This document discusses various cytogenetic techniques for gene location and transfer. It describes techniques for locating genes such as using structural and numerical chromosomal aberrations, chromosome banding, and in situ hybridization. Structural aberrations discussed include deficiencies, inversions, and translocations. Numerical aberrations discussed include aneuploids like trisomics, monosomics, and nullisomics. The document also describes techniques for transferring genes between species such as transferring whole genomes, whole chromosomes, chromosome arms, and through various types of interchanges. Specific examples of using these techniques in plants are provided.

1. Speaker :
Pratik Satasiya
A-2017-30-055
Cytogenetic Techniques for
Gene Location and Transfer
Credit seminar

2. Contents
• Introduction
• Techniques for Gene Location
1. Use of Structural Chromosomal Aberrations
2. Use of Numerical Chromosomal Aberrations
3. Use of Chromosomal Banding
4. Use of In situ hybridization techniques
• Techniques for Gene Transfer
1. Transfer of whole genome
2. Transfer of whole chromosome
3. Substitution of alien chromosome arm
4. Interchanges

3. Introduction
• Locating the gene means assigning the genes
to specific chromosome or chromosome arm
• Transfer of gene means transfer of particular
segment or whole chromosome or whole
genome across the species for improving its
economic use

4. Use of structural aberrations
1. Deficiencies
2. Inversions
3. Translocations
a) Use linkage between
marker and semi sterility
b) Overlapping
translocations
c) Use of B-A translocations
Use of aneuploids
1. Trisomic analysis
2. Monosomic and
nullisomic
analysis
Use of chromosome banding techniques
1. Q banding
2. G banding
3. C banding
4. N banding
In situ hybridization techniques
1. FISH
2. GISH

5. Deficiencies in Chromosome mapping
• Mackensen (1935) and Slizynska (1938) used deficiencies for locating genes (type of wing)
in Drosophila

6. Use of deficiencies in chromosome mapping in plants
• Female with recessive marker stocks crossed with
irradiated pollen from male carrying dominant
allele
• F1 heterozygote seed grown
• If……. Egg fertilized by normal pollen it will show
dominant phenotype
• If……. egg fertilized by pollen containing deficiency
at that concerned locus it will show recessive
phenotype instead of dominant
Here, the transmission of deficiency does not
impaired Why ??

7. • They used pollen of tomato variety red cherry’s pollen to irradiate with X rays to pollinate
homozygous recessive female
• Plants in M1 generation showing pseudo dominance used for cytological study
• Young flower buds from pseudo dominant plants collected and pachytene chromosomes
examined for deficiencies
• Cytological studies of 74 deficiencies of tomato chromosomes induced by radiation and
identified by the pseudo-dominant technique reveal the loci of 35 genes on 18 of the 24
arms of the complement

8. Cytogenetics, P.k gupta, 2016

9. Inversions for locating gene
• Inversions can be readily detected by a comparison of recombination frequency
between the plant with normal and inverted chromosome
• Existence- first detected by Sturtevant and Plunkett in 1926
• They prepared genetic maps for the genes se st p DL H ca located in the third
chromosome of Drosophila melanogaster (gene sequence: se st p DL H ca )
using less recombination within inverted segments

10. Interchanges in chromosome mapping
1.Using linkage between marker and semi sterility
• Translocations have been utilized in maize, barley and pea for determining linkage
groups/genes with specific chromosomes
• It is based on observed linkage between interchange breakpoint or semi-sterility &
marker gene located on one of the chromosomes
• involves crossing of translocation heterozygote and recessive marker stock
• F1 are selfed or backcross with recessive marker stock
• In F2 study of recombination between breakpoint or semi sterility & marker gene using
two point or three point test cross method

11. • on chromosome 8 marker gene j------japonica
• on chromosome 9 marker gene C----Coloured aleurone
sh-----shrunken
wx----waxy
•
• on chromosome 1 marker gene br-------brachytic dwarf
f---------fine stripped leaf
• on chromosome 7 marker gene ij-------iojap variegation
gl------glossy leaf 1

12. 2. Overlapping translocations procedure
• Method first proposed by Gopinath and Burnham in 1956
• Female produces four type of gametes
1. balanced translocation for one chromosome
2. balanced translocation for another chromosome
3. gamete with duplications
4. gamete with deficiencies(they are abortive)
• So, if gene located in interstitial region then progenies will have genotypes Aa and AAa
• From this when segmental trisomic (AAa) selfed, it will give trisomic ratio 17:1
• This technique extensively used in Drosophila to locate structural genes

13. 3. Use of B-A translocations
• Most efficient method for locating recessive mutants to the proper chromosome arm
• Roman and Ullstrup demonstrated the usefulness of the B-A translocation method
when they located a gene (hm) for reaction to Helminthosporium carbonurn in maize
• In 1947, Roman produced the first B-A translocations by X-raying mature pollen from
plants carrying B-type chromosomes
Production of a B-A translocation
Translocation heterozygote
Translocation homozygote

14. Cont…..
• Roman showed that nondisjunction of the B chromosome occurs at the
second division of the microspore
• It produces one hyperploid sperm with two B chromosomes and one
hypoploid sperm with none
Beckett, 1978

15. Use for locating gene
1. Cross of normal plant with recessive marker (susu) with
B-A translocation homozygote with dominant marker
(SuSu)
2. When hypoploid male nuclei fertilize the secondary
nuclei…….it will give sugary endosperm (susu) and
hyperploid with egg…….it will give F1 sugary seed
(SuSusu)
3. But in next generation only 6.4 % sugary seeds obtained
which is trisomic ratio as expected for the critical line

16. • They produced complex translocation TB-1L-3L by
crossing between two translocation
heterozygotes
One B-A translocation T1-B
Normal translocation T1-3
• They used this compound translocation to
locate the gene for anthocyanin
pigmentation
Birchler (1980) used this type of translocation
to locate the gene for Adh-1 (Alcohol
dehydrogenase 1) locus

17. Use of Aneuploid for locating genes
Aneuploidy
loss or gain of one or few
chromosomes as compared
to normal somatic
chromosome complement
Generally as a view of
locating the genes we
use nullisomic,
monosomic or trisomics
as per convenience

19. Trisomic Analysis
•Use of trisomics for locating the genes and preparing
chromosome mapping called trisomic analysis
•BRIDGES (1921) was first to use trisomics to locate the
gene ey (eyeless) has its locus in the fourth chromosome of
Drosophila melanogaster
•Similar identifications followed shortly in Datura: the gene “
White ” was identified with the trisomic type “ Poinsettia ”
( BLAKESLEE and FARNHAM 1923)

20. Analysis

21. Assigning linkage groups to chromosomes

23. Locating gene on specific chromosome arm

25. Monosomic & Nullisomic Analysis
Monosomic analysis
• Used for locating genes in Polyploid species and in maize (can tolerate
monosomic)
For dominant gene For recessive genes

26. Locating Dominant gene by absence of
expression in nullisomics
• When dominant gene located on chromosome
• Nullisomic for that chromosome will not show that character
• E.g.,
genes for red seed colour--- on chromosome 3D
gene for awn suppression -- on chromosome 4B and 6B
Wheat

27. Locating dominant gene by analysis of F1
• Cross whole set of monosomics using as female containing dominant marker with
normal male containing recessive for that trait
• Analyse ratio observed in F1

28. Locating recessive gene by analysis in F2
• Cross between whole set of monosomoics containing recessive trait with normal male
carrying homozygous dominant for that gene
• In F1 all plants show dominant phenotype for both critical and non critical lines
• In F2 ratios analysed

29. Locating hemizygous ineffective gene through
analysis of F3
• In some cases effect of aa differs from a-
but AA and Aa show dominance
• aa shows recessive character a- is
known as hemizygous ineffective
• Ex, spheroccocum character in wheat
• It shows same ratios in F2 for critical and
non-critical lines
• But in F3 study the ratio of progeny of
plants which showed dominance in F2

30. For digenic trait
• When character controlled by two genes,
it may have various types of interactions
• Critical lines have AAB- , A-BB, aab- , a-bb
• Non-critical lines have AaBb, AABB, aabb

31. Locating genes using intervarietal chromosomal
substitutions
• Use of whole chromosome substitutions
1. Monosomic series crossed with donor
variety
2. F1 monosomics selected cytologically
3. Selfed to get disomic for the univalent
chromosome of donor
4. Then disomics are back-crossed to
recipient to recover recipient
5. If this substitution leads to major
morphological changes then we can
conclude that gene located on that
chromosome

32. • They use intervarietal chromosome substitutions for locating characters like
awning, earliness, lodging, plant height, protein content, 1000 kernel weight
• They used recipient variety: Chinese spring
• Donor varieties: Thatcher, Hope and Timstein

33. Locating genes on chromosome arms
• Locating gene on one of two arms of chromosomes by using telocentric chromosomes in
form of
Monotelosomics - 20II + 1 tI
Monoisosomics - 20II + 1 iI
Ditelosomics- 20II + 1 tII
Monotelodisomics- 20II + 1 heteroII
• Example :
In Chinese spring wheat monotelosomics for chromosome arm 6BS are awned (Chinese
spring is awnless), so it concluded that awn inhibitor gene B2 is present on 6BL (Sears, 1962)

34. Use of chromosome banding techniques
• widely used in animals
• Due to lack of resolution of banding in
plant cells it is not widely used
• But now techniques available to study
and identify chromosomes, NOR regions,
facultative and constitutive
heterochromatin using these banding
techniques

35. • Q banding :
1. Use for chromosome identification
2.Karyotype study
• G banding :
1.In G banding instead of squash
preparation chromosomes air dried
and other modifications in staining
2.Not widely used in plants

36. • C banding
1. C banding techniqus developed for Vicia
faba, Secale cereale, Avena spp.
2. This techniques widely used for
identification and karyotype study of
various species
• N banding
1. Use for locating of NOR region
2. It stains more intensively a specialized
heterochromatin within C bands

37. • The meiotic behaviour of F1 hybrids of hexaploid Triticale that
differed in their genotypic or chromosomic constitution with
diploid rye was investigated using c banding
• Metaphase I configuration of the hybrid JM 78 X Canaleja showing
7 rye bivalents + 14 wheat univalents (C-banding staining)

38. Use of In situ hybridization for locating gene
• ISH is a type of hybridization that uses a labelled complementary DNA, RNA or
modified nucleic acids strand (i.e. probe) to localize a specific DNA or RNA sequence
• Probe- labelled nucleic acid (DNA or RNA seq.) molecule used to detect the presence
of its complementary strand by hybridization

39. Labelling of probes
• Radioactive labels
 32P
 35S
 3H
• Non-radioactive labels
 Indirect labelling
 Direct labeling
• Direct labelling
 Label is incorporated directly with nucleic
acid probe so that hybridization site could be
visualized immediately after hybridization
• Indirect labelling
 Label in the probe cannot be detected
directly.
 Secondary molecule called reporter is
required which recognizes the primary
molecule and binds to it.
 Like-Biotin, Dioxigenin,
Acetylaminofluorene fluorescent dye

40. FISH (Flourescent in situ hybridization)
• A technique for detecting and locating a specific
nucleic acid sequence in the specific region of the
chromosomes by hybridizing it with a probe having
fluorescent molecule

41. Procedure
1. Probe DNA-Characterization
2. Nick translation labeling of probe DNA
3. Purification of labeled DNA probe
4. Chromosome preparation
5. In situ hybridization
6. Detection of hybridization
7. Microphotography

43. • High-resolution cytogenetic maps to localize genetically mapped genes onto
chromosomes by fluorescence in situ hybridization (FISH)
• In this study, they developed a squash FISH procedure allowing successful detection of
single-copy genes on maize (Zea mays) pachytene chromosomes.
• Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences
separated by ∼100 kb can be resolved
• They localized nine genetically mapped single-copy genes on chromosome 9 in one FISH
experiment.

44. GISH (Genomic in situ hybridization)
Was developed in 1987 by M.D. Bennet and J.S. Heslop-Harrison for plants
Genomic in situ hybridization (GISH) is basically ISH that uses genomic DNA as a probe
GISH is used to estimate the amount of alien chromatin within chromosomes in
interspecific hybrids
Multicolor GISH (mcGISH), which employs several different genomic probes, can be used
to simultaneously visualize two or more genomes in a polyploid species

45. Procedure
1.Probe DNA- isolation and shearing of
probe DNA
2.Isolation and sizing the competitor DNA
3.Nick translation labeling of probe DNA
4.Purification of labeled DNA probe
5.Chromosome preparation
6.In situ hybridization
7.Detection of hybridization
8.Microphotography
Karyotyping of Himalayan rye
(Secale cereale)
(Chaudhary and Mukai, 2007)

46. • Pm62, a novel adult-plant
resistance (APR) gene against
powdery mildew, was
transferred from D. villosum
into common wheat in the
form of Robertsonian
translocation T-2BS.2VL

48. Techniques for gene Transfer
•Transfer of whole genome
•Transfer of individual whole chromosome
•Substitution of alien chromosome arms
•Interchanges
a) Through irradiation
b) Through recombination
i. Manipulation of dipolidizing system
ii. Without manipulation of diploidizing system

49. • Efforts made to transfer whole genome to 4x and 6x wheat from alien genera leads to
production of
Transfer of whole genome
• Used for transfer of resistance against
disease “ eye spot”
• Cultivar named ‘roazon’ developed by
back crossing
• Used for production of alien
addition lines for transfer of rust
resistance

50. Triticale, a successful man made cereal

51. Transfer of whole chromosome
(Alien addition and substitution lines)
• Alien addition lines and alien substitution lines produced
by
1. crossing of recipient species with donor
2. Produced F1 chromosomes doubled
3. Back crossed with recipient species
4. It gives pentaploid individuals
5. They back crossed with recipient species
6. It produces alien addition monosome or substitution monosome
7. Selfing of alien addition monosome gives alien addition line
8. Selfing of alien substitution gives alien substitution line

52. Sr
no
Alien additions Chromoso
mes
Scientists Achievements
1 Rye ------ Wheat 21II + 1II - Hardiness, disease
resistance
2 Aegilops,
Agropyron, -- Wheat
Daspyron
21II + 1II - A. junceum– salt tolerance
Disease resistance
3 Barley ----- Wheat 21II + 1II Islam et al., 1978
4 Wheat ---- Rye 7II + 1II Schlegel et al., 1986
5 O. officinalis ---- O. sativa 12II + 1II Jena and khush, 1986 Resistance to BPH
6 Avena hirtula --- A. sativa 21II + 1II Thomas, 1968 A. strigosa - rust resi.
A. barbata - PM resi.
7 Beta procumbens --- B. vulgaris 9II + 1II Savitsky, 1973 Resistance to cyst nema
8 G. sturtianum --- G. hirsutum 26II + 1II Altman et al., 1987 Cold tolerance
Disease resistance

53. Sr
no
Alien substitutions Chromosomes Scientists Achievements
1 Rye -- Wheat 20II + 1II O’ Mara,
1940
2 N. glutinosa -- N. tabacum 18II + 1II - Mosaic resistance
Var ‘samsoum’
3 A. strigosa --- A. sativa
A. barbata - A. sativa
20II + 1II -

54. Substitutions of alien chromosome arm
(Robertsonian translocations)
• Transfer of whole chromosome leads to addition of many undesirable
genes
• So we need to transfer part of chromosome which carry desirable genes
• It can be possible in alien lines by misdivision followed by re-joining of
arms
• A very important translocation is 1BL.1RS in wheat carries disease
resistance
e.g. Cultivars of ‘Veeri’ series of CIMMYT

55. Interchanges (using irradiation)
• Sears in 1956
• First useful transfer by irradiation
• Ae. umbellulata chromosome 6U to wheat
chromosome arm 6BL
• Transfer of leaf rust resistance gene Lr 9
• T. dicoccoides used as bridging species
• Irradiation of pollen with X rays

56. Interchanges through recombination
•Through manipulation of diploidizing system
1.Removal of 5B chromosome
2.Suppression of 5B effect by genome of Ae. speltoides or Ae. mutica
3.Utilizing recessive mutant of Ph1 locus on 5B
•Without manipulation of diploidizing system

57. • Used nulli 5B-tri 5D line of wheat to cross with Agropyron
• Absence of 5B allow pairing between homeologous chromosomes
• Transfer of segments of 3Ag (carrying Lr 24 gene) to 3D chromosome of
wheat
• Segments of 7Ag (Lr 19) to 7D chromosome of wheat

58. • g
Important translocations in Wheat

59. Recombination without manipulation of diploidizing system
• In groundnut dipolidizing system is weak or absent, so occasionally allosyndatic pairing
occurs which leads to useful translocations to transfer disease resistance from A.
monticola (Singh and Gobbons, 1986)
• In genus Brassica
club root resistance- B. camprstris to B. napus
self incompatibility transfer to forage rape
non shattering -- B. juncea to B. napus

60. Conclusion
• Both the classical and molecular cytogenetic techniques are important for the gene location
• Among the several cytogenetic techniques that can be employed in centromere mapping and in
determining the orientation of linkage maps, translocations and deficiencies have been used
profitably
• Deficiency and translocation are most commonly used for the location of genes in several crops like
tomato (deficiency), maize and barley (translocation)
• Monosomic and nullisomic analysis are widely used for locating the genes in polyploids such as
wheat
• Trisomics are used for gene detection in diploids

61. • Banding techniques are extensively used for karyotyping and gene location in plants
• Recently, In situ hybridization techniques (GISH and FISH) are extensively used in
determining physical location of genes with high degree of precision
• Fluorescence in situ hybridization (FISH), which uses repetitive DNA clones, is a
powerful tool for identifying chromosomes within a species or tracing intergenomic
chromosome rearrangements in polyploid species
• GISH is used to estimate the amount of alien chromatin within chromosomes in
interspecific hybrids

62. • Several techniques have been developed for transferring whole genome,
individual chromosome and segment of the chromosome
• Whole genome transfer used for the production of bridging species to
transfer specific gene and novel new species
• Alien addition and substitution lines widely used for transferring the
individual chromosome where whole genome transfer is not feasible
• Most important translocation like 1BL.1RS produced through interchanges
between chromosome of two different species