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Name : Espere, Jenalyn,
Gabriel, Allean, Score : ____ /20
Guma, John Norbert,
Montederamos, Josh,
Paras, Chariel Jetta,
Pilo, Benjie Mar

Program and year : BSA-2A

Date and Time of : Thursday, 8-11 am
Laboratory

AGRIBIO 2101-PRINCIPLES OF GENETICS

Exercise No. 9
LINKAGE, CROSSING OVER AND RECOMBINATION

INTRODUCTION

Linkage, crossing over, and recombination are fundamental concepts in genetics that
explain how genes are passed down and how genetic diversity is created. Linkage is the
phenomenon in which genes located close to each other on the same chromosome are
inherited together. Thomas Hunt Morgan discovered this concept in his experiments with
fruit flies (Drosophila melanogaster), which showed that certain traits did not assort
independently as predicted by Mendel’s laws. Crossing over occurs during meiosis, the
process by which cells divide to produce gametes. During this process, homologous
chromosomes exchange segments of genetic material, resulting in the formation of
recombinant chromosomes containing a combination of parental genes. The recombination of
genetic material is a major source of genetic variation in sexually reproducing organisms.

Thomas Hunt Morgan’s research on D. melanogaster revealed the first evidence of

genetic linkage and recombination. His experiments demonstrated that linked genes do not
assort independently, and that cross-over can result in new traits. Based on Morgan’s
findings, Alfred H. Sturtevant created chromosome maps that showed the relative positions of
genes on chromosomes according to recombination frequencies. This work established the
basis for modern genetic mapping techniques. Genetic mapping studies have used
recombination frequencies to determine the distances between genes on chromosomes.
These studies found that the physical distance between two genes increases the likelihood of
recombination. Research on linkage, crossing over, and recombination has also looked into
their roles in evolution and adaptation. By creating genetic diversity, these processes
contribute to the ability of populations to adapt to changing environments.
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OBJECTIVES

At the end of this activity, you will be able to:

1. Explain the significance of the mechanism of linkage, crossing over and

recombination.
2. Determine the linkage value, distance of genes through calculations and linkage
mapping and the coefficient of coincidence for DCOs.
3. Analyze the phenotypic ratio frequencies by testing with Chi-square hypothesis.

METHODOLOGY

Researchers was instructed by the instructor to make a laboratory report about

Linkage, Crossing Over, and Recombination, focusing on the analysis of two separate genetic
traits using the corn kernel model. In the first part of the study, the linkage involving two
genes (kernel shape: Gene R and kernel color: Gene S) was assessed through a testcross
procedure, examining the F2 progenies’ phenotypic ratios for compliance with the expected
1:1:1:1 segregation pattern, followed by applying the chi-square test for statistical
significance. Data on various kernel phenotypes were collected, and linkage values were
computed using the formula for distance between the two genes, resulting in linkage maps to
visualize gene association. In the second part, the methodology expanded to analyze three
genes in a trihybrid corn plant, leading to the assessment of a testcross to determine
segregation patterns, calculation of gene order based on progeny frequencies, and
determination of recombination frequencies to compute linkage values and coefficient of
coincidence, ultimately constructing a genetic linkage map. These steps were designed to
elucidate the principles of Mendelian genetics and provide insights into the inheritance
patterns observed in agricultural traits.

RESULTS & DISCUSSION

(Attached Worksheets)

According to Mendel, the postulate on independent assortment states that genes

segregate and independently assort to random pairing of alleles which would come to know
to be a result of crossing over: observed in the first meiotic division. In the event of crossing
over, alleles between two homologue chromosomes swap which takes course during
pachytene in prophase I, after synapsis (zygotene). By this, crossing over is the event at
which when chromosomes physically synapse, the alleles are shared, broken down and
recombined with the other allele—which other sources call recombination instead of
crossing over and the outcomes are identified to be the recombinant alleles (explained as
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Single Crossing over events). However, according to Klug and Cummings (1983), it has been
revealed by Thomas Morgan and Alfred Sturtevart that certain events set apart from
independent assortment and its impact to heredity and phenotypic expressions.

During recombination, certain chromosomes are observed to have not undergone

crossing over and the alleles are identified to have commonalities with the alleles of the
parental generation. This was discovered by Thomas Morgan during his experimentations
with Drosophila melanogaster as well as his contribution to the discovery of sex linkage
wherein traits are directly linked with sex chromosomes—one trait was observed for his
discovery. However, it was only when he analyzed crosses of two sex linked genes that he
hypothesized and insinuated the separation of genes and its frequency upon distribution (pp.
99). This provided the foundation for the case of linkage, crossing over and chromosome
mapping (pp. 98). The event of linkage is a complex circumstance wherein a set of genes are
distanced at close proximity that during recombination or crossing over, they are transmitted
as a single unit with very little chances of being separated unless the gap between genes are
so distinct that it is recombined with another gene. Another scenario occurs when the linked
genes are on opposite chromosomes, the frequency of recombinant alleles is thereby great.
Thus, linkage helps explain the cases for hereditary traits of offspring that are identical to the
parental generation and offspring that are uniquely different from their parents. Further,
linkage also provides a clearer viewpoint on the observed traits of children that have similar
traits from their grandparents which are different from their own parents.

To move along, as mentioned, linkage occurs either completely or incompletely.

Complete linkage occurs when there are no recombination events of genes, due to linkage of
genes on the same homologous chromosome (cis-form). In the case of incomplete linkage,
two linked genes on either homologues (trans-form) are recombined with one another which
results in new sets of genes that are uniquely different from the parental generation and are
inherited by the offspring.

Linkage is assured depending on the distance between genes on the map. Linkage
mapping is the basic analysis of the possibility of recombination to occur. The extent between
genes on the map is determined by calculating the linkage value of the genes which are based
upon the phenotypic recombinant frequencies attributed with the genes. The calculation for
linkage value is by summation of the population of crossover types divided by the total
number of the progeny and multiplied by 100. The resultant is the distance between genes
which is expressed in units of centimorgan, for recognition of T. Morgan’s discovery of linkage
and recombination by his student, A. Sturtevart on his works of the discovery of linkage
mapping (pp. 99). The genes are arranged according to their respective lengths from one
another, depending on the type of traits being analyzed. This mapping provides a better
understanding on the possibility of occurrence of recombinant and parental alleles expressed
by the phenotypes of the offspring, which on a broader perspective, explains the range of
genetic variations of the eukaryotic population. The greater the distance between alleles, the
higher the chances of producing offspring with recombinant alleles than those with parental
alleles, otherwise, the appearance of parental alleles in offspring are greater than those
recombinant alleles.
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However, additional studies reveal of multiple crossovers (incomplete linkage),
wherein multiple gene exchanges occur. In cases of three linked genes, double crossing over
(DCO) takes place—two simultaneous independent exchanges occurs. Yet, the chances of
DCOs are less likely to occur than SCO (pp.102). Though distinction of DCO is more difficult
and is less likely to occur, a common calculation if the occurrence were to take place is the
multiplication of the crossover types of two regions (loci) involved which results with
expected DCO whilst when there is an actual DCO occurrence, the formula goes, summation of
the total number of crossover types of both regions divided by the total number of the
population. Moreover, there are instances where certain crossover interfere on the
occurrence of another which is identified to be the strength of interference which is
determined by the coefficient of coincidence (CC) which is by dividing the actual DCO to the
expected DCO, only then the intensity of the interference is measured wherein an CC of 1.0,
indicates no interference whereas a CC of 0, complete interference happens.

In addition, Chi-square analysis can also be applied for linkage and recombination. By
testing hypotheses, either the phenotypic ratios observed in a given situation does exhibit
linkage, both for complete and incomplete linkage outcomes, or opposes the hypothesis and
no the genes are not linked. The results from worksheets demonstrate linkage between genes
of the phenotypic variations in specific instances as there are data evidences to prove the
variance between the frequencies of parental and recombinant alleles found in the offspring.

Answer to Guide Questions

Two Linked Genes

a.)
Ho: The observed phenotypic ratio fits with the expected phenotypic ratio of the
dihybrid cross and exhibits no linkage
Ha: The observed phenotypic ratio doesn’t fit with the expected phenotypic ratio of
the dihybrid cross and exhibits linkage
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b.) Test your hypothesis:
Condition: 1. If X² com < X² tab, accept Ho and reject Ha.
2. If X² com > X² tab, reject Ho and accept Ha.

Observed Expected
Deviation
Phenotypic Class Frequency Frequency D² X²=D²/E
(D=O-E)
(O) (E)

2674042.562
1. Red, plump 118 1753.25 -1635.25 1525.19
5

2. Red, 2821560.062
3433 1753.25 1679.75 1609.33
shrunken 5

2482988.062
3. White plump 3329 1753.25 1575.75 1416.22
5

4. White, 2625210.062
133 1753.25 -1620.25 1497.34
shrunken 5

n= 7013 X²com=6048.08

Expected Frequency: (1:1:1:1)

E1: 1(7013)/4= 1753.25
E2: 1(7013)/4= 1753.25
E3: 1(7013)/4= 1753.25
E4: 1(7013)/4= 1753.25

X²tab: df=n-1
=4-1
=3—7.815
X²com vs. X² tab
= 6,023.27 > 7.815

c.) State your conclusion:

- Condition 2, X²com > X² tab, therefore, reject Ho and accept Ha.

d.) Compute for the linkage value. Show your solution.

Total no. of crossover types

Linkage Value= ×100
Total no. of population
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118+133
Linkage Value= ×100 = 3.58 %
7013

e.) Make a linkage map

R S

3.58 cM

Three Linked Genes

a.) Show your hypothesis:

Ho: The observed phenotypic ratio fits with the expected phenotypic ratio of the
dihybrid cross and exhibits no linkage

Ha: The observed phenotypic ratio doesn’t fit with the expected phenotypic ratio of
the dihybrid cross and exhibits linkage

b.) Test your hypothesis:

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Condition: 1. If X² com < X² tab, accept Ho and reject Ha.
2. If X² com > X² tab, reject Ho and accept Ha.

Observed Expected
Deviation
Phenotypic Class Frequency Frequency D² X²=D²/E
(D=O-E)
(O) (E)

1. Plump,
colored
2728 2818.13 -90 8123.42 2.88
endosperm
, Non-waxy

2. Shrunken,
Non-
2518 939.38 1578.62 2492041.11 2653.93
colored
waxy

3. Shrunken,
Colored
118 939.38 -821.38 674665.11 718.20
endosperm
, Non-waxy

4. Plum, Non-
colored
114 939.38 -825.38 681252.14 725.21
endosperm
, Non-waxy

5. Plum,
colored
591 313.13 277.87 77211.74 246.58
endosperm
, waxy

6. Shrunken,
Non-
colored
604 313.13 290.87 84605.36 270.19
endosperm
, and Non-
waxy

7. Shrunken
colored
4 313.13 -309.13 95561.36 305.18
endosperm
, Non-waxy
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8. Plump,
Non-
3 104.38 -101.38 10277.91 98.47
colored
indivials

n= 6680 X²com=5,020.64

Expected Frequency: (27:9:9:9:3:3:3:1)

E1: 27(6680)/64= 2818.13
E2: 9(6680)/64= 939.38
E3: 9(6680)/64= 939.38
E4: 9(6680)/64= 939.38
E5: 3(6680)/64=313.13
E6: 3(6680)/64=313.13
E7: 3(6680)/64=313.13
E8: 1(6680)/64=104.38

X²tab: df=n-1
=8-1
=7—14.067
X²com vs. X² tab
= 5,020.64 > 14.067
c.) State your conclusion:
= Condition 2, X² com > X² tab, therefore, reject Ho and accept Ha.
d.) What is the correct gene sequence?
a.) Determine the genotype of each progeny.
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sh C Wx

Sh c wx
Sh C wx

sh c Wx
sh C wx

Sh c Wx

Sh C Wx
sh c cx

b.) Compute for the linkage value in Region I. Show your solution.

Crossover types (1st & 2nd gene)

Linkage Value= ×100
Total no. of population

591+604
Linkage Value= ×100=17.88 %
6680
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c.) Compute for the linkage value in Region II. Show your solution.

Crossover types (2nd & 3rd gene)

Linkage Value= ×100
Total no. of population

118+114
Linkage Value= ×100=34.7 %
6680

a.) Compute for the linkage value in DCO. Show your solution.

DCO
Linkage Value= ×100
Total no. of population

4+3
×100=0.105 %
Linkage Value= 6680

b.) Compute for the coefficient of coincidence. Show your solution.

Actual DCO
×100=0.105 %
CC= Expected DCO

(4+3)+(6680)
CC= =0.0000017
17.88×34.7
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c.) What is the implication of that CC value?
= It determines strength of interference of crossing over types.
d.) Compute for the interference. Show your solution.
Interference= 1-CC
= 1-0.0000017=0.9999986 or 1, thus there is no interference.
e.) What is the implication of the I value?
= It indicates if there are interference occurring or not.
f.) Make a linkage map of the three genes.

Wx Sh C

18 cM 35.78 cM

53.78 cM

CONCLUSION

In the final analysis of the activity, the mechanisms of linkage, crossing over, and
recombination are fundamental in understanding phenotypic expression and genetic
inheritance. Mendel’s principle of independent assortment, while foundational, is now
understood to be influenced by crossing over during prophase I of meiosis. This process
involves the exchange of alleles between homologous chromosomes, creating recombinant
alleles and contributing to genetic diversity.

Thomas Morgan’s experiments with Drosophila melanogaster revealed deviations

from independent assortment, establishing the concepts of linkage and genetic mapping.
Linkage occurs when genes are located close together on the same chromosome, reducing the
likelihood of recombination. Conversely, genes farther apart exhibit higher recombination
frequencies. Linkage mapping measures these distances in centimorgans, enabling
predictions about the inheritance of traits and providing insight into genetic variation.
Complete linkage results in no recombination, while incomplete linkage leads to
recombination and new allele combinations.

Double crossing over (DCO), though less frequent than single crossing over (SCO),
involves simultaneous recombination events and is analyzed using expected and observed
crossover frequencies. The coefficient of coincidence (CC) quantifies interference in crossover
events, with values ranging from 0 (complete interference) to 1 (no interference).
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Chi-square analysis further refines our understanding by testing hypotheses on
phenotypic ratios, distinguishing between independent assortment and linkage. Variations in
parental and recombinant allele frequencies validate the presence of genetic linkage.

These mechanisms and analytical tools collectively illuminate how traits are inherited,
the extent of genetic diversity, and the intricate interactions of genes during meiosis. This
comprehensive framework enhances our understanding of genetics and its applications in
fields such as medicine, agriculture, etc.

REFERENCES

Morgan, T. H. (1910). Sex Limited Inheritance in Drosophila. Science, 32(812), 120-122.

Retrieved from https://www.jstor.org/stable/1634791

Sturtevant, A. H. (1913). The linear arrangement of six sex-linked factors in Drosophila,

as shown by their mode of association. Journal of Experimental Zoology, 14(1),
43-59. Retrieved from
https://onlinelibrary.wiley.com/doi/abs/10.1002/jez.1400140104

Muller, H. J. (1916). The Mechanism of Crossing-Over. The American Naturalist, 50(592),

193-221. Retrieved from https://www.jstor.org/stable/2456134

Klug, W. and Cummings, M. (1983). Linkage, Crossing Over, and Chromosome Mapping.
Concepts of Genetics. (pp. 94-101, 109, 117). Charles E. Merill Publishing
Company.

Cabillo, C. (2024). Bio-22p-Lec2-MIDTERM. (pp. 194-195).