Sex-Linked Inheritance and Interaction of genes

• We were given three culture bottles with Drosophila

melanogaster , one set with red-eye color & two sets

of white -eyed mutants.

• Let one set of white eye mutant be named as “A” and

another set of white eye mutants is named as “B”.

• Before we set up the cross, we cultured the flies, so as

to increase the count of flies.

• The flies were sub-cultured for ten days.

• Approximately we got 150 flies.

Culture media

For Drosophila, a satisfactory standard culture medium

must be nutritious, inexpensive, have a high moisture

content and a firm texture, and be resistant to mould and

bacterial contamination. Most standard media contain

sugar source, a grain base, agar, a mould inhibitor and

yeast (Ashburner & Thompson,Jr 1978).

Culturing of D.melanogaster

Drosophila was one of the first organisms to be studied

genetically: its small size, short life cycle, high

reproductive rate, and ease of culture. Many different

species, and a large number and wide variety of

naturally-occurring and artificially-induced genetic

variants are available.

Now we should collect virgin females.

Importance of Virgin Females

All female flies used in controlled genetic crosses mustbe “virgins”. Female flies are capable of mating as earlyafter emerging from the pupae stage and arepolyandrous, that is, capable of mating with severalmales. Once mated, females can retain viable sperm forseveral days and this will confuse the results of asubsequent controlled mating. To prevent this, all adultflies are removed from the culture bottle prior, so thatall newly hatched flies will remain virgin.

Experiment 1

After collecting the virgins we have to keep for aging.

Aging is done to know whether the collected virgins are

contaminated or not.

Crosses are set up. In direct cross, 5 virgin red-eyed

female is crossed with 5 white-eyed males(A).

In reciprocal cross, 5 virgin white-eyed females(A) are

crossed with 5 red-eyed males.

We allowed the cross to develop for 8 days.

Then F1 progenies were isolated and phenotypes were

examined.

The observed results are as follows:

Direct cross

Reciprocal cross

Reciprocal cross

Phenotype Red eye female Red eye male

No. of flies 90 72

F1 Generation

Phenotype Red eye female white eye male

No. of flies 75 63

DIRECT CROSS

RECIPROCAL CROSS

By observing F1 progeny of direct and reciprocal cross,

we could analyze that there is no symmetry i.e. they are

asymmetric.

In direct cross all F1 progeny red eyes(100%) is seen

but whereas in reciprocal cross both red and white eyes

are seen(appx. 50:50).

As it shows asymmetry it is not present on autosome,

so it may be present on sex chromosome.

In direct cross, we could only find red eyed F1

progenies. So we suggest that red is the dominant

character.

In reciprocal cross only F1 males were white-eyed. This

shows sex bias, hence it is present on sex chromosome.

As only males are affected and females are not affected,

we could predict that the gene is present on X

chromosome

This may be because females consist of two X

chromosome and male only I X chromosome.

May be red-eye is dominant over white-eyed.

By the end of F1 generation, we couldn’t identify the

given flies were either in homozygous or heterozygous

condition.

How to identify whether F1 progeny

are in homozygous or heterozygous

condition?

To confirm whether the given parental flies are

in homozygous or heterozygous condition we

have to conduct test cross

Test cross was conducted by using virgin F1

female with recessive male parent.

DIRECT CROSS RECIPROCAL CROSS

Date RED WHITE RED WHITE

24/10 0 1 3 1 2 3 5 1

25/10 5 7 5 1 4 1 6 4

26/10 6 4 3 5 7 6 2 2

28/10 4 7 6 4 5 7 7 6

29/10 3 2 2 5 7 3 3 4

39 35 45 40

74 85

TEST CROSS

By the end of the test cross both direct and reciprocal

cross, we got all possible progenies i.e.,

1. Red-eyed females

2. Red-eyed males

3. White-eyed females and

4. White-eyed males.

Obtained ratio was found

to be 1:1

Whether the character present on one gene or multiple gene?

F1 selfing was carried out so as to confirm how many

genes were controlling the eye trait.

Five F1 females was crossed with five F1 males for

both direct and reciprocal cross.

In direct cross, we got F2 progeny in the ratio of

Red : White

2:1

In reciprocal cross, we got F2 progeny in the ratio of

Red: White

1:1

DATE RED EYED FEMALE RED EYED MALE WHITE EYED MALE

24/10/13 34 20 20

25/10/13 40 23 18

26/10/13 35 22 14

28/10/13 39 20 19

29/10/13 35 15 13

183 100 84

DIRECT CROSS F2 PROGENIES

DATE RED EYED FEMALE

RED EYED MALE

WHITE EYED FEMALE

WHITE EYED MALE

24/10/13 35 34 34 37

25/10/13 40 32 30 45

26/10/13 35 39 31 43

28/10/13 42 27 35 35

29/10/13 39 32 25 30

189 164 155 190

RECIPROCAL CROSS F2 PROGENIES

CHI SQUARE :DIRECT CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 283 275.25 7.8 60.8 0.221

WHITE 84 91.75 7.5 56.25 0.65

367 0.87

F2

CHI SQUARE :RECIPROCAL CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 353 347 4 16 0.04

WHITE 345 349 4 16 0.04

698 0.08

F2

CHI SQUARE :TEST CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 39 37 2 4 0.10

WHITE 35 37 2 4 0.10

74 0.2

Direct cross

CHI SQUARE :TEST CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 45 42.5 2.5 6.25 0.14

WHITE 40 42.5 2.5 6.25 0.14

85 0.28

Reciprocal

ResultChi squarevalue (3.84)

Accept or Reject of data

Direct cross 0.87 accepted

Reciprocal cross 0.08 accepted

Test cross(direct) 0.20 accepted

Test cross (reciprocal) 0.28 accepted

Experimental 1

Interpretation

We got 75% of red eyed

F2 progenies which

confirms that red eye is

dominant over white eye.

We got 50:50 F2

progenies both red eye

and white eye.

NORMALRECIPROCAL

In direct cross, F2 Ratio is appx. 3:1 which is comparable to Mendel’s monohybrid ratio but in reciprocal cross, F2 ratio is appx. 1:1 which is comparable to normal test cross ratio.

Both F1 and F2 progeny did not follow Mendelian ratio, hence the patter of inheritance is Non-Mendelian.

In reciprocal cross, the white eye character passed from female to F1 male. Further it was inherited to F2 female.

Here we can see inheritance from grandmother to granddaughter through son. Therefore criss-cross pattern of inheritance is seen

Criss-cross mode of inheritance is a significant

character of X-linked inheritance.

So, we confirm that the genes controlling the eye colour

present on X chromosome.

Experiment 2

After collecting the virgins we have to keep for aging.

Aging is done to know whether the collected virgins are

contaminated or not.

Crosses are set up. In direct cross, 5 virgin red-eyed

female is crossed with 5 white-eyed males(B).

In reciprocal cross, 5 virgin white-eyed females(B) are

crossed with 5 red-eyed males.

We allowed the cross to develop for 8 days.

Then F1 progenies were isolated and phenotypes were

examined.

The observed results are as follows:

Phenotype Red eye female Red eye male

No. of flies 97 68

F1 Generation

Phenotype Red eye female Red eye male

No. of flies 83 61

DIRECT CROSS

RECIPROCAL CROSS

F1 interpretation

Only red eyed females and males as the F1 progenies

there by we could postulate that red eye trait is dominate

over white eyed.

By comparing direct and reciprocal cross we could see a

similarity i.e. symmetry in phenotype of F1 progenies.

Symmetry in F1 progenies, show that the trait

present on autosome.

There is no sex bias seen between the two sexes

as seen in previous experiment.

To confirm whether the given parental flies are

in homozygous or heterozygous condition we

have to conduct test cross

Test cross was conducted by using virgin F1

female with recessive male parent.

Dates Red Scarlet Brown White

24/10/13 7 4 6 6

25/10/13 6 5 3 3

26/10/13 3 6 5 5

28/10/13 5 2 2 3

29/10/13 2 7 4 4

23 24 20 21

TEST CROSS (direct)

Total Number of Progeny = 86

Dates Red Scarlet Brown White

24/10/13 5 4 6 6

25/10/13 6 5 3 3

26/10/13 3 6 5 6

28/10/13 5 2 6 3

29/10/13 2 3 4 4

25 20 24 22

TEST CROSS (reciprocal)

Total Number of Progeny = 91

• The test cross ratio of both normal and reciprocal

cross was coinciding with normal mendelian test

cross ratio i.e. 1:1:1:1.

• The test cross progeny of both direct and reciprocal

cross showed 4 types of phenotype i.e. normal red

eyed, brown eyed, scarlet eyed and white eyed.

• We could find two recessive character expressed

independently.

• May F1 was in heterozygous hybrid condition

therefore could produce double recessive progeny,

resulting in scarlet and brown eyed mutant.

• To analyze the number of gene controlling the trait F1

selfing was carried out using 5 F1 females and 5 F1

males.

• In the F2 progenies are same for both direct and

reciprocal cross.

• In F2, we obtained 4 types of progenies with different

phenotype like red eyed, brown eyed, scarlet eyed

and white eyed.

• In the F2 progenies, the ratio obtained was appx.

9:3:3:1 which is normal Mendel’s ratio.

NORMAL CROSS OK × bw/st

Date Red Brown Scarlet white

24/10 14 15 5 3 7 5 0 0

25/10 15 12 6 7 6 5 2 0

26/10 13 11 5 4 5 6 1 3

28/10 17 13 3 6 6 4 2 2

29/10 21 11 6 4 5 3 1 2

142 49 52 13

RECIPROCAL CROSS OK × bw/st

Date Red Brown Scarlet white

24/10 12 12 3 0 0 5 0 0

25/10 17 16 2 5 6 4 2 3

26/10 11 14 6 5 6 5 1 2

28/10 12 14 5 4 4 6 1 4

29/10 15 16 3 3 4 5 2 0

139 50 45 15

CHI SQUARE :DIRECT CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 142 255×9 =143.4316

122-123.75= 1.75

3.062 3.062 =0.024123.75

SCARLET 49 255x3 =47.8116

49-47.81=1.19 1.416 1.416 =0.02947.81

BROWN 52 255×3 =47.8116

43-41.25 = 1.75 3.062 3.062 =0.07441.25

WHITE 14 255x1 =15.9316

13-15.93=-1.93 3.724 3.724 =0.23315.93

0.36

F2

CHI SQUARE : RECIPROCAL CROSS

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 139 255×9 =143.43716

139-143.437=4.43

19.68 0.13

SCARLET 50 255x3 =47.812516

50-47.812= 2.19

4.79 0.10

BROWN 45 255×3 =47.81216

45-47.812= 2.81

7.89 0.16

WHITE 15 255x1 =15.93716

15-15.937=0.937

0.877 0.054

0.44

F2

CHI SQUARE :TEST CROSS (DIRECT)

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 23 86×1 =21.54

1.5 2.25 0.10

SCARLET 24 86x1 =21.54

2.5 6.25 0.28

BROWN 20 86×1 =21.54

1.5 2.25 0.10

WHITE 21 86x1 =21.54

0.5 0.25 0.01

0.49

CHI SQUARE :TEST CROSS (RECIPROCAL)

PHENOTYPE OBSERVED(O)

EXPECTED(E)

d = O-E d2 d2/E

RED 24 91×1 =22.754

1.25 1.56 0.06

SCARLET 22 91x1 =22.754

0.75 0.56 0.024

BROWN 23 91×1 =22.754

0.25 0.062 0.10

WHITE 22 91x1 =22.754

0.75 0.56 0.024

91 0.15

ResultChi squarevalue (7.82)

Accept or Reject of data

Direct cross 0.36 accepted

Reciprocal cross 0.44 accepted

Test cross(direct) 0.49 accepted

Test cross (reciprocal) 0.15 accepted

Experimental 2

Reference

• http://www.ou.edu/journals/dis/DIS60/Ramachandra%20171.pdf

• www.googleimages.com

• www.wikipedia.com

• www.scienceencyclopedia.com

• http://www.nature.com/scitable/topicpage/thomas-hunt-morgan-and-sex-linkage-452