UNIT 4 – Linkage, Crossing Over and Mapping in Eukaryotes (Q&A) | MZO-002 MSCZOO | IGNOU

SAQ 1

a) What is gene mapping? How do the linked genes help in gene mapping?

Gene mapping is the method used to determine the location of genes on a chromosome and the distance between them. It helps in identifying the exact position of a gene responsible for a particular trait or disease. The concept started with the work of Thomas Hunt Morgan in the early 1900s when he studied Drosophila melanogaster (fruit fly) and observed that some traits are inherited together. This was because the genes responsible for those traits were located close to each other on the same chromosome. This phenomenon is known as linkage.

There are two main types of gene mapping:

1. Genetic Mapping (Linkage Mapping):

• Genetic mapping uses the frequency of recombination or crossing over between genes to estimate their distance on a chromosome. It gives a relative position of genes rather than their exact physical location.

2. Physical Mapping

• Physical mapping uses molecular biology techniques to determine the exact nucleotide sequence of DNA and the exact physical distance between genes. It gives accurate location of genes in base pairs.

How Linked Genes Help in Gene Mapping?

Linked genes are genes located close to each other on the same chromosome. Because of their close position, they tend to be inherited together and do not follow Mendel's law of independent assortment. This special relationship helps in gene mapping in several ways:

1. Recombination Frequency Shows Distance

During meiosis, crossing over can occur between homologous chromosomes. If two linked genes are close, crossing over between them is rare, resulting in low recombination. If they are far apart, crossing over happens more often. By measuring how often recombination occurs, scientists estimate the distance between genes.

2. Creating Genetic Maps

Recombination frequency is converted into map units or centiMorgans (cM). One percent recombination equals one centiMorgan. Using these distances, genes can be arranged in order on chromosomes to create genetic linkage maps.

3. Determining Gene Order

By comparing recombination frequencies among three or more linked genes, scientists can determine their linear order on the chromosome.

4. Grouping Genes

Genes on the same chromosome form linkage groups. This helps in assigning genes to specific chromosomes.

5. Tracking Disease Genes

In human genetics, linked markers help locate genes related to diseases by studying inheritance patterns in families.

b) Differentiate between linked genes and unlinked genes?

Genes are specific sequences of DNA that code for proteins and determine traits in an organism. During the study of chromosomal theory of  inheritance, scientists found that not all genes behave the same way. Some genes tend to be inherited together while others assort independently. Based on this behavior, genes are divided into two types: linked genes and unlinked genes. This concept is very important in genetics because it helps in understanding how traits are passed on and how gene positions can be mapped on chromosomes. These differences are explained based on specific criteria:

1. Based on Chromosomal Location

Linked genes are located close to each other on the same chromosome. Because of their close physical proximity, they usually move together during meiosis and are inherited as a group. For example: In Drosophila melanogaster (fruit fly), the genes for eye color and wing shape are located close to each other on the X chromosome.

Unlinked genes are either located on different chromosomes or are far apart on the same chromosome. Their distance is so great that crossing over happens frequently, making them behave as if they are on separate chromosomes.

2. Based on Inheritance Pattern

Linked genes do not follow Mendel's law of independent assortment. They are inherited together more often than not, unless crossing over occurs between them.

Unlinked genes follow Mendel's law of independent assortment and are passed to offspring independently of each other.

3. Based on Recombination Frequency

Linked genes show recombination frequency less than 50%. The closer the genes are on a chromosome, the lower the chance of crossing over between them. This helps in estimating the distance between them.

Unlinked genes show 50% recombination frequency. Crossing over between them occurs so often that it appears as if the genes are assorting randomly.

4. Based on Use in Gene Mapping

Linked genes are very important in gene mapping. The recombination frequency between them helps scientists to construct linkage maps. The lesser the frequency, the closer the genes are on the chromosome.

Unlinked genes do not help in determining distance between each other as their independent assortment gives no specific information about gene location.

5. Based on Historical Observations

Linked genes were first discovered by Bateson and Punnett in 1905 while studying flower color and pollen shape in Lathyrus odoratus (sweet pea). They observed that some traits did not follow expected Mendelian ratios and concluded that certain genes were linked.

Unlinked genes were explained by Gregor Mendel during his experiments with pea plants. He worked with genes located on different chromosomes and observed independent assortment of traits like seed color and seed shape.

SAQ 2

a) How is three factor cross different from two factor cross?

In classical genetics, different types of crosses are used to study inheritance patterns and gene linkage. Two of the most commonly used crosses are the two-factor cross and the three-factor cross. To understand how they differ from each other, we need to compare them based on certain defined criteria as mentioned below:

1. Based on Number of Genes Studied

Two-Factor Cross: In this cross, inheritance of only two genes is studied at a time. These genes may or may not be located on the same chromosome.

Three-Factor Cross: In this method, inheritance of three genes is studied together. These three genes are usually located on the same chromosome and are studied to find their relative positions.

2. Based on Purpose of the Cross

Two-Factor Cross: The main purpose is to identify whether the two genes are linked or independently assorted. It also helps in calculating the recombination frequency between the two genes.

Three-Factor Cross: It is mainly used for gene mapping, to find the linear order of the three genes and to determine how far apart they are from each other using crossover data.

3. Based on Gametes and Phenotypic Classes

Two-Factor Cross: This cross generally produces 4 types of gametes depending on the arrangement of alleles. As a result, 4 phenotypic classes are commonly observed in the offspring.

Three-Factor Cross: This type of cross produces 8 types of gametes, because three genes can be assorted in more combinations. So, the progeny usually shows 8 different phenotypic classes.

4. Based on Detection of Double Crossovers

Two-Factor Cross: It is not able to detect double crossover events between genes. So, it may give an incomplete picture of the recombination events.

Three-Factor Cross: This method can clearly identify both single and double crossover events. This is helpful in getting more precise genetic distances.

5. Based on Gene Order Determination

Two-Factor Cross: Since only two genes are involved, we can only study their linkage or recombination frequency, but not their linear order.

Three-Factor Cross: This is specially designed to find the exact sequence or order of the three genes on a chromosome, which is essential in genetic mapping.

6. Based on Accuracy in Mapping

Two-Factor Cross: Gene mapping is possible, but it is limited in accuracy. It gives rough estimation of distance between two genes.

Three-Factor Cross: Provides highly accurate gene mapping by using recombination frequencies from both single and double crossovers, giving a clear picture of gene positions.

b) If the organism with the genotype Ab/aB produces 10% each of the crossover gametes, AB and ab in a test cross, what is the distance between A and B gene loci?

Given:

Genotype of organism: Ab/aB

This is a repulsion (trans) heterozygote, meaning A is with b on one chromosome and a is with B on the other chromosome.

The organism is test crossed (i.e., crossed with ab/ab).

The crossover gametes are:

• AB = 10%
• ab = 10%

Total crossover frequency:

Crossover gametes are produced only due to recombination. In this case:

• AB and ab are the recombinant gametes
• Ab and aB are the parental (non-recombinant) gametes

So,

Crossover frequency = AB + ab = 10% + 10% = 20%

Distance between A and B gene loci:

In genetics, 1% recombination = 1 map unit (centiMorgan or cM)

So,

Distance between A and B = 20 cM

Answer: 20 cM

c) The distance between the genes A and B is 15 map unit, B and C 8 map unit and A and C 23 map unit. In an individual of genotype AbC/aBc, what will be the order of gene? What will be the expected percentage of gametes with the genotype ABC?

Given:

A–B = 15 map units

B–C = 8 map units

A–C = 23 map units

Check if A–B–C fits:

A–B + B–C = 15 + 8 = 23 

So, gene order is: A–B–C

Genotype of individual:

AbC / aBc

This is a double heterozygote and the arrangement of alleles shows coupling and repulsion between different loci.

Parental chromosomes:

• AbC (from one parent)
• aBc (from another parent)

Now we determine the expected frequency of ABC type gamete.

To get ABC, recombination must occur in both segments:

• Between A and B
• Between B and C

So, this is a double crossover product.

Double crossover frequency = (distance A–B) × (distance B–C)

= (15/100) × (8/100)

= 0.15 × 0.08 = 0.012 = 1.2%

But since there are two possible double crossover gametes (ABC and abc),

Each will appear in half of the double crossovers

So, expected percentage of ABC gametes = 1.2 / 2 = 0.6%

Final answers:

Gene order: A–B–C

Expected percentage of ABC gametes: 0.6%

d) What is interference? How does it affect the double cross over recombinants?

Interference is a genetic feature that controls how crossovers happen during meiosis. When a crossover takes place between two genes on a chromosome, it affects the chances of another crossover happening nearby. Usually, it reduces the possibility of a second crossover in the nearby region. This means crossovers do not occur completely independently. Because of this effect, we see fewer crossovers near each other than what we expect by simple probability. This is called positive interference.

This happens because the chromosome structure becomes less favorable for another crossover after one has already occurred. It is a natural control system to avoid too many crossovers in a small region.

How does interference affect double crossover recombinants?

Double crossovers happen when two separate crossover events occur between three genes. For example, suppose we have three genes A, B and C. A crossover may happen between A and B, and another between B and C. If we know the recombination frequency between A–B and B–C, we can calculate the expected number of double crossover recombinants by multiplying the two frequencies.

But due to interference, the actual number of double crossover recombinants is usually less than expected.

To measure this, we use two terms:

1. Coefficient of coincidence (c): This is the ratio of the observed number of double crossovers to the expected number.
2. Interference (I): It is calculated as 1 minus the coefficient of coincidence (I = 1 – c)

If interference is 1, it means no double crossovers occurred at all. If it is 0, there is no interference and crossovers happen freely. So interference directly reduces the number of double crossover gametes and changes the accuracy of genetic distance calculations.

e) How is recombinant percentage calculated?

Recombinant percentage (also called recombination frequency) tells us how often recombination happens between two genes during meiosis. It is used to find out the genetic distance between two loci on the same chromosome.

When two genes are close to each other, the chance of recombination between them is less. But when they are far from each other, recombination happens more often. The recombinant percentage shows how much recombination took place in a genetic cross.

This value is calculated by comparing the number of recombinant offspring (those with new trait combinations) to the total number of offspring.

Formula:

If two genes assort independently (either on different chromosomes or very far on the same chromosome), the maximum recombination frequency is 50%.

If two genes are linked (close on the same chromosome), the recombinant percentage will always be less than 50%.

This percentage is also directly used to calculate genetic map distance:

Example:

Suppose a cross between two individuals gives a total of 1000 offspring. Out of these, 80 offspring show new trait combinations. These 80 are recombinant offspring.

Now apply the formula:

Recombinant percentage = (80/1000) × 100

Recombinant percentage = 0.80 × 100 = 8%

This means the recombination frequency is 8%, and the two genes are 8 map units apart on the chromosome.

f) Why is the frequency of double crossover overly low?

The frequency of double crossover is usually much lower than expected. This is mainly due to a natural genetic mechanism called interference, which controls the distribution of crossover events during meiosis. Crossover is essential for genetic recombination but is also tightly regulated to prevent instability in the genome. The lower frequency of double crossovers can be explained by the following reasons:

1. Physical Constraints of Chromosomes

Chromosomes have a limited length and physical structure. When a crossover happens at one region of a chromosome, the local chromatin structure and spatial arrangement become less favorable for another crossover nearby. This physical limitation reduces the chance of two crossovers occurring very close to each other on the same chromosome segment.

2. Crossover Interference

One of the main reasons for reduced double crossovers is the phenomenon called interference. Interference is the effect where the occurrence of one crossover decreases the probability of another crossover happening close to it on the same chromosome. Because of interference, crossovers tend to be spaced apart rather than clustered together. This reduces the overall frequency of double crossovers.

3. Limited Number of Recombination Hotspots

Recombination does not occur randomly but at specific sites called recombination hotspots. These hotspots are limited in number and location along the chromosome. If two hotspots are far apart, a double crossover between two closely linked genes becomes less likely. This limits the frequency of double crossovers.

4. Structural and Functional Constraints

Certain regions of chromosomes, such as centromeres and heterochromatin, are less prone to recombination. These regions restrict where crossovers can occur. As a result, the overall chances of having two crossovers in the same chromosome segment are reduced.

5. Biological Control Mechanisms

The cell has evolved mechanisms to control crossover events to ensure proper chromosome segregation during meiosis. Too many crossovers or closely spaced crossovers might disrupt chromosome stability. Therefore, biological systems regulate crossover frequency and spacing, indirectly limiting double crossover frequency.

g) Illustrate with an example how can pedigrees be used to study the linkage analysis in humans?

In humans, we cannot do experimental crosses as we do in animals or plants. So to study the inheritance of genes, especially disease-causing genes, we use pedigree analysis. A pedigree is a family tree that records the appearance of a trait across generations. It helps in observing how a gene or a disease is passed from parents to children. Pedigrees are essential in linkage analysis because they help track how a genetic marker and a trait are inherited together.

Linkage analysis is the study of how close two genes (or a gene and a marker) are on the same chromosome. If they are physically close, they will show co-segregation, which means they will be inherited together more often than expected by chance. This happens because crossing over is less likely to occur between them. In humans, we track this through pedigrees across generations using molecular markers like SNPs or microsatellites.

Example: Using a pedigree to find linkage of a disease gene

Let us consider a pedigree of a family affected by a rare autosomal dominant disorder. This pedigree includes three generations. Scientists decide to test a known DNA marker (say, a microsatellite on chromosome 4) in all family members.

• The marker has two alleles: M1 and M2

• All affected individuals carry the M1 allele

• All unaffected individuals carry M2 allele

• The same pattern is observed in multiple generations

• This suggests that the disease gene and marker M1 are linked

Now, if any recombination occurs, we may see an affected person without M1 or an unaffected person with M1. Let's say:

• Total informative meioses = 20

• Recombinant cases = 2

• Recombination frequency = (2/20) × 100 = 10%

• So, map distance between marker and disease gene = 10 cM

This analysis tells us the disease gene lies close to the marker. Such studies are used in mapping human disease genes like the Huntington's disease gene, which was mapped using pedigree-based linkage analysis.

Thus, pedigrees help study inheritance in real families and identify linked genes without controlled crosses. They are especially useful in human genetics and medical research.

TERMINAL QUESTIONS

1. State the reason for the following:

a) The linkage map is not a physical map.

A linkage map shows the order of genes on a chromosome based on how often crossing over happens between them during meiosis. It uses recombination frequency to measure distance, expressed in map units or centiMorgans (cM). But this distance does not represent the actual physical space between genes.

The reason linkage maps are not physical maps is because:

1. Recombination frequency is not equal to physical distance:

Genes that are physically far apart can sometimes have low recombination if crossing over is rare in that region. Similarly, genes close to each other can appear farther if crossing over is frequent.

2. Recombination rates vary in different chromosome regions:

Some chromosome parts have "hot spots" with high recombination, while others have "cold spots" with little or no recombination. This variation affects the linkage distance.

3. Interference and multiple crossovers change recombination frequency:

Interference reduces the number of double crossovers observed, making recombination frequency less accurate in showing real distances.

4. Physical map shows actual DNA length:

Physical maps are created by measuring DNA base pairs or using molecular tools like DNA sequencing or restriction mapping. They give exact physical distances in units like base pairs or kilobases.

b) It is easier to study the linkage relationship for X-linked genes as compared to autosomal genes in humans.

Yes, in humans, it is easier to study linkage relationships for X-linked genes than for autosomal genes. This is mainly because of the unique pattern of inheritance of X-linked genes and the simpler genetic structure found in males.

In humans, females have two X chromosomes (XX), while males have one X and one Y chromosome (XY). Due to this difference, X-linked genes show specific inheritance patterns which help researchers in easily observing and tracking the recombination events across generations. Also, the way X-linked traits are expressed in males provides a more direct way to study linkage relationships.

Here are the following reasons that explain why the study of linkage relationship for X-linked genes is easier as compared to autosomal genes in humans:

1. Hemizygosity in males:

Males have only one X chromosome. So, any gene located on this chromosome is expressed directly. Whether the gene is dominant or recessive, it shows up in the phenotype without being masked. This direct expression avoids complications like heterozygosity which are common in autosomal genes.

2. No recombination in male X chromosome:

In males, the X chromosome does not pair or recombine with the Y chromosome during meiosis, except in a very small region. This means genes on the X chromosome in males are transmitted without recombination, making it easier to observe linkage between markers and genes.

3. Predictable inheritance pattern:

X-linked traits follow a known pattern. Fathers pass their X chromosome to all daughters and never to sons. Mothers pass their X chromosomes to both sons and daughters. This regularity in inheritance makes it easier to follow how genes are passed on and where recombination has occurred.

4. Simpler genetic background:

Since males carry only one X chromosome, the chance of detecting linkage between X-linked genes is higher due to lack of gene interaction. In contrast, autosomal genes often involve more complex dominance and interaction patterns.

5. Relatively smaller size of X chromosome:

The X chromosome is smaller than most autosomes and has fewer genes. This makes it technically easier to map genes, compare distances, and study recombination frequencies with fewer variables.

c) Three factor mapping is generally considered for linkage map preparation.

Linkage mapping is a method used in genetics to determine the relative positions of genes on a chromosome. It is based on the principle that genes located close to each other on the same chromosome tend to be inherited together. To prepare a linkage map, geneticists rely on recombination frequencies between genes. Among various mapping techniques, three-factor mapping is the most informative and widely used when constructing linkage maps. It involves the use of three linked genes in a single cross and allows for a deeper understanding of gene arrangement and recombination patterns. It gives more detailed and accurate information than a simple two-point test cross.

Here are the key reasons why three-factor mapping is generally preferred:

1. Accurate Gene Order Detection

In two-factor crosses, we can only know whether genes are linked and how far apart they are, but we cannot determine the actual sequence or order of multiple genes. In three-factor mapping, we can clearly find the correct linear order of three genes on a chromosome by identifying the double crossover classes. This is not possible in two-point crosses, where ambiguity in gene order may remain.

2. Detection of Double Crossovers

Double crossovers are important because they give information about the gene that lies in the middle of the three. In a three-point test cross, we compare the parental and recombinant classes and identify the double crossovers, which help in locating the middle gene precisely. This improves the quality of the linkage map. Two-factor mapping often misses double crossovers, which leads to underestimation of distances.

3. Measurement of Interference and Coincidence

Only in three-point crosses can we calculate the coefficient of coincidence (COC) and interference (I). These values show how much one crossover can affect the occurrence of another in nearby regions. This gives deeper insight into the behavior of recombination and chromosome structure. Two-point crosses do not allow this type of analysis.

4. More Reliable Genetic Distances

Three-factor crosses give more reliable and refined distances between genes because they involve more recombination classes (single and double crossovers). This helps in constructing a more accurate linkage map, especially when genes are close together. In two-point mapping, recombination frequency can become less reliable due to undetected multiple crossovers.

5. Simultaneous Study of Multiple Genes

Three-point test crosses allow geneticists to analyze three genes at once, saving time and effort. This is efficient compared to doing several two-point crosses between each pair. It also minimizes experimental errors and provides consistent results across one data set.

2. In Drosophila, yellow (y) body, vermilion (v) eyes and singed (sn) bristles are X-linked recessive traits. Wild type females, heterozygous for these three X-linked genes, were test-crossed with yellow, vermilion and singed males. The following progenies were obtained:

a) Determine the order of genes.

b) Find the distance between the genes and construct the linkage map.

c) Find the coefficient of coincidence and interference.

a) Determining the Gene Order

To determine the gene order, compare the double crossover (DCO) progeny with the parental types.

• Parental Types (highest frequency):
• yellow, singed = 333
• vermilion = 340

• Double Crossovers (lowest frequency):
• yellow = 4
• vermilion and singed = 4

Now compare the DCO with the parental to find which gene is in the middle.

• From parental (yellow, singed) to DCO (yellow), vermilion must be the gene that changed.

• From parental (vermilion) to DCO (vermilion and singed), yellow changed.

So the gene that switches its position between parental and DCO is the middle gene. Hence, the gene order is:

y (yellow) - v (vermilion) - sn (singed)

b) Find the distance between the genes and construct the linkage map.

To find distances, we use the recombinant classes:

• Single crossovers between yellow and vermilion (y-v):
• yellow, vermilion = 105
• yellow, vermilion and singed = 59

       →Total = 164

• Single crossovers between vermilion and singed (v-sn):
• singed = 98
• yellow, vermilion and singed = 59

      → Total = 157

• Double crossovers:
• yellow = 4
• vermilion and singed = 4

      → Total = 8

Distance between yellow and vermilion (y-v) = [(164 + 8) / 1000] × 100 = 17.2 cM

Distance between vermilion and singed (v-sn) = [(157 + 8) / 1000] × 100 = 16.5 cM

Linkage Map:

y — 17.2 cM — v — 16.5 cM — sn

c) Find the coefficient of coincidence and interference.

• Expected DCO frequency = (distance y-v × distance v-sn) / 100

     = (17.2 × 21.1) / 100 = 2.838%

     = 28.38 individuals (out of 1000)

• Observed DCO = 4 + 4 = 8

• Coefficient of Coincidence (C) = Observed DCO / Expected DCO

     = 8 / 28.38 = 0.28

• Interference (I) = 1 – C = 1 – 0.28 = 0.72

So, there is strong interference (72%), meaning most expected double crossovers do not actually occur.

3. From the following three factor test cross data between heterozygous female and homozygous recessive male:

a) Determine the order of genes.

b) Find the distance between the genes and construct the linkage map.

c) Find the coefficient of coincidence and interference.

(a) Determine the order of genes.

Identify parental types

Parental combinations are most frequent → look for the two highest numbers:

• + b + = 368
• a + c = 347

These are the non-recombinant (parental) types.

So, two original chromosomes were:

• + b +
• a + c

Identify double crossover (DCO) types

DCOs are least frequent:

• + b c = 10
• a + + = 8

Now compare these with parentals to find the middle gene.

From parental (+ b +) to DCO (+ b c), only the c gene changed.

From (a + c) to (a + +), only c changed.

So, c is the middle gene

Final gene order is: a – c – b

(b) Find the distance between the genes and construct the linkage map.

To calculate distances, we count recombinants for each gene pair.

Distance between a and c

Recombinants between a and c are:

• a b + = 68
• + + + = 67
• + b c = 10
• a + + = 8

Total = 68 + 67 + 10 + 8 = 153

Recombination frequency = (153 / 1000) × 100 = 15.3 cM

Distance between c and b

Recombinants between c and b are:

• a + c = 347
• + b + = 368
• + b c = 10
• a + + = 8

Total = 347 + 368 + 10 + 8 = 733

But a + c and + b + are parentals, not recombinants.

So only:

• a b c = 78
• + + c = 54
• + b c = 10
• a + + = 8

Total = 78 + 54 + 10 + 8 = 150

Recombination frequency = (150 / 1000) × 100 = 15.0 cM

Linkage Map:

a ––15.3 cM–– c ––15.0 cM–– b

(c) Find the coefficient of coincidence and interference.

Observed identify double crossover (DCO) = 10 ( + b c ) + 8 ( a + + ) = 18

Expected Identify double crossover (DCO)

Expected DCO = (Recombination frequency a–c) × (Recombination frequency c–b) × total progeny

= (15.3 ÷ 100) × (15 ÷ 100) × 1000

= 0.153 × 0.15 × 1000

= 22.95 ≈ 23

Calculate coincidence (C) and interference (I)

• Coefficient of Coincidence (C) = Observed DCO ÷ Expected DCO = 18 ÷ 23 ≈ 0.78

• Interference (I) = 1 – C = 1 – 0.78 = 0.22

Read More:

• UNIT 1 – Mendel's Laws (Q&A) | MZO-002 MSCZOO | IGNOU

• UNIT 2 – Gene Action and Interactions (Q&A) | MZO-002 MSCZOO | IGNOU

• UNIT 3 – Gene Structure and Function (Q&A) | MZO-002 MSCZOO | IGNOU