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Crossing Over & Gene Linkage – Thomas Hunt Morgan’s Fruit Fly Experiment
Linkage of Chromosomes
The tendency of the genes present on a chromosome to be inherited together is called linkage. The genes showing such property of inheritance are known as linked genes.
Linkage is a tendency of genes on a chromosome to remain together and passed as such in the next generation. It results in the more parental type. The strength of linkage between two genes increases if they are closely placed on a chromosome. Linkage also helps to maintain a new improved variety. Genes whose loci are nearer to each other are less likely to be separated into different chromatids during chromosomal crossover and are therefore said to be genetically linked.
Discovery of Linkage
T.H. Morgan in 1911 from his works on the fruit fly Drosophila melanogaster discovered the phenomenon of linkage. However, before Morgan, Bateson, and Punnett in 1906 from their hybridization experiments on sweet peas, Lathyrus odoratus, observed a deviation of independent assortment of genes as suggested by Mendel. They suggested coupling and repulsion as the cause behind this exception to an independent assortment of genes. Their finding and proposition of coupling and repulsion became a clue to the discovery of linkage.
Morgan’s Experiments on Fruit Fly (Drosophila Melanogaster)
Based on two dihybrid experiments on fruit flies and their results, Morgan could be able to discover linkage. The characters considered by Morgan were body colour and the shape of wings. The normal body colour of a fruit fly is grey which is determined by a dominant gene say G. The contrasting feature black is determined by the recessive allele of G say g. On the other hand normal wing shape in the fly is long which is determined by the dominant gene L and its recessive allele l determines vestigial wing features. In consideration of these characters, hybridization experiments as were carried out by Morgan may be shown in the following manner.
Explanation of the Result:
In the above-noted experiments, the F1 progeny were hybrids expressing only dominant features and therefore, they were grey long by character. But in the F, grey long flies of the first experiment, the G and L genes came from one parental organism. On the other hand in the second experiment the F, hybrids obtained the G gene from one parent and the L gene from the other parent. In this consideration, it may be said that one parent contributed G & 1 to the hybrid and the other parent contributed g & L to the hybrid. Therefore, in the first case G & L remained in a coupling association, but in the second case, G & L remained in a repulsive association.
In both the first and second cases F: hybrid grey long flies were crossed with black vestigial flies. Being test cross by nature, in both cases four types of progeny should appear in 1 : 1 : 1 : 1 ratio. But it was found that the ratio of the progeny types was 41.5 : 8.5 : 8.5 : 41.5. Not only that the result indicated that in both the crosses parental type of progeny (Grey long & Black vestigial in 1st experiment and Grey vestigial and Black long in second experiment) appear in far greater number than the recombinant type of progeny. On the basis of such result, Morgan proposed that coupling or repulsion could not affect the results from the test crosses, rather the close association of the two genes (G&L) could be the cause for the deviation from the expected ratio. He also pointed out that the dose association of the genes is linked and this association may be of two types namely coupling and repulsion. Based on this concept Morgan also proposed the linkage theory which may be stated as under-
Linkage Theory
- Genes present over a chromosome are oriented in linear order and genes of the same chromosome show linkage.
- Linked genes show a tendency to be inherited together.
- Coupling and repulsion are two different aspects of the same phenomenon of linkage.
- Genes located closer over the chromosome show strong linkage force and genes located distantly over the chromosome show weak linkage force.
Types of Linkage
On the basis of linkage theory, as proposed by Morgan, linkage may be categorized into two types complete linkage and incomplete linkage.
(a) Complete Linkage:
When the genes of a chromosome are very closely located and because of a strong linkage force the linked genes cannot be separated from each other. As a result, such genes can be inherited from generation to generation only in parental combination. The linkage between such two or more genes is termed complete linkage.
Example: The fourth chromosome of Drosophila melanogaster is a dot and all the genes present over it are completely linked. Besides in males Drosophila genes of all the chromosomes show complete linkage. In the case of complete linkage, only the parental type of progeny may be produced.
(b) Incomplete Linkage:
In spite of the tendency of being inherited together, if the linked genes get separated at times, the linkage is called incomplete linkage. In case of incomplete linkage along with the production of a parental type of progeny, some recombinant progeny are also produced. The genes present over the first, second, and third chromosomes of fruit fly females show incomplete linkage. As shown in the experiments of Morgan, the genes determining body colour and wing shape in fruit fly females due to incomplete dominance produced recombinant progeny by 17%.
Linkage Group
Genes located on the same chromosome are said to belong to the same linkage group. So, each chromosome represents one linkage group. The total number of linkage groups in an organism is equal to the number of chromosomes in one haploid set. In the case of Drosophila melanogaster four linkage groups can be distinguished into three large and one small linkage group corresponding to the four pairs of chromosomes. 23-linkage groups are present in humans corresponding to 23 pairs of chromosomes. Pea plant has 7-linkage groups, corresponding to the 7-pairs of chromosomes.
Crossing Over of Chromosomes
The phenomenon of segmental exchange between the non-sister chromatids of the paired homologous chromosomes at meiosis is called crossing over. During meiosis cell division the homologous chromosomes in the cell come in pairing and then a segmental exchange may occur between them. Because of this, the gene combination of a chromosome may be broken resulting in a new combination of genes. This phenomenon is called crossing over. Crossing over is an antagonistic phenomenon to linkage and it can separate the linked genes.
With the example as stated before showing the location of the two genes over the chromosome, the incidence of chromosomal exchange with the formation of recombinants may be exhibited. Morgan (1912) 1st used the term ‘Crossing Over’. Production of recombinant progeny from parents may occur due to crossing over. It also promotes the development of variation in living organisms.
Mechanism of Crossing Over
During meiosis at the pachytene substage of prophase-I crossing over occurs, but before the occurrence of crossing over some events are essential in a cell. Such important events are the Pairing of homologous chromosomes at pachytene, Achievement of tetrad stage at pachytene and segmental exchange, Separation of two homologous chromosomes through diplotene, and Terminalization.
1. Pairing of Homologous Chromosomes:
Only after pairing of the homologous chromosomes in the cell does crossing over may occur. At prophase-I of meiosis during leptotene substage, the chromatin material appears as fine threads which during zygotene become somewhat thickened and after this, the homologous partners come in pairing. The paired homologous chromosomes together are called bivalents. Such pairing of bivalents may occur due to the formation of a synaptonemal complex between them. The pairing of homologous chromosomes is known as Synapsis.
2. Formation of Tetrads and Exchange of Parts:
After synapsis, the bivalent at the pachytene substage forms tetrad when each member in the bivalent is longitudinally splitted except at the centromere region. At this stage, the chromosome becomes more thickened and the bivalent appears more prominent. After the attainment of the tetrad stage, the non-sister chromatids participate in the process of exchange of parts. Because of this fact, one chromatid of the bivalent gets recombined and the other maintains an original combination of genes. During this process of exchange of parts of the broken ends of the chromatids are ligated. For breaking the chromatids the enzyme endonuclease and for ligating the enzyme ligase help in the process.
3. Segregation of Two Homologous Chromosomes through the Diplotene Substage:
As crossing over is accomplished, homologous chromosomes cannot remain together further and they start moving apart from each other. Following pachytene, this behaviour of the chromosomes is found at the diplotene substage. However, though the synapsed chromosomes show a movement apart from each other, the chromatids which take part in the process of exchange remain connected at one or more points. These points of attachment between the homologous chromatids are called chiasmata (singular chiasma).
4. Terminalization:
As the chromosomes go apart more and more, during diakinesis the chiasmata also move towards the terminal ends of the chromosome. This phenomenon is called terminalization. After the process of terminalization, the cell enters into diakinesis. Following diakinesis through metaphase-I, complete separation of the homologous chromosomes may be achieved at anaphase-I.
Types of Crossing Over
Crossing over may occur at one or more points on the homologous chromosomes. On the basis of this, crossing over may be categorized into three types, namely single crossing over, double crossing over, and multiple crossing over.
(a) Single Crossing Over:
When crossing over occurs only at one point of the paired homologous chromosome, it is called a single crossing over. As a result of single crossing over 50% parental progeny and 50% recombined progeny may be formed. Crossing over in this case occurs at one point between two given genes. Hence, it is also called a two-point crossing over. On the basis of the number of progeny produced in a two-point crossing over the distance between two genes may be determined.
(b) Double Crossing Over:
When crossing over occurs at two points on the paired homologous chromosomes, it is called double crossing over. In double crossing over three genes may be assigned between which two crossing-overs may be possible. Hence, it is also known as a three-point cross. As the result of a three-point cross or double-crossing over, three types of recombinant progeny may be formed in addition to a pair of parental progeny. These cross-over types or recombinant progeny are known as Single crossing over type-I, Single crossing over type-II, and Double crossing over type. The double crossing-over type represents simultaneous crossing-over at two adjacent points.
(c) Multiple Crossing Over:
When the paired homologous chromosomes crossing over occurs at more than two points, it is called multiple crossing over. Types of recombinant progeny produced out of multiple crossing over depends on the number of points at which multiple crossing over occurs.
Chromosome Mapping
The concept of linkage and the number of recombinant progeny produced due to crossing form the basis for locating genes over a chromosome. This is known as chromosome mapping or linkage mapping. In the investigation of the linkage relationship between genes, scientists would be able to discover that when the genes are incompletely linked they may be separated from each other by crossing over and forming the recombinant progeny.
Cross-over products or recombinants produced due to crossing over between two genes always appear to be constant by frequency and percentages between two pairs of linked genes but may differ depending on their nearness or farness. Such difference in the crossing-over frequency between different pairs of genes becomes relative to the force of linkage between the corresponding genes. This means that when the force of linkage between two genes is less, it permits more crossing over between these genes. Because of this, the number of recombinant progeny also becomes more. On the contrary, when the linkage force between two genes becomes high, it does not permit more crossing over to occur. As a result frequency or percentage of recombinants also becomes low.
This truth was first realized by a student of Morgan named Sturtevant. He also suggested that if the frequency of recombinants or percentage of crossing over product is an indication of the magnitude of linkage force between two genes, then the same may be used as an index of the distance between them over a chromosome. Based on this principle Sturtevant proposed the method of chromosome mapping or the construction of a linkage map. According to him the percentage of recombinants produced due to crossing over between two genes over a chromosome is the distance between the genes under consideration. He advocated the use of a simple map unit or m.u. to denote this distance. However, cM (Centi-Morgan) also may be used instead of m.u. to show respect to a famous geneticist and discoverer of linkage, Morgan.
Principles on Which Chromosome Map is Based:
- Genes are arranged over a chromosome in linear order.
- Genes wider in location over the chromosome show more crossing-over, while genes closer by location show lower crossing-over frequency.
- The magnitude of crossing over is the index of distance between two genes and the percentage of crossing over is taken as a measure for this distance which is expressed by map unit (m.u.) or cM.
- Because the crossing-over percentage cannot be equal to or more than 50%, in chromosome mapping the genes usually closer and showing a crossing-over percentage less than 50% are taken into consideration for mapping. Smaller distances (in cM) between genes are added to obtain a higher map distance between genes. Mapping of many genes over a particular chromosome may be done by this method.
- Genes very widely located over a chromosome cannot be considered in chromosome mapping because there may be multiple crossing over between them and this virtually alters the actual percentage of crossing over.
- If the percentage of crossing over between two genes becomes 50%, then genes are out of linkage and they can be assorted independently.
Explanation:
Suppose two genes a and b are present over a chromosome and the crossing-over percentage between a and b is 15%, then, the distance between a and b is 15 m.u. or 15 cM. A cross-over frequency of 1% between 2 genes is defined as 1 m.u. A linkage map for these two genes a and b may be shown as under.
Method of Chromosome Map Preparation in Consideration of Three Genes:
In most of the cases for the construction of a linkage map, three genes are taken into consideration. The results of crossing over are analyzed in consideration of three genes and on the basis of that location of the genes and their distance are determined.
Suppose over a chromosome there are three genes namely p, q, and r. For constructing a linkage map for these three genes one cross is to be established where one heterozygote for these three genes (PQR/pqr) is crossed with a homozygous recessive (pqr/pqr) organism. From such a cross possible progeny types and their number (in an arbitrary manner) may be shown in the following manner.
Given progeny number give the percentage as under.
1. S.C.O. type-I = \(\frac{160}{1000}\) × 100 = 16%
2. S.C.O. type II = \(\frac{74}{1000}\) × 100 = 7.4%
3. D.C.O. type = \(\frac{6}{1000}\) × 100 = 0.6%
(Simultaneous C.O. between p & q and q & r)
As the double-crossing over represents simultaneous crossing over at two different points between three genes, the actual S.C.O type-I% should be (16+0.6) or 16.6% and the actual S.C.O type-II% comes to (7.4 + 0.6) or 8%. Therefore, the relative distance between p and q is 16.6 m.u, and that between q and r is 8 m.u. On the basis of this, a linkage map for these three genes may be drawn as under.
Information Obtained from Chromosome Map:
- Actual position of more than one gene over a chromosome.
- The relative distance between two genes and the linkage force between them.
- Type of linkage between two genes.
- Expected number of specific progeny types from one heterozygote parent.
- Linkage group for some given genes.
Some Problems on Gene Mapping
1. Test cross was done on three heterozygous organisms. The progeny produced from these crosses is indicated below. Show gene mapping in these cases.
Solution:
(a) AB/ab × ab/ab
AB : 230
ab : 246
NCO Progeny: 476
Ab : 35
aB : 40
CO Progeny: 75
Total = 551
∴ Total Number of progeny = 230 + 246 + 35 + 40 = 551
Out of the 551 progeny, 35 + 40 = 75 represents recombined progeny.
So, Hence recombination frequency = 75/551 = 0.136 or 13.6%
Therefore, the Distance between a and b is equal to 13.6 m.u.
(b) BC/bc × bc/bc
BC : 136
bc : 132
Bc : 16
bC : 18
Total = 302
∴ Total number of progeny = 136 + 132 + 16 + 18 = 302
Out of the 302 progeny, 16 + 18 = 34 represent recombinant progeny.
Hence recombination frequency = 34/302 = 0.1125 or 11.25%
Therefore, the distance between b and c is equal to 11.25 m.u.
(c) AC/ac × ac/ac
AC : 198
ac : 202
NCO Progeny : 400
Ac : 60
aC : 70
CO Progeny : 130
Total = 530
∴ Total number of progeny = 198 + 202 + 60 + 70 = 530
Out of 530 progeny, 60 + 70 = 130 represents recombined progeny.
Hence recombination frequency = 130/530 = 0.245 or 24.5%
Therefore, the distance between a and c is equal to 24.5 m.u.
So, the Distance of the a, b, and c gene and linkage map is
2. Crosses of AB/ab and BC/bc produce progeny in the following manner. Explain how this is possible.
Prepare a linkage map for these genes.
Solution:
From the first cross, it appears that Ab (35) and aB (30) progeny represent the recombination classes.
In this case, the total number of progeny is 270 + 275 + 35 + 30 = 610.
Out of the 610 progeny, 30 + 35 = 65 represents the recombined progeny.
Hence, the recombination frequency is 65/610 = 0.106 = 10.6%.
However, in the second cross, the total progeny comes to 144 + 162 + 148 + 158 = 612.
In this case, recombined classes Be (148) and bC (158) come to 148 + 158 = 306.
This is 306/612 = 0.50.
Therefore, the recombination frequency is 50%.
This indicates that there is no linkage between b and c.
Hence, a linkage map for the genes may be given as 
3. In Drosophila three genes a, b & c present on the X chromosome. The normal alleles of the genes are a+, b+, and c+ and these are dominant over a, b and c. abc/abc female fly being mated with a+b+c+ male produced F2 progeny. Find out the order of these genes and construct a linkage map. Detect the amount of interference between the genes.
Solution:
The F2 progeny appear in the following form:
As the genes are present on the X chromosome and the female was abc/abc, the F, female, and male were of the following gene combination.
Among these a+bc+ (6) and ab+c (4) appeared as DCO-type progeny which showed a shifting of position when they are compared with the parental type. Hence, gene b remains in the order. Therefore the gene order on the X chromosome would be a — b — c.
When the progeny are arranged in order and the frequency of the NCO and crossover types are calculated we get the following figure.
∴ SCO I in actual = 16.67 + 0.36 = 17.03% and SCO II = 10.22 + 0.36 = 10.58%.
Hence, the distance of b from a is 17.03 m.u., and the distance of c from b is 10.58 m.u.
Therefore, the map distance may be indicated below:
In the present case expected DCO frequency comes to = 0.1703 × 0.1058 = 0.0180
Observed DCO frequency = 0.0036
∴ Coefficient of coincidence = \(\frac{0.0036}{0.0180}\) = 0.2
Interference = 1 – coefficient of coincidence
Therefore, in this case, interference = 1 – 0.2 = 0.8 or 80%.