Title: Exploring Advances in Cytogenetics and Molecular Cytogenetics Description: Delve into the intricate world of cytogenetics and its cutting-edge counterpart, molecular cytogenetics, through this insightful presentation. Understand the profound relationship between chromosome structure, behavior, and gene function, with a particular focus on their relevance to crop improvement programs. Key Points: Introduction to Cytogenetics: Explore the fundamental principles of cytogenetics, its historical significance, and the recent influence of molecular tools, leading to the emergence of molecular cytogenetics. Importance in Crop Improvement: Uncover the pivotal role of molecular cytogenetics in crop improvement programs, offering insights into the structural and functional organization of genomes within chromosomes. Karyotyping: Gain a comprehensive understanding of karyotyping, its significance in identifying chromosomal abnormalities, and its applications in studying evolutionary relationships among different taxa. Chromosome Identification and Sorting: Learn about the techniques involved in the identification and sorting of individual chromosomes, crucial steps in cytogenetics research for various crops. Chromosome Banding Techniques: Explore different chromosome banding techniques, such as G-Banding and C-Banding, and understand their applications in detecting structural rearrangements. CHIAS (Chromosome Image Analyzing System): Get insights into the CHIAS software and its role in mapping and identifying chromosomes automatically. Flow Cytometry: Discover the applications of flow cytometry in detecting and measuring physical and chemical characteristics of cells, with a focus on its relevance in chromosome research. In Situ Hybridization: Explore the technique of in situ hybridization, particularly the fluorescent variant, and its applications in precise localization of specific DNA segments. Genomics and Whole Genome Sequencing: Delve into the realm of genomics and whole-genome sequencing, understanding the approaches like BAC to BAC and Whole Genome Shotgun. Case Study: Uncover a case study involving the identification of a Wheat-Psathyrostachys huashanica ditelosomic addition line, showcasing the practical applications of the discussed techniques. Conclusion: Summarize the key takeaways from the presentation, emphasizing the role of these techniques in advancing precision breeding and crop improvement.

1. GP 691: 1(1+0)
DOCTORAL SEMINAR I
Advances in Molecular Cytogenetics:
Potential for Crop Improvement
K. Modunshim Maring (2003305002)
Reg. No. : D/PBG/337/2020-21
PhD Scholar(2nd Year)
Department of Genetics & Plant Breeding
DRPCAU, Pusa
Seminar In-Charge :
Dr. Nilanjaya
Dr. S. K. Singh
Department of Genetics & Plant Breeding
DRPCAU, Pusa
1

2.  Cytogenetics is an area of research that relates structure, behaviour
and function of chromosomes with the genes that these
chromosomes carry.
 Over that years, research in this discipline has been greatly
influenced by molecular tools that have been extensively utilized
for crop biotechnology, leading to the emergence of a new area of
cytogenetics research called "molecular cytogenetics".
 The emergence of genomics as the new area of research allow
complete sequencing of Arabidopsis genome.
INTRODUCTION
2

3. IMPORTANCE
 Molecular cytogenetics involves an understanding of the structural
and functional organization of genome within the chromosomes. This
would also facilitate isolation and/or manipulation of genes, which
are important for crop improvement.
 With the availability of molecular maps, it is now possible to clone
mapped genes through the techniques involving map based cloning
and/or microdissection/microcloning.
 It provides useful information for understanding relatedness of a crop
with its wild relatives, that make an important genetic resource for the
improvement of this crop.
3

4. Advance techniques and approaches used in molecular
cytogenetics
Karyotyping
Chromosome Banding
Fluorescence In Situ Hybridisation (FISH)
Multicolour FISH (Mcfish)
Genomic In Situ Hybridisation (GISH)
Flow Cytometry
Pulse Field Gel Electrophoresis (PFGE)
Microdissection
Microcloning
Bacterial Artificial Chromosomes (BACs)
4

5. KARYOTYPING
Particular chromosome
complement of an
individual or a related group
of individuals, as defined by
the chromosome size,
morphology and number is
known as a “Karyotype”.
Karyotype
5

6. IDEOGRAM
 When the haploid set of chromosome of an organism are ordered in a
series of decreasing size, it is said to be an idiogram.
Ideogram/
Karyogram
6

7. LOW/HIGH RESOLUTION KARYOTYPE
7
18
LOW
LOW
HIGH
HIGH
7

8. Advantages of Karyotyping
 Reveals structural features of each chromosomes
 Help in identification of chromosomal aberrations
 Help in studying the chromosome banding pattern
 Aids in studying evolutionary changes.
8

9. CHROMOSOME IDENTIFICATION/SORTING
 Identification of individual chromosomes is the first step of
cytogenetics research in any crop.
 In early 70’s, chromosome can be precisely identified based on the
differences in size and morphology of chromosomes that could be
resolved either at mitotic metaphase or at pachytene of meiosis.
 In barley and bread wheat, pachytene analysis is not feasible.
Chromosome banding techniques and CHIAS provided reliable
methods.
9

10. CHROMOSOME BANDING TECHNIQUES
 Banding techniques refers to the staining of
chromosome that give rise to pattern of bands
along the length of chromosome.
 Paris conference in 1971 defined band as a
part of a chromosome which is clearly
distinguishable from its adjacent segments by
appearing darker or lighter with various
banding methods.
 It resolved the intraspecific variations of DNA
organization within chromosome.
10

11. TYPES OF CHROMOSOME BANDING
Types of banding Staining Summary
G-Banding Giemsa stain,
AT-rich regions stain darker than GC-rich regions,
Q-Banding Quinacrine florescent dye,
stains AT-rich regions
R-Banding Reverse of G-Banding, stains GC-rich regions, Heat treatment
preferentially melt the DNA helix in AT-rich regions.
C-Banding Stains heterochromatic regions close to the centromeres
Silver Staining Silver nitrate stains the NOR(Nuceolar Organiser Region)
11

12. Ideogramatic representation of G-(left) and R-(right)
banded karyotypes
12

13. GENERAL PROCEDURE OF STAINING
The dividing cell like meristematic cell of root or shoot tip are treated with
chemical that disables spindle fiber formation.
Such mitotically arrested cells are then immersed in a hypotonic solution
that causes cell to take up water and burst.
On preparation for microscopic examination such cell are squashed on a
microscopic slide, such that the chromosomes are spread out and can be
observed by staining.
13

14. NAMING OF CHROMOSOMAL LOCATION BASED ON
BANDING PATTERNS
17q12.1
17ptel
14

15. ADVANTAGES AND DISADVANTAGES OF BANDING
TECHNIQUES
ADVANTAGES DISADVANTAGES
It allows the identification of
chromosome deletions, duplications,
translocations, inversions.
It can only detect rearrangements
that involve more than 3 Mb of
DNA. Banding techniques are
limited to mitotically active cells.
15

16. CHIAS (CHROMOSOME IMAGE ANALYSING SYSTEM)
 First developed in 1980.
 Latest version- CHIAS IV.
 CHIAS is software is that used for
mapping and identifying chromosome
automatically.
 It is possible to get quantitative traits
for all chromosome within 25
minutes.
16

17. Application of the CHIAS for straightening cation-treated
bend chromosome
Figure : No EDTA treatment in ‘a’ and ‘c’, Treatment with EDTA in ‘b’ and ‘d’
Integrity of condensed chromosome in the nucleus maintained by Ca and Mg ions.
Their depletion leads to chromosome bending
17

18. Steps for
straightening bent
chromosome images
18

19. IN SITU HYBRIDIZATION (ISH)
 ISH-precise localization of a specific
segment of nucleic acid through the
application of a probe.
 Initially developed using radio-active
probes by Gall and Pardue (1969).
 Original techniques
 Highly sensitive
 Time consuming & Combersome.
19

20. ISH USING FLUORESCENT LABELED PROBE
In situ hybridization
Fluorescent in situ
hybridization
Genomics in situ
hybridization
Greater Safety
Stability
Ease Of Detection
Biotin
Labeled
20

21. FLUORESCENT IN SITU HYBRIDIZATION (FISH)
 FISH is a laboratory technique for
detecting and locating a specific
DNA sequence in metaphase or
interphase cells
 Fluorescent dye are utilized to label
the probe.
 Detection is done with the help of
fluorescent microscope.
21

22. SCHEMATICS OF FISH PROCESS
22

23. DETECTION OF FLUOROPHORE
23

24. APPLICATIONS
 Detect chromosomal aberrations or diagnosis of genetic disorders
A) Normal B) Abnormal (translocation)
Interphase FISH
X
Y
24

25. Advantages and Disadvantages of FISH
Advantages Disadvantages
Can be applied to both dividing and non-
dividing cells
Cannot detect small mutations
Simultaneous detection with multiple
probes
Probes are not yet commercially available
for all chromosomal regions
For studies of chromosomal changes and
gene mapping
25

26. McFISH (Multicolored FISH)
 Labelling of probes to multi-color
detection and the creation of multi-color
FISH images.
 Multi-probe detection increased the
speed of chromosome analysis.
26

27. GENERAL PROCESS OF McFISH
Process 1. Probe labelling
Process 2. Chromosome denaturation and
hybridization
Process 3. Multi-color detection after in situ
hybridization
Process 4. Detection of FISH signals
27

28. Distribution of the repetitive DNA probes on the
somatic metaphase chromosome using McFISH
TYPES OF PROBES USED:
•Satellite DNA (CL1,2,3,4)
•Ribosomal RNA
•Centromere-like repeat (CL17)
•Telomere repeat
(Deng et al., 2016)
28
Unstained chromosomes
A
B
C
D
E
F
G
H
I
J
K
L
A

29. GENOMIC IN SITU HYBRIDIZATION (GISH)
 Modification of FISH which allows distinguishing the genomes in a cell.
 Whole genomic DNA as a probe.
Figure: GISH intergeneric rapeseed hybrids. (a) Hybridisation of the two addition
chromosome in two metaphases from nematode-resistant BC3 individual from a B.
Napus x R.sativus. (b) Identification of the monosomic addition chromosome in a
phoma-resistant BC3 individual from B. Napus x S.arvensis.
DAPI (Blue)
29

30. APPLICATIONS
 GISH allows characterization of the genome and chromosome of
hybrid plants and recombinant breeding lines.
 Helps in assessing phylogenetic relationships between species of
plant.
 Cytological identification of foreign chromosome in interspecific
hybrids at the molecular level.
 Detection of parental genomes in natural allopolyploid species.
30

31. FLOW CYTOMETRY
 Technique used to detect and measure physical and chemical
characteristics of a population of cells or particles.
 It represents an ideal means for the analysis of both cells and
subcellular particles, with a potentially large number of
parameters analyzed both rapidly, simultaneously, and
quantitatively.
 Tool for the understanding of fundamental mechanisms and
processes underlying plant growth, development, and function.
31

32. Working of Flow Cytometer
32

33. PULSE FIELD GEL ELECTROPHORESIS
Charles Cantor
&
(David C. Schwartz)
1984
•Separation of large DNA molecules
•Voltage is periodically switched among three directions
•Longer time
•For small DNA molecules,
•Voltage in one directions but shorter time
33

34. HOW DOES PFGE WORK?
Cells are taken on agar plate
Cells mixed with agarose and pour into plug mold
Cells are broken open with biochemicals or lysed so
that DNA is free
DNA is loaded into the gel and electric field is
applied which separates DNA fragment as according
to their size.
Gel is stained to visualise DNA under UV light
34

35. APPLICATIONS
 PFGE is a technique used for the separation of
large DNA molecules by applying to a gel
matrix an electric field that periodically changes
direction.
 It is considered as gold standard for
epidemiologiocal studies of pathogenic
organisms.
 Used for gene mapping in microbes.
 Used for genotyping or genetic fingerprinting.
DNA Fingerprinting
35

36. MOLECULAR MAPS
 The availability of deletion stocks and molecular techniques like PFGE,
BAC, FISH, etc has provided powerful tools to generate physical maps
of major crops plants genomes.
 Gene mapping
 describes the methods used to identify the locus of a gene and the distances
between genes.
 describes the distance between different sites within a gene.
 The essence of all genome mapping is to place a collection of molecular
markers onto their respective positions of the genome.
36

37. COMPARATIVE GENOME ANALYSIS
 The availability of molecular genetic/physical maps have made possible for
comparative genomic studies of chromosomes in several groups of plants including
Brassicaceae, Poaceae, Fabaceae and Solanaceae.
 Comparative genomics is the field of research
in which the genomic features of different
plants are compared.
 Evolutionary relationship between the plants.
•Whole genome alignment is a typical
method in comparative genomics
37

38.  Among a number of genes that have already
been studied through comparative genome
analysis , one important example is
 Gibberillin-insensitive dwarfing genes, which
have been found to be orthologous between
wheat and maize (Peng et al., 1999)
Figure: Near-isogenic dwarf wheat lines:
left- tall control;
centre- semi-dwarf Rht-B1b;
right- semi-dwarf Rht-D1b.
38

39. (Peng et al., 1999)
39
GAI
Rht-D1b D8

40. MICROCOLINEARITY
 The studies on molecular maps across related plant species, though
revealed significant conservation of gene content, gene order and gene
homology, had their own limitations. For instance, only up to about one
marker per 10 centimorgan genetic distance is available for comparative
analysis, thus making it difficult to analyse small deletions, duplications
and inversions involving only a few centimorgans.
 In view of these limitations, instead of mapped markers, DNA sequences
representing small regions of genomes have been used for comparative
analysis, sometimes resolving what is described as microcolinearity
40

41. Applications
 It has also been shown that microcolinearity may occur not only
between related species but also between more distantly related
species.
41

42. Chromosome Microdissection and Microcloning
 Initially developed in 1981 on Drosophila polytene chromosomes
 Individual chromosomes as well as chromosome segments representing
satellites, chromosome arms and centromeres could also be dissected out
and utilized for a variety of purposes including development of probes
and tagging of important genes.
 Chromosome microdissection followed by microcloning is an efficient
tool combining cytogenetics and molecular genetics that can be used for
the construction of the high density molecular marker linkage map and
fine physical map.
42

43. PROCEDURE
Material Fixation
Preparation of Chromosome Samples
Microdissection of Target Chromosome
PCR Amplification of the Target Chromosome DNA
Probe Labeling of the Target Chromosomes DNA
Characterization of DNA from Microdissected Chromosome by FISH
Construction of Single- Chromosome Library
43

44. APPLICATIONS
 An efficient and direct approach for isolating DNA from specific
chromosomes and/or specific chromosome sections.
 The isolated DNA is used for genomic research including:
(1) genetic linkage map and physical map construction
(2) generation of probes for chromosome painting
(3) generation of chromosome -specific expressed sequence tags libraries
44

45. GENOMICS AND WHOLE GENOME
SEQUENCING
 Two approaches of whole genome
sequencing
1) The BAC to BAC approach - Slow
2) Whole Genome Shotgun (WGS) -
Faster
45

46. BAC TO BAC APPROACH
Cutting of genome (150,000 bp)
Insertion into BAC
Fingerprinting
BAC is broken into 1500 bp pieces
and placed M13
Sequencing of M13 libraries
Computer Program
(PHRAP)
46

47. WHOLE GENOME SHOTGUN (WGS)
Genome sheared into
2000 bp &10,000 bp long
Two fragments inserted
into plasmid
Sequencing of both
Plasmid libraries
Assembling by computer
algorithms
47

48. The genome sequence and structure
of rice chromosome 1
Figure : Physical map of rice chromosome 1.
Positions of the BAC contigs are indicated by
black bars. Purple numbers indicate the physical
distances that were calculated on the basis of the
nucleotide sequence length of each contig. The
centromeric region is shown as a red circle. The
green numbers show the gap sizes as measured
by using FISH.
48

49. 49

50. A B C
50
A B
C D
Common wheat x P. huashanica
DT23

51. CONCLUSIONS
 Utilizing the approaches of genomics and comparative genomics in
model plants and major crop plants, molecular cytogenetics in future
will also facilitate the discovery and isolation of many genes of
agronomic importance, which are not amenable to the conventional
Mendelian approach of genetic analysis .
 The results of majority of these molecular cytogenetic studies have
been shown to be relevant to crop improvement programmes, so that
in future these will be extensively utilized in what is popularly
described as precision breeding..
51

52. REFERENCES
Jain, H. K. and Kharkwal, M. C. (2004). Plant Breeding – Mendelian to Molecular Approach. New
Delhi, Narosa Publishing House.
Liehr, T. (2021). Molecular Cytogenetics in the Era of Chromosomics and Cytogenomic Approaches.
Frontiers in genetics, 12.
Shibata, F. and Hizume, M. (2016). Multi-Color Fluorescence in situ Hybridization. The Japan Mendel
Society, 80(4): 385–392.
Tan, B., Zhao, L., Li, L., Zhang H., and Wei Zhu, W. (2021). Identification of a Wheat-Psathyrostachys
huashanica 7Ns Ditelosomic Addition Line Conferring Early Maturation by Cytological Analysis
and Newly Developed Molecular and FISH Markers. Frontiers in plant science, 12.
Zhang, Y. X., Deng, C. L. and Hu, Z. M. (2016). The Chromosome Microdissection and
MicrocloningTechnique. Methods of Molecular Biology, 1429: 151-60.
52

53. Thank You