it includes FISH, GISH and their recent modifications such as comparative genome hybridization, chromosome painting, spectral karyotyping, multicolour FISH, fiber FISH and Q-FISH

1. welcome
1

2. Seminar
SHREYAS A
PGS18AGR7783; Sr. M.Sc.
DEPT. OF GENETICS AND PLANT BREEDING
MODERN CYTOGENETIC TOOLS
IN CROP IMPROVEMENT
2

3. Contents
1 • Introduction
2
• Modern cytogenetic tools
3
• Fluorescence In situ
Hybridization
4
• Genomic In situ
Hybridization
5
• Applications in crop
improvement
6
• Conclusion
3

4. Introduction
 Swiss botanist Nageli (1840), described thread-like
structure, named “Transitory Cytoblasts”.
 Cytogenetic technique for chromosome identification
 Advent of banding techniques
Q-banding
G-, R-, C- and NOR banding
In situ Hybridization
Traditional methods Modern Methods
Traditional methods
4

5. In Situ Hybridization
 Technique to visualize nucleic acid probes on
target
 Location of nucleic acid can be determined in vivo
 Developed by Pardue et al.,(1969) and John et al.,
(1969) independently
 Radioisotopes -labels for nucleic acids
 Autoradiography - detect hybridized sequences.
5

6. Labeling Methods
Nick Translation
It is effective for total genomic DNA or large cloned
inserts.
Random-Primer labeling( or Primer
Extension)
It is used to produce uniformly labeled probes.
PCR based labeling
The advantage of PCR labeling, over random-primed
labeling is the incorporation of a higher number of
labeled nucleotides along the amplified DNA strands.
6

7. Labels
Radioactive labels Non-radioactive labels
Radioactive labels are the
isotopes which emit β-
particles and are detected
by autoradiography.
E.g. 35S , 32P , 3H
Procedures are of two
types:-
 Direct ISH
E.g. TRITC, FITC,
AMCA
 Indirect ISH
E.g. Biotin, Digoxigenin,
Fluorophor
7

8. Example- Detection by Biotin
• Biotin, is first introduced
enzymatically into NA probe.
• Probe hybridized to target NA.
• Then avidin, conjugated to same
signal generating system, (say
FITC) is introduced.
• Detected by green colored
fluorescene of FITC.
• To enhance the signal strength
avidin can further be detected by
biotin- antiavidin conjugate.
• Then again avidin, conjugated to
some signal generating system is
introduced.
8

9. Probes
Gene/locus specific probes
 Both in metaphase and interphase stages.
Centromere probes
 Useful for determining the number of copies of a particular
chromosome
Whole chromosome probes
 Made from flow-sorted or micro dissected chromosomes
 Determine composition of marker chromosomes, confirm the
chromosome rearrangements
Telomeric probes
9

10. 10

11. 11

12. Steps in ISH
Denaturation of nucleic acid
(Specimen & probe)
Washing
In situ Hybridization
Visualization
Analysis
Detection of system
Probe selection &
labelling
Preparation of
biological
specimen
7

13. AAATTCCCGGGTTGG
TTTAAGGGCCCAACC
Complementarity
Hypothetical representation of ISH
DNA
Probe
denaturation renaturation
Hybridization
13

14. Modern cytogenetic tools
 FISH and GISH Techniques
 Modification of in situ hybridization technique.
 Fluorescence in situ hybridization (FISH).
 Fluorescent molecule is deposited at the site of in situ
hybridization, location of genes or DNA can be
visualized.
 Total genomic DNA is used as probe in (GISH)
14

15. Advent of FISH technique
 Rudkin and Stollar (1977): described FISH,
detected rRNA genes in Drosophila by using
Fluorescent antibody.
 Pinkel et al (1986): Directly labeling DNA probes
with biotin molecules which can be detected by
fluorescent labeled molecules.
15

16. Fluorescence In situ Hybridization
 FISH uses fluorescent molecules
to “paint” DNA or chromosomes.
 Use of short sequences of single-
stranded DNA (probes).
 Probes are labeled with fluorescent
molecule.
 See the location of those sequences
of DNA by fluorescent
microscope.
16

17. Principle
“FISH is based on the ability of single-stranded
DNA (probes) to hybridize to complementary
DNA sequence”
 Target either in metaphase or interphase.
 Probe is either directly labeled or indirectly labeled.
 Labeled probe and the target DNA are denatured and
hybridized.
17

18. Steps in FISH
 Probe DNA-
Characterization
 Nick translation labeling of
probe DNA
 Purification of labeled
DNA probe
 Chromosome preparation.
 In situ hybridization
 Washing
 Fluorescent microscope
18

19. 8-hydroxyquinoline (1–4 h)
Fixed
methanol and glacial acetic acid (-20˚C)
Roots wash
citric acid-sodium citrate (20 min)
Digested
mixture 1% cellulase and 20% pectinase (1–1.5 h at 37˚C)
Root tips -squashed
45% acetic acid
Post fixed
ethanol: glacial acetic acid,
Dehydration
absolute ethanol and air-drying.
19
Flow Diagram – chromosome preparation
(Robert et al., 2005)

20. Chromosomes prepared
RNase, Proteinase treatment
Probe labelled and Purified
Dissolved in Hybridization Mixture
Denaturation of Probe and Chromosomal DNA (750C 10 min)
In situ hybridization (370C)
Post Hybridization Washes ( 420C 10 min)
Incubation with Fluorochrome
Mounted in Medium containing Fluorescent Counterstain
Viewed under a fluorescence microscope
Flow Diagram – FISH methodology
18

21. 21

22. Detection methods
DAPI is a fluorescent dye that binds DNA and
stains.
 Labeled probes are immunogenic and detected by
antibodies raised against the label.
E.g. Anti digoxigenin.
Signal generating system: which is conjugated
to the antibody or steptavidin.
22

23. Fluorescent microscopy
An upright fluorescence microscope
(Olympus BX61)
An inverted fluorescence microscope
(Nikon TE2000)
23

24. Uses of FISH
Identification and characterization of
numerical and structural chromosome
abnormalities.
Detection of microscopically invisible
deletions.
Parental diagnosis of the common aneuploids.
24

25. Advantages
 Higher spatial resolution and speed.
 High efficiency of hybridization and detection.
 Whole chromosomes, chromosomal segments or
single copy sequences can be highlighted.
 Physical location along chromosomes.
 Hybridization with multiple probes enable detection
of translocation products.
25

26. Disadvantages
 Limited number of commercial probes available.
 Probe must be available - given sequence of DNA.
 Unsuspected variation in nuclear organization
cannot be detected.
 Need specialised camera and image capture
system.
 Only provide information about the probe being
tested.
26

27. A novel, simple and rapid Nondenaturing FISH (ND-
FISH) technique for the detection of plant telomeres.
(Cuadra et al., 2009)
Objectives:
Detect telomere region in different crops species with
no prior denaturation of the chromosomes.
Effect of RNase A treatment prior to ND-FISH
detection of barley telomeres.
27

28. 28

29. Effect of RNase A treatments on barley telomeres.
RNase A (Metaphase)
29

30. Genomic in situ hybridization (GISH)
30
 Genomic in situ hybridization (GISH) Is a cytogenetic
technique that allows the detection and localization of
specific nucleic acid sequences on morphologically
preserved chromosomes using genomic DNA of donor
specie as probe.
 An unlabeled DNA of parental species is used as
competitor DNA.
 GISH for plants…was developed in 1987 by M.D. Bennett and
J.S. Heslophorizon

31. GISH essentially involve eight steps: -
31
 Probe DNA
 isolation and shearing of probe DNA
 Isolation and sizing the competitor DNA
 Nick translation labeling of probe DNA
 Purification of labeled DNAprobe
 Chromosome preparation
 In situ hybridization
 Detection of hybridization
 Microphotography.
DNA from a test organism
that is denatured and then
used in vitro hybridization
experiment s in which it
competes with DNA
(homologous) from a
reference organism; used
to determine the
relationship of the test
organism to the reference
organism.

32. Steps in GISH
 Probe DNA- isolation and shearing of probe
DNA.
 Isolation of competitor DNA.
 Nick translation labeling of probe DNA.
 Purification of labeled DNA probe.
 Chromosome preparation.
 In situ hybridization.
 Detection of hybridization.
 Microphotography.
32

33. BREEDING
Parent AA Parent BB
Total DNA cut and labeledTotal DNA cut and labeled
Natural / controlled
hybrid AB
Chromosome spread
Denaturation
hybridization
Detection
Microscopic observation
Chromosome A
chromosome B
Principle of genomic in situ
hybridization. (Courtesy of CIRAD)
(Julian Osuji et al., 1999)
33

34. GISH as Tool to Study the
interspecific hybrid
N. sylvestris(2n=2x=24) N. tomentosiformis 2n=2x=24)
Kostoff hybrid (2n=4x=48)
(digoxigenin-labelled probe,
FITC signal, green)
(biotinlabelled probe,
Cy3 signal, pink)
(Michael et al.,2010)
X
34
F1
Colchicine treatment

35. 35
Genomic In Situ Hybridization Identifies Parental
Chromosomes in Somatic Hybrids of
Diospyros kaki and D. glandulosa
( Young et al., 2002)
Objective:
GISH could distinguish parental chromosomes
of the somatic hybrid between Diospyros kaki
and Diospyros glandulosa.

36. 36
Photograph of GISH using total DNA probes from D. kaki and
D. glandulosa. D. kaki was detected with rhodamine revealed
reddish orange colour and D. glandulosa detected with FITC
showed yellow colour.

37. Types in GISH
 cenGISH- centromeric genomic in situ hybridization
 mcGISH- multicolour genomic in situ hybridization
 GISH, for analyzing interspecific, intergeneric
hybrids and allopolyploid species as well as
introgression, addition and substitution lines.
37

38. Recent modifications
 Comparative Genome Hybridization
 Chromosome painting
 Spectral Karyotyping
 Multicolour FISH
 Fiber FISH
 Q-FISH
38

39. Comparative Genomic Hybridization
(CGH)
 CGH involves two-colour FISH
 CGH involves the differential labelling of test and
reference DNA to measure genetic imbalances in
entire genomes.
 Fluorescent molecular technique that identifies DNA
gain or DNA loss
39

40. 40

41. 41

42. Heterochromatin
treshold 0.8 treshold 1.2
chromosome number
number of
chromosomes in
analysis
gain loss
fluorescent
ratio profile
Identification of aberrations
Minimaly 10 metaphases should be processed.
Florescent ratio profile is compared to the fixed tresholds (15-20% from ratio 1). The
ratio profile that deviates 15 % - 20 % from ratio 1.0 is typically regarded as aberrant.

43. Advantages of CGH
Identifying abnormal regions in the genome.
Does not require fresh sample.
 Provide information on whole genome in
single test.
Disadvantages:
Reciprocal translocations or inversions can not
be detected.
43

44. Spectral Karyotyping
 SKY for characterising numerical and structural
chromosomal aberrations.
 Permits the visualizations of all chromosomes at a
time, ‘painting’ each pair of chromosomes a different
fluorescent color.
 Followed by spectral imaging and chromosome
classification.
 Produces a colour karyotype of the entire genome.
44

45. Display Image
Picture analyse using Sky View
Classified Image
The objective of the Sky View spectral karyotyping software is to
automatically classify and karyotype chromosomes in the Display image,
thereby overcoming the ambiguity inherent in the display colors.
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12 12
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7
12 12

46. 46

47. Spectral Karyotyping
Advantages:
Mapping of chromosomal breakpoints.
Detection of translocations.
Characterization of complex rearrangements.
Disadvantages:
Very expensive equipments.
The technique is labour intensive.
Does not detect structural rearrangements
within a single chromosome. 47

48. Multicolor FISH
 Nederlof et al., (1989) to achieve triple
hybridization and detection using centromere
specific probes.
 Employment of several different Fluorochromes.
 Each Chromosome is specifically marked with an
individually different coloring materials.
 Assigning pseudocolours using computer software.
 Simple and complex translocations, interstitial
deletions, insertions.
48

49. Identification of chromosomes in two Chinese spruce
species by multicolor fluorescence insitu hybridization.
(Hizume et al., 1999)
49
To access the performance of multicolour FISH for
chromosome Identification in Picea．

50. 50

51. 51

52. 52

53. Fiber FISH
To reveal the fine detail of DNA structure
Centromeric DNA elements and associated
proteins to be revealed at high resolution
Permitting physical ordering of DNA probes to
a resolution of 1000 bp
Assessment of gaps and overlaps
53

54. High resolution FISH of rice somatic chromosomes,
pachytene chromosomes, the nucleus, and
extended DNA fibers. (chromosome 12 )
telomere signals (green) and the subtelomeric
tandem repeat TrsA (red). (Nobuko et al., 2010) 54

55. Q-FISH
 Q-FISH combines FISH with PNA-conjugated probes
and computer software to quantify fluorescence
intensity.
 Used routinely in telomere length research.
Advantages:
 Highly reproducible results
 Possible to acquire fluorescence data on thousands of
cells
55

56. Applications FISH and GISH in crop improvement
56

57. Chromosome mapping
FISH - utilized in many plants to identify
chromosome accurately.
Using species-specific repetitive sequences,
ribosomal genes and even unique sequences.
 FISH - used for the physical mapping of
ribosomal genes, microsatellite and transposable
DNA sequences on sugar beet chromosomes.
(Schmidt et al., 1996)
57

58. Phylogenetic Analysis
 Phylogenetic and taxonomic studies for determining
and testing genomic relationship of wild and
cultivated plants.
 Classified 11 diploid specie of Allium into 5 types, A
to E based on chromosomes localization and
distribution patterns of 5S rRNA genes by means of
FISH. (Lee et al., 1999)
58

59. Chromosomal localization of 5S rRNA gene loci and
the implications for relationships within the Allium
complex.
(Lee et al., 1999)
Objectives:
• To study on the physical mapping of the 5S rRNA
genes on the chromosomes of various diploid and
alloploid species of Allium by fish technique.
• To understand the origin of 3 alloploid species from
the distribution patterns of the 5S rRNA genes of the
diploid species.
59

60. 60

61. Characterization of genome
 characterization of the genome and
chromosomes of hybrid plants, alloploid species
and recombinant breeding lines.
 Study the pedigree of ancestry
 Multicolor GISH - discriminating each genome in
natural or artificial amphidiploids used to distinguish
3 genomes of hexaploid wheat.
(Mukai et al., 1993)
61

62. Simultaneous discrimination of the three genomes in
hexaploid wheat by multicolour fluorescence in situ
hybridization using total genomic and highly repeated
DNA probes .
(Mukai et al., 1993)
Objectives:
• To discriminate simultaneously the three genomes in
Triticum aestivum cv.Chinese Spring with different
colours using multicolour fluorescence in situ
hybridization technique.
62

63. 63
The hybridization sites of the A genome probe were detected by
yellow fluorescence, while those of the D genome probe were
detected by orange fluorescence. The B genome chromosomes were
neither labeled yellow nor orange but appeared faint brown as a
result of cross-hybridization of the A and B genome probes

64. Repetitive DNA sequences
 Following repetitive DNA sequence that have value
in chromosomes and Genome identification.
 rRNA genes.
 Tandem repeats.
 Telomeric seq.
 Centromeric seq.
 Microsatellites.
64

65. Detection of Chromosomal aberration
 FISH can provide a rapid & accurate identification of
most common trisomics and structure abnormalities.
 In many polyploid species, there are intergenomic
translocations shown by GISH.
 Translocation events demonstrated by molecular in
situ hybridization and chromosome pairing analysis
in highly asymmetric somatic hybrid plants
(Hinnisdaels et al., 1992)
65

66. Detection of Alien Chromatin
 Interspecific & intergeneric crosses aim at transfer
desirable trait from wild into cultivable species.
 Alien chromosome, chromosome Segments, and
genes can be identified and characterized by GISH
and FISH.
 Intergenomic translocations and the genomic
composition of Avena maroccana revealed by FISH.
(Leggett et al., 1994)
66

67. Analysis of Somaclonal variations
 Tissue culture phases may impose stress, and induce
chromosome breakage and DNA transposition,
leading to karyotyping changes.
 Examination of chromosome distribution of 5S and
18S-26S rRNA is useful in identifying the types of
genomic changes. (Hudakova et al., 2001)
67

68. Conclusion
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69. 69