Different ways of mapping the genes using cytological tools, Difference between conventional and cytological mapping. Case studies.

1. LOCATION AND MAPPING
OF CHROMOSOMES USING
CONVENTIONAL AND
CYTOLOGICAL MEANS
NOOR E MUJJASSIM
PALB 8078

2. 2
Contents
Introduction to Genetic map
Steps in construction of linkage map
 Application and limitation.
 Cytological map construction
 Deletion, interchange, monosomics and
trisomics, monotelodisomics.
 Conclusion

3. A map - Graphical representation that provides information
about the location of sites and the spacing between them.
Genetic map is the schematic representation of the various
genetic markers in specific order in which they are located
in a chromosome as well as the relative distances between
these markers.
Map

4. Why to map the genes ?
Information gained regarding
 chromosome organization
 gene function and evolution
Mapping of a gene is often first step in
 identifying gene responsible for
phenotype
 identifying mutants
 studying gene function

5. GENETIC/ LINKAGE MAP
• Linkage map is a schematic representation of relative location and
position of genetic markers on the chromosomes as determined by
frequency of recombination between all possible pairs of genetic markers.
• Depiction of the genes linked together on a straight line.
• measured in centi morgans (cM)
• map unit- 1 mu = 1% recombination between linked genes
• also known as a chromosome map. 5

6. • A linkage or genetic map of any plant or animal species denotes the
linear order of markers on a particular chromosome, which is determined
by the number of crossover events and recombination frequencies
between markers (Sturtevant, 1913).
• During segregation if a block of genes or chromosomal segments do not
assort independently, they are described as being linked. This is the basis
on which a genetic map is developed.

7. • Recombination among polymorphic loci and the likelihood that
recombination events occur between two points of a chromosome depends
in general on their physical distance.
• The closer they are located to each other, the more they tend to stay
together after meiosis.
• With the increase of the distance between these points on the chromosome,
the probability for recombination increases and genetic linkage tends to
disappear.
• This is why genetic linkage can be interpreted as a measure of physical
distance.
7

8. Steps in linkage map construction
1. Assigning markers onto chromosomes or linkage groups.
2. Finding the relative order of the markers.
3. Estimating the relative distances b/w the markers in map units
8

9. principle in assigning the markers on
chromosomes
• All those markers between whom the recombination frequency is less than
0.5 should be present on same chromosome or single chromosome.
• Punnett and bateson (1905), who examined two other traits (flower colour
and pollen shape) in pea plants.
• If traits are co-inherited more often than expected by chance, they are
linked.
9

10.  Genetic maps of chromosomes are based on the average number of
crossovers that occur during meiosis.
 Recombination frequencies less than 15 % estimate map distance
directly; however greater than 15% underestimate map distance because
multiple cross over events do not always produce recombinant
chromosomes.
 an average of one chiasma during meiosis is equivalent to 50 centi
morgan of genetic map distance.
10

11. MAP CONSTRUCTION
pre requisites:
Divergent parents.
Mapping population
Probes (c dna clones or genomic dna clones)
software.
11

12. DNA
isolation
Keep the tubes in
PCR
Complimentary strand
synthesis
Amplified product separated
by gel electrophoresis
Screen in mapping
population
Identify polymorphic markers
Score the data
Feed into software
Linkage map

13. Once the markers are assigned to linkage group,
then what next?
 Identifying the best possible order of markers:
1.Minimum sum of adjuscent RF (SURF)
2.Minimum product of adjuscent RF (PUF)
3.Maximum sum of adjuscent LOD Score(SALOD)
4.Least square method
5.Maximum likelihood method
13

14. Finding the relative distance between ordered
markers;
• By using mapping functions;
1.Morgan’s mapping function
2.Haldane mapping function
3.Kosambi mapping function
14

15. Factors on which linkage map depends;
 Size of the mapping population.
 Number of markers.
 Recombination fraction threshold.
 LOD score
 Criteria used to arrive at best possible order
 Mapping function used
15

16. BASIC AND APPLIED USES OF GENETIC MAPS
(a) Study genomes of related species with common markers,
(b) Tag important traits of interest with associated markers and follow the
transmission and selection of the former in breeding cycles, and
(c) With the advent of recombinant DNA technologies, genetic mapping can
be carried to its logical conclusion, positional cloning (isolation) of a gene
solely on the basis of its chromosomal location without regard to its
biological function.
16

17. LIMITATIONS OF GENETIC MAP
 The resolution of genetic map depends on the
number of cross overs that have been scored
 Genetic maps have limited accuracy.
 Certain genomic regions more sensitive to
recombination
 Markers must be polymorphic for genetic
mapping 17

18. Contents
• Introduction
• Techniquesfor Gene Location
1. Useof Structural ChromosomalAberrations
2. Useof Numerical ChromosomalAberrations
3. Useof ChromosomalBanding
4. Useof In situ hybridizationtechniques
• Techniquesfor GeneTransfer
1. Transfer of whole genome
2. Transfer of whole chromosome
3. Substitution of alien chromosomearm
4. Interchanges

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

20. Use of structuralaberrations
1. Deficiencies
2. Inversions
3. Translocations
a) Uselinkage between
marker and semisterility
b) Overlapping
translocations
c) Useof B-Atranslocations
Use ofaneuploids
1. Trisomicanalysis
2. Monosomic and
nullisomic
analysis
Use of chromosome bandingtechniques
1. Qbanding
2. Gbanding
3. Cbanding
4. Nbanding
In situ hybridizationtechniques
1. FISH
2. GISH

21. Deficiencies in Chromosomemapping
• Mackensen (1935) and Slizynska (1938) used deficiencies for locating genes(type ofwing)
in Drosophila

22. Use of deficienciesin chromosome mapping in plants
• Female with recessive marker stocks crossed with
irradiated pollen from male carrying dominant
allele
• F1heterozygote 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

23. • 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 cytologicalstudy
• 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 thecomplement.

24. Cytogenetics, P.kgupta, 2016
35 genes
on 18
chromoso
me arms

26. Inversions for locatinggene
• Inversions can be readily detected by a comparison of recombination frequency
betweentheplantwithnormalandinvertedchromosome.
• Existence-firstdetectedbySturtevantandPlunkettin1926
• They prepared genetic maps for the genes se st p DL H ca located in the third
chromosomeof Drosophila melanogaster (gene sequence: se st pDLHca) using
lessrecombinationwithininverted segments.

27. Interchanges in chromosomemapping
1.Using linkage between marker and semisterility
• Translocations have been utilized in maize, barley and pea for determining linkage
groups/genes with specificchromosomes
• It is based on observed linkage between interchange breakpoint or semi-sterility &
marker gene located on one of thechromosomes
• involves crossing of translocation heterozygote and recessive markerstock
• F1are selfed or backcrosswith recessive markerstock
• In F2study of recombination between breakpoint or semi sterility & marker gene using
two point or three point test crossmethod.

28. Chromosome mapping using translocation in maize
Chomosome Marker genes mapped References
1 br ( brachytic dwarf)
f ( fine striped leaf)
an ( anther ear dwarf)
bm2 ( brown midrib leaf)
Burnham ( 1948)
7 V5 (virescent)
Ra ( ramose tassel ear )
gl ( glossy leaf -1 )
Ij ( iojap variegation )
Burnham ( 1948)
8 j (japonica) Burnham ( 1934)
9 C ( coloured aleurone)
sh (shrunken)
wx (waxy)
Burnham ( 1934)

29. • 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

30. 2. Overlapping translocationsprocedure
• Method first proposed by Gopinath and Burnham in1956
• Femaleproduces four type of gametes
1. balanced translocation for onechromosome
2. balanced translocation for anotherchromosome
3. gamete with duplications
4. gamete with deficiencies(they areabortive)
• So,if gene located in interstitial region then progenies will have genotypes AaandAAa
• From this when segmental trisomic (AAa) selfed, itwill give trisomic ratio 17:1
• Thistechnique extensively used in Drosophila to locate structuralgenes

31. Interstitial trisomic region

32. 3. Use of B-Atranslocations
• Most efficient method for locating recessive mutants to the proper chromosome arm
• Romanand Ullstrup demonstrated the usefulness of the B-Atranslocation method
when they located agene (hm) for reaction to Helminthosporium carbonurn in maize
• In 1947, Romanproduced the first B-Atranslocations by X-raying mature pollen from
plants carrying B-typechromosomes
Production of aB-Atranslocation
Translocation heterozygote
Translocation homozygote

33. Cont…..
• Romanshowed that nondisjunction of the Bchromosome 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

34. Use for locatinggene
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

35. Use of Aneuploid forlocatinggenes
Aneuploidy
loss or gain of one or few
chromosomes ascompared
to normal somatic
chromosome complement
Generally asaview of
locating the geneswe
usenullisomic,
monosomic or trisomics
asper convenience

37. TrisomicAnalysis
• Use of trisomics for locating the genes and preparing chromosome mapping called
trisomicanalysis
• BRIDGES (1921)was first to usetrisomics to locate the gene ey (eyeless) hasits locus
in the fourth chromosomeof Drosophilamelanogaster.
• Similar identifications followed shortly in Datura: the gene “ White ” wasidentified
with the trisomic type “Poinsettia ” (BLAKESLEEandFARNHAM1923)

38. Analysis

39. Assigning linkage groups tochromosomes

40. Locating gene on specific chromosomearm

42. Monosomic & NullisomicAnalysis
Monosomic analysis
• Usedfor locating genesin Polyploid speciesand in maize (can tolerate
monosomic)
For dominant gene For recessive genes

43. Locating Dominant gene by absenceof
expression in nullisomics
• When dominant genelocated on chromosome
• Nullisomic for that chromosome will not show that character
• E.g.,
genesfor red seedcolour--- on chromosome3D
genefor awn suppression -- on chromosome 4Band6B
Wheat

44. Locating dominantgeneby analysis ofF1
• Crosswhole set of monosomics using asfemale containing dominant marker with
normal male containing recessive for thattrait
• Analyse ratio observed in F1

45. Locating recessivegene by analysis in F2
• Crossbetween whole set of monosomoics containing recessive trait with normal male
carrying homozygous dominant for thatgene
• In F1 all plants show dominant phenotype for both critical and non critical lines
• In F2ratiosanalysed

46. Locating genes usingintervarietal chromosomal
substitutions
• Useof whole chromosomesubstitutions
donor1. Monosomic series crossed with
variety
2. F1monosomics selected cytologically
3. Selfed to get disomic for the univalent
chromosome of donor
4. Then disomics are back-crossed to
recipient to recoverrecipient
5. If this substitution leads
morphological changes then
conclude that gene located
to major
we can
on that
chromosome

47. Locating genes on chromosomearms
• Locating gene on one of two arms of chromosomes by using telocentricchromosomes in
form of
Monotelosomics - 20II +1tI
Monoisosomics - 20II +1iI
Ditelosomics- 20II +1tII
Monotelodisomics- 20II +1heteroII
• Example :
In Chinesespring wheat monotelosomics for chromosome arm 6BSare awned (Chinese
spring is awnless), soit concluded that awn inhibitor gene B2is present on 6BL(Sears,1962)

49. The long arm of chromosome 5A of common wheat carries the gene Q which is
responsible for the suppression of speltoid effect and for squarehead spike
characteristic of the variety Chinese Spring (SEARS 1954).

50. Materials and Methods
 Chinese Spring wheat ditelosomic for chromosome arm 5AL was crossed to the
substitution line 'spelta-5A'.
 Spikes of the F1 plants (2n=41+t) were speltoid and nonsquare head.
 The F1 plants were selfed as well as test-crossed as male parents to Chinese Spring.
Results and Discussion
 The test-cross progeny consisted of 44 plants of which 26 were of vulgare-type and 18
of spelta-type. This phenotypic segregation conforms to a 1:1 ratio, indicating independence
of the Q locus and the chromosome 5A centromere.
 The chromosome constitution and phenotype of the test cross plants are summarized in
Table 1.

52.  The F2 progeny of the selfed monotelodisomic F1 consisted of 227 plants of which
166 were of spelta-type (nonsquarehead) and 61 were of vublgare-type
(squaerhead). This segregation was a very good fit for a 3:1 ratio (X2=0.424;
p=0.50-0.70).
 A random sample of 80 F2 plants were used for meiotic studies to determine their
chromosome constitution.
CONCLUSION
 The data of both test cross and F2progenies indicated that the Q locus is
genetically independent of the chromosome 5A centromere.
 Hence it can be concluded that the gene Q is located 50 or more crossover units
from the centromere, i.e. it has a distal location on the long arm of chromosome
5A.

53. Conclusion
• Both the classical and molecular cytogenetic techniques are important for thegene 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 genesinseveral crops like
tomato (deficiency), maize and barley (translocation)
• Monosomic and nullisomic analysis are widely used for locating the genesin polyploids suchas
wheat
• Trisomics are usedfor gene detection in diploids