-- -

EXPERIMENT 13 VERIFICATION OF
MONO1317BRID
MENDELIAN RATIO
AND CHI-SQUARE
ANALY §IS

13.1 Illtroduction
Objcc~ivcs
13.2 Malclials Required
- 13.3 Procedure
13 4 Gbsclvat~onsand Rcsults
13.5 Discussion of Rcsulls
13.6 x2 A11aJys1's

13.1 INTRODUCTION

In Unit I of LSE-03, you havc studicd Mcndcl's laws of Inheritance, i.c., Lhc
law, of scgrcgalion, and lhe law of indcpcndenl assorlmcnl. This and thc
following 1;lbornlorp cxcrcise pcrlain to Lhcsc two l ~ w srcspcclivcly. Beforc
beginning Lllis exercise, ]el us, rec;lpilulatc bricfly the law 01scgrcgalion. You.
may rccall hat Mcndcl uscd varictics or lincs of garden pca (Pisum sativum),
Lhat exhibited pairs of contrasting characters, c.g., Lhc long slcm lines (or the tall
planls) and h e short stem lincs (01.~ h short
c plants) as parcnts (P gcncralion)
in his crosses. Each line was purc brecding, so Lhal plants from that line always
bred tnle Ior Ulc cliaracter bcirlg studicd, i.c., tall x tall produced only LaU,
progeny. Whcn two lines with con~raslingcharnclcrs wcre crossed (c.g., La11 x
shorl) Lo produce a firs1 filial gcncralion, all Lhe progcny werc 01onc phcnolypc,
which was thc samc as onc of Lhe parcnts (c.g., tall). This charactcr was said Lo
be doi?zinarzt,and thc cliaracter that did not appcar in Lhc F, (e.g., shorl) was
_ callcd recessive. Thcre had becn no blcnding 04 thc two cliaraclcrs. Whc11 Lhc F,
HYIS nlloci1lcd LO scli-, Lhe progeny in thc ncxt gcncralion (F,) wcrc in the ratio of
approxi~~lalely 3 donlir~nlliLO I rcccssivc plicnotypcs (e.g., 3 tall : 1 sllorL). What
:wcre his conclusiot~sIrom Lhc abovc cxpcrimcnts? (i) Each parent conlaincd Lwo
unit factors (gencs) of wllich onc was conl~ibutcdLO cach mcmbcr 01thc F,
progeny. (ii) Each gcnc could exisl in Lwo allcmatc forms or ullcle,~,one 01
wllich was Ibr thc dominant characlcr (i.c., T) and dclcllnincd thc phcnotgpe of
Lhc F, with thc oUxr bcing for ~ l i crecessive characlcr (i.c., t). (iii) Each mcnlbcr
of the F, co~~laincd onc 01each rrllclc (TL) and was hclcrozygous, wl~crcascnch
parcnt conlaincd Lwo idcnlical allclcs (TT in Ihc u11 parcnl; tt i n Ule shorl
parent) and was homozygous. (iv) Thus, thc dominant phcnotypc (tall) rcsulled
from LWO diIfcrent gcnotypcs, the liomozygous onc (TT) or ~ h chcrcrozygous one
(TL),whcreas Lhc rcccssivc phcnolypc was only dctcl~nincdby onc l~omozygnus
gcnotype(tt). (v) When Ihc F, plants produccd pollcn and cggs, Lhcy wcre
clearly of Lwo typcs, occurring with equal frcqucncy and conlainin$ cilhcr onc
allele for Lhe dominant characler (T) or onc allclc for thc rcccssivc charactcr (L),
i.e., ~ I CLWO alleles in thc I?, scgregatcd clearly from each oll~crwhcn gamclcs
were Iom~ed.(vi) Thc pollen and cgg nuclci or Lhc Lwo gcnolypcs T and L Iuse
 Laboratory Course-I at random to produce thc F, zygotes. Thus, Lhe F, genotypes would be of three
types in the ratio of 1 homozygous for alleles for the dominant character : 2
heterozygous : 1 homoiygous for allelcs for the rccessive character ( i . ~ . 1TT
, :
2Tt : ltt). This gives Lhc I=, phcnotypic ratio of 3 dominant : 1 recessive (3 tall :
1 short). Let us now procecd to do thc cxcrcise.

Objectives

After doing this laboratory exercise you should be able to:

e demonstrale Mcndel's law of scgrcgation in a monohybrid cross;
r correlate the cxpcrimental steps with thc natural processes occurring;
analyst the ratio obtaincd with regard to its goodncss of fit, by using
Chi-Square tcst.

Prior Reading for this Experiment

1. Unil 1, Gcnetics (LSE-03)

2. Unit 16, Malhcmatical Mcll~ods(MTE-03)

13.2 MATERIALS REQUIRED

1) 3 containcrs/250 ml plastic beakers

2) 5 0 red bcads

3) 50 ycllow bcads

13.3 PROCEDURE

You will have to work in pairs for this investigation.

Step 1 : Placc 50 rcd bcads in onc containcr to rcprescnt the gametes of a tall
parcnt (T). Placc SO ycllow bcads in the othcr container to represent
See Pig. 13.1 (a) the gamctes of a dwarf parent (t). Wc assume that bolh thc gametes
are from parcnts that breed m e for the chracteristic, namely, stem -
p. 100
hcigl~t,whose inherilance is bcing studied in this experiment.
Stcp 2 : Withdraw a bead from each container. Each bead withdrawn
rcpresents a gamete containing a single allele of a pair. Placc the
See Fig. 13.1 (by beads togcthcr. This rcpresents the proccss of ferlilisalion, by which
the paired alleles of gene in the offspring arc re-established.

Step 3 : Just as in Stcp 2, continuc to withdraw pairs of beads as above, and

See Fig. 13.1 (c)
nrrangc on the tablc.
m a t would be the gcnotype of the.individuali of thc F,
.
gcneration? ..,...... ....................... . ... ................. .............. . ...

Stcp 4 : T o simulate thc gametes of this F, generation, place SO bcads (25 rcd
See Pig. 13.1 (cl) and 25 ycllow) i n cach conlainer. Onc container rcprcscnts Ihc fcmalc
r .r- .
84
 gametes, and the other represents the mf e gametes produced by the Monohybrld Mendelinn Ratlo
F, generation.

Step 5 : Shake each container vigourously for 30 seconds, taking care the
beads do not f d l off.

Step 6 : To produce the F, generation, withdraw one bead from each container See Fig. 13.1 (e, P)
with your eyes closed and place them together. Your partner should
note the combination of genes obtained.
This represents the genotype of an F2individual.

Step 7 : After noting the genotype of each pair, discard the pair of beads into
a spare container.

.Step 8 : Repeat steps 6 and 7 until all beads have been paired and their
combinations noted.

Step 9 : Calculate the ratio of the phenotypes of the F2 individuals.

Step 10: Record the ratios obtained by other groups in your batch and calculate
the average ratio.

13.4 OBSERVATIONS AND RESULTS

A. F, Generation

a) Total number of F, individuals ........................

.....................
b) Phenotypes of F, individuals ................................................
c) Genotypes of F, individuals .................................................
B. F2 Generation

- a) Total number of F, individuals .................,... ........................

b) Phenotypes of F2individuals ................................................
c) Number of individuals,in each
phenotypic class ........................,.................
d) The phenotypic ratio .................................................
e) Genotypes of F, individuals ................................................
f) Number of individuals in each
genotypic class .

g) The genotypic ratio

 Group Average Phenotypic classes

Group 1
Group 2
Group 3
........
.........
Group n
Total
Average .............. .............

13.5 DISCUSSION OF RESULTS

1) In thc iibovc exercise which character was dominant?

2) Was the F, homozygous or hcterozygous?

3) Whcn F, plants wcrc sclf-pollinatcd how many different types of pollen and
egg nuclei werc produced? What were lheir genotypes and what were the
frequencies of thc genotypes? I

4) Why wcre the bcads shaken in step 5, and wilhdrawn with closcd eycs in
slcp ti?

................................................................................................
5) How does your ratio, and the group average ralio compare with Mendcl's
prcdiction? Explain any differences?
 Monohybrid Mendelian Rntlo

...............................................................................................
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6) Make a Punnctt square to show thc outcome 01F, x F, cross.

7) LVlly is it callcd a monohybrid cross?

................................................................................................

8) WhaL would be thc monohybrid tcst cross of thc cxarnple takc:n in this
expcrimenl?
9,

9) What would bc ~ h cback cross for ~ h ccxaniplc taken in this cxpcrimcnl?

Laboratory course-I 10) Can you record and present the results in another manner?

11) Explain how this practical acts as a model for breeding and inheritance in
pea?

12) In the diagram given below, write as to which step (described in

experimental procedure above) cornsponds to the indicated stage.

AA X (i) .......................# ?............,..... ..

I
a

Qametogenesls

(HI ...................... ?.............................

Fertilisatlon

(Self-

Gametogeneals

~ e n b t ~ratio
~lc 1 AA 2 Aa ' 1 ea
L-4-J
Phenotypic ratlo 3- . 1 '
 13) Supposing a random sample of F, seeds obtained were sown and their Monohybrid Mendelian Ratio
mature plants were allowed to self. What proportion of plants would
produce (i) only tall plants (homozygous), (ii) only dwarf plants
(homozygous) and (iii) a mixture of tall and dwarf (heterozygous)
plants?
. . . . . . . _ _ . . . , . . . . . . . , . . . _ . . . . . . . . . . . . . . . . . . . . . ..... . . . . - - - .

13.6 x2 ANALYSIS

By using the X' (Greek letter. chi pronounced as 'Lye squared') test, you can
evaluate your results of this experiment. This is a statistical test that is
"
frequently used to determine whether the data obtained experimentally provides
, a ' g o d fit', or appmximation to the expected or theoretical data. Basically this
. test can be used to determine whether any deviations from the expected values
are due to chance. Chance alone can cause the actual observed ratio to vary
from the calculated ratio for a genetic cross. For example, for a monohybrid
cross you would get very rarely an exact ratio of 3 : 1. The observed results
differ, but there comes a point when the difference is so great that the observed
data is not in conformity with the expected one. The chi-square test indicates
this point. In this exercise, for example, the hypothesis is that the F, data do not
differ significantly from a 3 : 1 ratio. This type of formulation is called null
. hypothesis, because we will test that nothing has happened to disturb the
expected ratio significantly. Consider an alternative hypothesis, such as the F,
data does differ significantly fmm a 3 : 1 ratio because gametes with different
genotypes are produced in unequal numbers. Such a hypothesis would be
imprecise and would not lead to an exact numerical prediction. From this
example it should be clear that the null hypothesis being tested should always
be stated for the test.

1 The formula for this test is:

xZ = chi-square

C = sum of

d = difference between the expected and observed results, often termed as

deviation (o - e)

e = expected results

o = observed results

Let us see how we can apply this formula. As you know, in a monohybrid cross
a 3 : 1 ratio is expcclcd. Supposing you count a total of 160 plants, out of
which 120 are tall and 40 arc dwarf. But anothcr student counts 116 tall plants
and 44 dwarls. Thcn thc valuc Tor thc chi-squarc tcst would be calculated as
.(own in Tablc 13.1.
 Laboratory Course-I Table 13.1: Calculation of tlie Chi-square Value

Observed Expected difference Partial Chi-square

Phenotype number (0) number (e) (d = o - e) d
2
d2/e -
Tall 116 120 4 16 16/120 = .I33

Dwarf 44 40 4 16 16/40 = .400

Ed2
-C
= .533
x2 = .533

The next step is to look up lhis Chi-square value (x2)

in the Table 13.2, bat
indicates whcthcr thc probability (P) is that thc diflerences noted arc duc only to
chance in ~ h cform of random sampling error or whether it would be bettcr to
explain the results on the basis of a different prediction or hypothesis.

Table 13.2: Critical VaIues of Chi-square for 1-30 degrees of freedom that are eql~alledwith or exceeded
with particular probabilities (P). Figures at the top of the table indicate levels of significance.

In Tablc 13.2, thc notation df rcfcrs to the degree of freedom, which in tlus
cxpcriment would be dctcnnincd by the number of phcnotypic trails studied. In
our example, we havc two classcs, tall and dwarf plants. As indicated in the
tablc for Lhe value of df, i.e., we nced to know the valuc of C-I. You might
.have followed that the dcgrec 01fscedom (df) is calculated by using the formula
C-I, whcre C is the Lord numbcr of classcs. In L1us casc C is 2 Illcrcforc, dr is
equal to 1 (i.e., 2 - 1 = 1). Therefore, you should look for xZvalue in the firs1
row (i.e., in I), of Lhc Tablc 13.2. The valuc .533lics between 5 0 and .30
probabilily valucs. This mcarls that by random chance, this difrcrcnce betwecn
90

--
P
the actual count and the expected count would occur between 30 and 50% of Monohybrld Mendelian Ratlo
the time. In biology, it is generally accepted that a P value greater than 0.05 is
acceptable, while a P value lower than 0.05 would indicate that the results
cannot be due to random sampling and, therefore, do not fit the original
prediction (hypothesis).

Do a Chi-square analysis of your results by filling in Table 13.3.

Table 13.3 Chi-Square Analysis of Results

Observed Expected difference Partial Chi-square

Phenotype number (0) number (e) (d = o - e) d' dale

(dl'
I-=e

P (from Table 13.2) = ....................

Do your results support Mendel's prediction? ...............................................
If no, can you account for this? ....................................................,.... ........
B) Do Chi-square analysis for your entire batch?

Observed Numbers
Phenotype
Group 1 Group 2 Group 3 - --- -- Group n Total

Tall

Dwarf

Total no. of
Individuals ....................
Chi-square analysis

Observed Expected difference. Partiat Chi-square

Phenotype number ( 0 ) number (e) (d = o - e) d' dZ/e

Tall

Dwarf
-. - ,

I --- --
Laboratory Course-I xZ= .....................;
C-1 = a.........

P (from Table 13.2) = .....................

Do lhcsc rcsults support Mcndcl's prcdiclion? ................................................

......................................................................................................
on thc abovc rcsul ts?
Co~~lrnent ...................................................................

......................................................................................................
Wl~nlare thc advanlagcs or taking a bigger sample? .....................................