Genome mapping of Drosophila

2. Group Agricultural Biotechnology
Division
Rubina Bukhari
Andleeb Anjum Qazalbash
Najida Irfan
Members:
Waqar Khan
Asma Zia
Arzish Javaid

3. Outline
• Introduction, Life Cycle and Role as
Model Organism
• Meiosis in Drosophila
• Polytene Chromosome
• Morgan’s Experiment with Drosophila
• Deletion Mapping
• Recombination Mapping

4. Introduction
• Drosophila derived from the Greek word drosos
means dew loving
• They belong to the Drosophilidae family and are
agricultural pest most frequently known as fruit
flies
• Cinderella of genetics
• They feed primarily on unripe or ripe fruits
• Most Drosophila spp. are small, about 2–4 mm
long
• Drosophila's wings can beat 220 times per
second
• Drosophila contains one of the most advanced
forms of eye among insects, i.e., compound eye

5. Drosophila
Chromosomes

6. Radiation caused harmful genetic defects in the offspring of exposed humans –this was at
the advent of man's attempts to harness and exploit nuclear fission
Irradiated flies looked normal, their offspring frequently showed the effects of mutation
Using Drosophila in the 1920s, Muller discovered that x-rays caused a massive increase in
the mutation rate of genes, and could actually break chromosomes
Establish that they were found within chromosomes (long before it was even established
that DNA is the genetic material).
Morgan greatly refined the theory of inheritance first proposed by Gregor Mendel, by
using Drosophila to define genes
Father of Drosophila research “Thomas Hunt Morgan”

7. » The genome of the fruit fly has been entirely sequenced and annotated
» It has over 14,000 genes spread over four chromosomes, although only
three of them contain the bulk of the genome
» Two-thirds of the known disease-causing genes in humans have been
identified in the fly
» The genome information of Drosophila allows targeted tissue-specific
overexpression and downregulation of disease-inducing genes that
may be used to determine the medicinal/pharmacological effects of
various plants/plant-derived components by examining their influence
on disease progression and rescue
» The GAL4-UAS binary system in flies is a sophisticated tool used to
upregulate and downregulate a gene
Genetic study

8. Life
Cycle

9. Drosophila as a model organism -
Advantages
»Mutations of any gene in D. melanogaster can be easily generated
within a month using the clustered regularly interspaced
palindromic repeats/CRISPR-associated (CRISPR/Cas9) system,
allowing the creation of a large number of mutant and transgenic
fly lines
»Additionally, the life cycle of a fly is short. Within 10 –12 days at
25°C, a single viable mating pair can generate hundreds of off
springs that are genetically identical to their parents
»Moreover, Drosophila is a very small insect (approximately 3 mm
in size), very easy to handle, and requires very little space in the
laboratory

10. Black
soybean seed
coat extract
BSSCE upregulated the
expression of iron
metabolism genes (Fer1HCH,
Fer2LCH, dZIP13 and Mvl),
which were downregulated by
Pb
Drosophila
role against
pb toxicity
Pb upregulated the mRNA level
of SOD1, SOD2, CAT and Nrf2 and
BSSCE restored them to a relatively
normal level
RNA isolation,
cDNA synthesis
and
quantitative
real time RCR
(q-PCR)
BSSCE in
promoting
iron
absorption

11. Why use Drosophila as a model to
study meiosis?
 Ease of care and handling
 Short generation time (10 days at 25°)
 Large brood sizes (one female can lay >75 eggs per day)
• Provided insight of chromosome sites (including the centromere and
the telomeres)
• Constructed the first meiotic map based on recombination frequency
• Genetic mutants easily created, maintained, and shared among
members of the Drosophila
• Maintaining classical mutant alleles, due to the availability of a wide
variety of balancer chromosomes

12. Ovarioles develop oocytes in temporal order, with
stem cells at one end, and mature oocytes at the
other
Each ovariole is divided into two main sections.
The germarium, has stem cells and early meiotic
prophase
The vitellarium, has oocytes arrested in late meiotic
prophase concerned with
 Development
 Growth of the oocyte
 The germarium (D) is composed of germline stem cells
(pink) in their niche
 Daughters of the germline stem cells (peach) divide to
form a cyst
 (E); the pro-oocytes (gray) initiate SC formation in
zygotene of prophase I
 Follicle cells (light green), daughters of follicle stem cells
(dark green), encapsulate the cyst at the posterior of the
germarium.

13. Oocyte development: stages 1–10
Germline stem
cell (asterisk)
Cystoblast,
16-cell
interconnected
cyst
Synaptonemal
complex (SC)
Unpaired
centromeres
(blue) in two-cell
cysts
Clustering in
eight-cell cysts
16-cell cysts in
region 2A
(prophase I )
Double-strand
break (DSB)
formation

14. • One nucleus chosen as the oocyte nucleus
• Nurse cells are formed by other cells
Stage 1
• Chromosomes are reorganized and
condense to form the karyosome.
Stage 2-3
• Euchromatic SC begins to disassemble in
midprophase
Stage 5
• Euchromatic SC will completely absent
• Centromeres remain clustered
Stage 7-9
• Chromosomes briefly decondense
Stage 10
• Oocytes and transcription is upregulated
before chromosomes recondense
Stage 11

15. • Karyosome recondenses
Stage 11
• Preparation for germinal vesicle breakdown
Stage 12
• Germinal vesicle breakdown
• Tubulin recruited to the chromosomes organized into a bipolar
spindle
• Dynamic movements of Achiasmate chr. toward s spindle poles
in prometaphase I,
Stage 13
• Achiasmate chromosomes congress to join the chiasmate
chromosomes
• Biorentation of chr. at metaphase plate
• Oocyte arrest metaphase 1 until activation
Stage 14

16. Meiosis I spindle assembly
• Upon GVBD, tubulin is recruited to the chromosomes
• Meiotic spindles are anastral (acentriolar) in Drosophila oocytes
• It is the chromosomes that recruit tubulin and organize the
direction of the developing bipolar spindle
• The spindle composed of antiparallel microtubules at the central
spindle that interact laterally with the chromosomes kinetochore
microtubules and other polar spindles

17. Meiosis II
• Drosophila oocytes remain arrested at metaphase I up to 2 days
• Drosophila oocyte activations as well as resumption of meiosis is a passage
through the oviduct than fertilization
• After activation, anaphase I and meiosis II completed within ∼20 min
• Ca2+ signaling plays an important role in activation
• The progression from anaphase I to meiosis II is dependent on the calcineurin
pathway
• Upon activation, the spindle rotates to align at a right angle with the anterior–
posterior axis of the oocyte and the CR of the spindle elongates
• As chromosomes move toward the spindle poles in anaphase I, the center of the
spindle pinches in between the chromosomes and an aster of microtubules
• Loss of the centrosomal protein centrosomin leads to an aberrant central aster
between the developing prometaphase II spindles, illustrating the requirement for
centrosomal proteins for the formation of the central aster
• After prometaphase II, the sister chromatids segregate away from each other on
the twin spindles to rapidly complete the second meiotic

18. Polytene Chromosomes
»Specific interphase chromosomes consisting of
thousands of DNA strands
»They are very large and display a characteristic
band–interband morphology
»They provide a high level of function in certain
tissues such as salivary glands of insects.
»First reported in insects by E.G.Balbiani in 1881
»Reported in Drosophila by Bulgarian geneticist
Dontcho Kostoff in 1930
VF Semeshin

19. Polytene Chromosomes
Formed in
• Cells that undergo transient nuclear envelope breakdown
without subsequent chromosome separation
• Pathologically from perturbations in cell cycle regulation
o Muscular dystrophy patients
o Spontaneous abortions
o Many tumor types
• Can be formed by treatment with mitosis blocking drugs
Significance
• Good study model for chromosomes
• Large size & prevalence → easy visualization of structural
features

20. Found in:
• Dipteran flies
• Collembola arthropods
• Ciliophora protozoan
• Mammalian trophoblasts
and antipodal
• Suspensor cells in plants
Chromonemata
0.5 mm
20 μm
Repeated division of the chromosome
in the absence of cytoplasmic division
Endomitosis
Dark Bands
more DNA and
less RNA
Light Bands
more RNA and
less DNA
Function:
• ↑ Nuclei’s volume => Cell expansion
• High level of gene expression
o Endoreduplication of larval
salivary glands => large quantities
of adhesive mucoprotein (“glue”)
before pupation
o Tandem duplication located near
the centromere => Bar phenotype
of kidney-shaped eyes
Interbands
• Interacts with the active
chromatin proteins &
nucleosome remodeling
• Binding sites for RNA pol II
• Initiate replication
Zykova et al.
I. F. Zhimulev et al.

21. sections of the genome are not
replicated during S-phase
gene poor regions & repetitive
regions
(heterochromatin)
Merit:
focus cellular resources
elsewhere
Consequences:
• DNA breaks
• Accumulation of deletions,
translocations & inversions
genes involved in cell adhesion &
neurogenesis
specific loci are replicated additional times
compared to the rest of the genome
follicle cells in the Drosophila ovary
chorion genes (egg shell) Stormo B.M., Fox D.T., et al

22. Puffing
The bands of polytene chromosomes become
enlarged at certain times to form swellings called
puffs
Uncoiling of individual chromomeres
Indicate the site of active genes
where mRNA synthesis takes place
Balbiani rings
Formed of DNA, RNA and a
few proteins
Sites of transcription
Contains RNA
polymerase and RNPs
Stormo B.M., Fox D.T., et al

23. Morgan discovery of linked genes
» Characteristics of linked genes
• Genes that are close chromosome are often inherited together
• Linked genes usually don’t separate during crossing over prophase I of
meiosis
» Used Drosophila in his experiment because:
• Mature in 2 weeks
• Produce large number of offspring
• Only have 4 pairs of chromosomes
• One pair is sex chromosome

24. Experiments for linked genes
» Morgan crossed fruit flies for two traits
» Homozygous dominant gray bodies and normal wing size (GGWW) with
homozygous recessive black bodies and small wings (ggww)
» Expected ratio:
• ¼ gray normal
• ¼ gray short
• ¼ black normal
• and ¼ black short
GGWW × ggww
(F1) GgWw × ggww

25. »Instead of getting testcross ratio he observed following
frequency:
• 41.5 % gray/normal
• 41.5 % black/short
• 8.5 % gray/short
• 8.5 % black/normal
»Concluded that both the genes are on the same
chromosome which indicates that genes for body color
and wing size were linked on one chromosomes.
Deviation from testcross ratio

26. Morgan sex linked experiment
» In 1910, Morgan published details of his research in an article titled “Sex
Limited Inheritance in Drosophila”
» Sex Lined genes on sex chromosomes
» Discovered the 1st sex linked gene in fruit flies
• Crossed red eyed female (WW) with white eyed (ww) recessive male
• In F1 all were red eyed
• Then red eyed female crossed with red eyed male
• 3:1 red eye to white eye (only male had white eyes)
» Hypothesized: Eye color must be a sex-linked gene

27. Morgan’s experimental cross

28. Deletion mapping
• Specialized genetic mapping technique that enables scientists to
determine the location of a specific gene on a chromosome
• This technique is useful when the location of alleles, variants of a
recessive gene, are known to be located within a specific region, but
their specific location is unknown
• The process of deletion mapping is that a donor strain, which carries a
point mutation in the gene of interest, cannot restore the wild-type
function of the gene when crossed with a recipient deletion mutant
strain when both the point mutation and a region of the deletion
coincide i.e. affect the same base pair. If the point mutation of one
strain does not coincide with the deletion mutation in the other strain,
then the restoration of wild-type function can occur

29. Deletion mapping methodology
• Designing a deletion series
• Generating deletion mutants
• Phenotypic analysis
• Correlating phenotypes with deletions
• Fine mapping and identification

30. Examples
• Eye development genes: Deletion mapping has been
employed to study genes involved in eye development in
Drosophila. For example, the eyeless (ey) gene is crucial for
eye formation. Deletion mapping experiments helped identify
the region of the genome containing the ey gene. Further
analysis of smaller deletions within that region allowed
researchers to pinpoint the exact location of the ey gene and
understand its role in eye development.
• Other examples include circadian rhythm genes and memory
regulation genes.

31. Deletion Mapping and in situ hybridization
• A form of complementation analysis can be used to determine whether a given mutation maps
within a given deletion or outside it. Essentially the deletion stock and the mutant stock are
crossed to generate flies carrying one chromosome of a pair of homologues carrying the
deletion and the other carrying the mutation (mutation/deletion). If flies of this genotype have
the mutant phenotype (the deletion fails to complement the mutation) the mutation must fall
within the deleted region so the fly has no wild type copy of the gene. Conversely if flies of this
genotype show a wild type phenotype(the deletion complements the mutation) the mutation
must map to somewhere outside this deletion.
• Collections of overlapping deletions for many regions of the Drosophila genome have been
isolated and mapped.

33. Deletion mapping problem
• Locations of six deletions mapped on
Drosophila chromosome
• Recessive mutations a, b, c, d, e, f, and g
are known to be located in the same
regions as deletions
• “m” and “+” represents mutation and
wild, respectively
• Order of seven mutant genes on the
chromosome?
Location
of
six
deletions
Mutations
Results
g c b
a e d f
Gene order:

34. Recombination
Mapping

35. First genetic map
» Alfred Henry Sturtevant proposed that the "proportion of crossovers could be used
as an index of the distance between any two factors"
» Sturtevant drew first chromosomal linkage map for the genes located on the X
chromosome of fruit flies
» In Sturtevant's gene map, six traits are arranged along a linear chromosome
according to the relative distance of each from trait B. Traits include yellow body
(B), white eyes (C, O), vermillion eyes (P), miniature wings (R), and rudimentary
wings (M)

36. Mapping
through
two-point
test cross

37. Recombination Frequency =
No. of recombinant progeny
Total no.of progeny
× 100
=
110+90
390+410+110+90
× 100 = 20 cM
Gene Mapping
Genotype Counts
vg+ b+ 390
vg b 410
vg b+ 110
vg+ b 90
1000
20 cM
vg b

38. Why are three-point testcrosses
important?
» Simultaneous analysis of 3 points (genes)
» By solving a three point cross you can determine two
important things:
• Order of the genes on a chromosome
• Distance between each pair of genes
Three types of
crossover with three
linked loci

39. Three-point testcross
can be used to map
linked genes
st e+ ss
Progeny
number
283
278
50
52
5
3
43
41
755
Parents
DCO

40. Recombination Frequency (RF)
Recombination frequency is the frequency with which a single
chromosomal crossover will take place between two genes during
meiosis
Unit:
Map unit (m.u.) OR centimorgan (cM)
Formula:
RF =
No.of recombinants
Total progeny
× 100

41. • RF (st-ss) =
50+52+5+3
755
× 100 = 14.57%
• RF (st-e) =
50+52+43+41
755
× 100 = 24.64%
• RF (e-ss) =
43+41+5+3
755
× 100 = 12.19%
 The map distance is equal to the frequency of recombination
• Thus, st and ss are separated by 14.57, st and e by 24.64, and e and
ss by 12.19 cM
Linkage Map:

42. Interference
The detection of the double recombinant classes shows that
double crossovers must occur. When crossover in one region
affects the probability of a crossover in a nearby region the
interaction is called interference.
Interference(I) = 1 − c.o.c.
c.o.c. =
Observed number of double recombinants
Expected number of double recombinants
• Coefficient of coincidence =
5+3
0.14×0.12×755
=
8
12.68
= 0.631
• Interference = 1-0.631 = 0.369 = 0.369×100 = 36.9%

44. Task 1
In fruit fly, cherub wings (ch), black body (b), and cinnabar
eyes (cn) result from recessive alleles that are all located on
chromosome 2. A homozygous wild-type fly was mated with
cherub, black, and cinnabar fly, and the resulting F1 females
were test-crossed with cherub, black, and cinnabar males.
The following progeny were produced from the test cross:
Q1. Calculate the recombinant distance between the three
loci.
Q2. Find out the linear order of the genes on the
chromosome.
Q3. Determine the coefficient of coincidence and the
interference for these three loci.

45. Task 2
Find out the order of
the genes and
calculate the
interference

46. Task 3
a. Draw the alleles in their proper
positions on the chromosomes
of triple heterozygous
b. If appropriate calculate
interference

47. References
• https://onlinelibrary.wiley.com/doi/full/10.1002/9780470015902.a0001183.pub2#:~:tex
t=Polytene%20chromosomes%20are%20gigantic%20interphase,formation%20directly%
20under%20the%20microscope
• Zhimulev, I.F. and Koryakov, D.E. (2009). Polytene Chromosomes. In eLS,
(Ed.). https://doi.org/10.1002/9780470015902.a0001183.pub2
• https://en.wikipedia.org/wiki/Polytene_chromosome#:~:text=7%20External%20links-
,Function,high%20level%20of%20gene%20expression.
• Polyteny: still a giant player in chromosome research - Scientific Figure on
ResearchGate. Available from: https://www.researchgate.net/figure/Common-features-
of-polytene-chromosomes-While-polytene-chromosomes-are-found-in-
a_fig1_318902685 [accessed 17 Jun, 2023]
• Lobo, I., & Shaw, K. (2008). Thomas Hunt Morgan, genetic recombination and gene
mapping.

48. THANK YOU