impose stress, and induce instability
desired genes or gene clusters from alien species into (chromosome breakage and the DNA transposition)
otherwise superior cultivars of crop plants. leading to karyotyping changes. Genetic instability
Genomic divergence in Gibasis spp. has been may be associated with the fraction of repeated
investigated using GISH24. Despite the similarity of the sequences of DNA present in the plant genome30.
karyotypes and close taxonomic affinity of Analysis of genetic variation in the regenerated plants
G. karwinskyana and G. consobrina, probing with is necessary for identification and utilization of the
genomic DNA distinguished the chromosomes, and only proper somaclonal variation for crop improvement.
the region proximal to each nucleolus organizer was Examination of the chromosomal distribution of 5S
strongly conserved between the two chromosome sets. and 18S-26S rRNA is useful in identifying the types
The 5S ribosomal RNA (rRNA) genes of higher of genomic changes that might occur during in vitro
plants are organized into clusters of tandem repeats culture31.
with thousands of copies at one or more positions in Physical map showing the localization of 5S and
the genome. Each repeat consists of a highly 18S-26S rRNA genes was constructed by bi-colour
conserved 5S rRNA coding region of approximately FISH in amphidiploid Allium wakegi cultivar and an
120 base pairs in length and of NTS regions that vary amphidiploid tissue culture regenerant10. A rhodamine
in size between 100 and 700 base pairs. Most repeats labelled 5S rDNA and a biotin labelled 18S-26SrDNA
appear to be uniform in a species. On the basis of the were used as probes. The signals of 5S rDNA were
high degree of stability during the course of evolution, detected on the intercalary region of short arm in
comparative studies of the nucleotide sequences of chromosomes 9 (two region) and 15 (one region). The
rRNA genes provide a means for analyzing signals of 18S-26S rDNA were detected on the
phylogenetic relationship over a wide range of terminal region of short arm of chromosome 10 and at
taxonomic levels25. The variation in sizes and same regions of chromosomes 6 and 14 including the
sequences of the NTS of the 5S rRNA gene was satellite. In an amphidiploid regenerant, homologous
found to be useful for the phylogenetic reconstruction chromosomes were identified by chromosomal
of species26, and to discover differences between localization of rRNA gene families.
cultivars in barley and wheat, and between the Using digoxigenin-labelled 5S rRNA and biotin
breeding lines in maize27. labelled 18S-26S rDNA gene probes. Lee et al32
Lee et al28 classified eleven diploid species of compared the FISH patterns of regenerated
Allium into five types, A to E, based on the autotetraploid plants with the diploid wild type in
chromosomal localization and distribution patterns of Allium cyaneum. The physical localization of rRNA
the 5S rRNA genes by means of FISH. Data on the genes in tetraploid regenerants corresponded with that
amphidiploids with genomes of type B and C, A. of diploid species. Thus, the results of FISH
deltoid-fistulosum is an allotetraploid resulting from a suggested that tetraploid regenerants originated from
cross between a B type species and a species of an exact doubling of normal diploids.
unknown type. Do and Seo29 demonstrated the
In a karyotype analysis of somaclonal variants of
phylogenetic relationship among Allium subgenus
A. tuberosum (2n=4X=32), the chromosomal
Rhizirideum species based on the molecular variation
positions of rRNA genes were physically mapped33.
of 5S rRNA genes. The size of this region was 120
Both normal A. tuberosum and aneuploid regenerant
base pairs in all the species and the sequences were
(At 30) exhibited two sets of 5S rDNA sites, one on
highly conserved. The size of non-transcribed spacer
the proximal position of the short arm of chromosome
(NTS) regions varied from 194 bp in A. deltoid-
3, and the other on the intercalary region on the long
fistulosum to 483 bp in A. sativum. Snowdon et al19
arm of chromosome 6. There was one 18S-5.8S-26S
applied GISH for identification and characterization
rDNA site in the terminal regions on the short arm of
of parental genome components in oilseed rape
chromosome 8 including secondary constriction and
(B. napus) hybrids.
satellite. However, At 30 showed only 3 labelled
Analysis of Somaclonal Variations chromosomes 8 indicating that this was one of the lost
Somaclonal variations arising in tissue culture have chromosomes of At 30. Do et al34 further identified
been looked upon as a novel source of genetic the lost chromosomes in three aneuploid somaclonal
variation for crop improvement. Tissue culture phases variants of A. tuberosum. Chromosome compositions
DEVI et al.: FISH & GISH : MODERN CYTOGENETIC TECHNIQUES 311
of these variants were confirmed as being fixed lines addition lines of L. elongatum showing resistance to
during two years of greenhouse cultivation. The 5S Cephalosporium graminicum38. Pickering et al39
rRNA gene signals in all variants as well as the wild characterized progeny from H. vulgare X H.
type were detected as two sets while only one bulbosum crosses by GISH, confirmed the presence of
18S-5.8S-26S rRNA gene site was located. The three a monosomic alien substitution plant and established
lost chromosomes of At 29 variant (2n=29) were all the presence of a distal H. bulbosum introgression on
chromosome 2, the two for At 30 (2n=30) were chromosome 3 HL.
chromosome 7 and 8, and At 31 (2n=31) was missing F1 hybrid of a cross T. aestivum cv. Olmil X S.
one of the chromosome 2. cereale cv. Paldanghomil was backcrossed to T.
aestivum cv. Olmil as a male parent and progenies
Detection of Alien Chromatin were advanced to BC1F6 generation40. The presence of
Interspecific and intergeneric crosses have been rye chromatin was identified in 32 plants put of 467 in
made in plants with the aim of transferring desirable BC1F5 by GISH technique. The analysis showed that
traits, such as disease and pest resistance from wild or the mode of rye chromatin in these plants was almost
related species into cultivated species. Following telocentric. FISH at meiosis of wheat lines in BC1F6
hybridization if the donor has at least one genome in generation also depicted the presence of rye
common with the recipient, and then the chromatin. Zhang et al41 used GISH to investigate
recombination between the homologous genome in meiotic crossing-over in hybrids between H.
common with the recipient, then recombination bulbosum and H. vulgare and FISH with an
between homologous genome can readily take place oligonucleotide sequence (CCT) 10 followed by
and, through several cycles of backcrossing and GISH to map introgressions in selfed progeny from
selection, the desired trait can be transferred. If the hybrids. GISH established that pairing is intergenomic
donor and the recipient genomes are not homologous, and pairing frequency exceeded recombination.
the preferred method of handling such hybrid is to
continue backcrossing and chromosome screening to Detection of Chromosomal Aberration
produce series of addition or substitution lines of the The identification of structural abnormalities by
genome of the donor parent. Chromosome medicated routine and high-resolution cytogenetic studies plays
alien gene transfers through hybridization have an important role in diagnosis and treatment of
resulted in the genetic improvement of many crops. disease. However, this analysis is relatively gross and
Recently, development of direct gene transfer only permits the visual diagnosis of aberrations of
methods have further helped to engineer genes of single chromosome bands on the order of seven
importance into crops. million or so base pairs. ISH technique has felicitated
In plant breeding programme, alien chromosome, the diagnosis and identification of chromosomal
chromosome segments, and genes can be identified aberrations particularly for human and animal
and characterized by FISH and GISH. They can be chromosomes. Using chromosome-specific DNA
visualized and counted in wide hybrids and libraries, it permits the identification of small
amphidiploids, not only in high quality metaphase chromosome aberrations, which are not readily
spreads, but also within interphase nuclei. detected by standard high resolution banding
Subsequently, alien chromosomes can be followed techniques. Therefore, this technique may be used in
through backcrosses and recombinant lines. FISH prenatal and postnatal cytogenetic studies. For
technology has been used to identify partial example, women who have an increased risk of
amphidiploids derived from crosses of wheat with carrying abnormal fetuses can undergo cytogenetic
Thinopyrum intermedium and Lophopyrum elongatum analysis of fetal cells to rule out chromosomal
with the resistance to BYDV35 and wheat streak aberrations but it requires time. In that case, FISH can
mosaic virus36. provide a rapid and accurate identification for the
Highly repetitive sequences such as those in 5S loci most common autosomal trisomics and sex
have been used to identify certain addition lines of Ae. chromosome abnormalities. With mFISH analysis all
umbellulata6. Sequential C banding and GISH have 24 human chromosomes can be hybridized using
been used to identify addition lines of Haynaldia fluorochrome labelled chromosome specific DNA
villosum37 and substitution lines of Lophopyrum libraries. The advantage of this staining method is the
ponticum38, while FISH was used to characterize demonstration of structural aberrations, which cannot
312 INDIAN J BIOTECHNOL, JULY 2005
many species. FISH or PRINS (primed in situ including identification of parents or ancestors in
labelling) overcomes these limitations by providing unknown crosses or in polyploid species, information
specific labelling patterns useful for discrimination of about genomic regions which have diversified
similar chromosomes. Additional cytogenetic between species, and enabling clear identification of
landmarks can be obtained using species- or genus- pairing partners at meiosis and evolutionary
specific satellite repeats that are often amplified to translocations between genomes in polyploid and
high copy numbers and form discrete bands or spots hybrids. Application of FISH to somaclonal variants
on chromosomes. These repeats frequently occur at a is a useful tool for identifying and understanding
higher number of genomic loci and may therefore chromosomal changes during the tissue culture
produce signals characteristic for each chromosome process.
within the karyotype. Successful painting of a specific However, there are some limitations of these
plant chromosome wthin its own genome was techniques. Multicolour FISH can only be used
reported by Vega et al60. Dissected isochromosomes successfully on polyploids with at least one known
for the long arm of chromosome 5 of the wheat B progenitor species. Closely related genomes in certain
genome (5BL) were amplified and used as probes. allopolyploids cannot be discriminated using GISH
Hybridization signal data suggested that chromosome technique. Multicolour FISH is less sensitive and
and homoeologous group-specific sequences are more shows a lower degree of detection resolution than
abundant in 5BL than in genome specific sequences. single colour FISH due to multiple exposure
FISH had been used61 to analyse the structure of the photographs65.
rye B chromosome. GISH demonstrated high level of Advances in mammalian genetics involving the use
similarity between A and B chromosomes of rye. The of techniques outlined above provide promise for
B-specific repeat families D1100 and E 3900 were future progress in plant molecular cytogenetic
analysed in terms of their physical location and research. There is no doubt that the applications of
possible contiguity. these techniques will multiply in coming years and
Accurate identification of individual chromosomes enable the investigation of even more difficult
of Secale montanum Guss was achieved using problems of biology.
simultaneous and (or) successive FISH and
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