A variety of methods are available for detecting mutations, both in plants and animals.
Some of most important methods are described below.
1. CLB method for detecting mutations:
This method perfected by Muller has been used to detect mutations in X chromosomes of Drosophila. Even though it is primarily meant to detect radiation induced mutations, it can be used in the detection of spontaneous mutations as well.
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Three characters on the X chromosome are chosen to study the mutation. These are – C = Cross over suppressor. This represents a long inversion in the X chromosome, suppressing the cross over in the inverted portion.
Cross over may still occur, but cross over gametes rarely survive due to deficiencies and duplications. L = Lethal gene. This is a recessive character. Hence homozygous females (l) and homozygous (1/y) males do not survive.
Image Source: wingx.ch
B – Bar eye. This is a dominant gene. This is used as a phenotypic marker for the flies required mating in the F, generation.
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The working mechanism of this is illustrated in the fig. A CLB male is mated to a normal male which is subject to radiation.
The idea is to find out whether there are any lethal mutations sex linked chromosome of the male. Y chromosome is regarded as neutral as for as genes on x chromosomes are concerned.
Therefore any X chromosome of male which is irradiated I can be followed in subsequent generations and lethal, if any can be detected.
In the X generation (F1 generation; called X, because the individuals are X 1 irradiated X chromosome of the male goes to the daughters, who have another X from the mother; hence in this generation any mutation induced cannot be detected. The X progeny will be as follows.
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A cross is now made between the normal male (with X also normal because it is obtained from female and the CLB female.
This CLB female has one of X chromosome from the male parent which is irradiated and the other is CLB this (X, or F2) generation all the males get one of the X chromosome, they (50%) because there is already a lethal gene.
If they get the irradiated chromosome, and if a new lethal mutation is induced then the other 50% also will die Hence if there is a lethal mutation on the X chromosome in the F2, no male progeny will survive and all will be females.
This is a clear cut evidence for the presence of a lethal mutation on the irradiated X chromosome. In the X2 generation the progeny will be as follows.
Females:
50% CLB; 50% Normal (with an irradiated X chromosome)
Males:
50% CLB (die); 50% carry irradiated X chromosome; die if there is a lethal mutation.
2. The Muller 5 method:
Like the CLB method this is also devised by Muller. The experiment involves crossing a homozygous Muller 5 female Drosophila with an irradiated male.
The Muller 5 female Drosophila has two marker genes – apricot eye (wa/wa) and Bar eye (B/B) in a long inversion so that there is no cross over. A homozygous apricot (eyed) bar eyed female is crossed with an irradiated male.
As shown in the figure, the F, generation female offspring which gets the irradiated X from the male parent is crossed with a wild male.
If a lethal mutation has occurred in the X chromosome, no wild types of male would survive, because they would get the irradiated X.
The absence of wild males in the F2 is an indication of lethal mutation. Muller was awarded Noble prize in Medicine in 1946 for this and many other discoveries that he had made in Genetics.
3. Detection of Autosomal mutations (Balanced lethals):
Autosomal mutations can be detected by making use of balanced lethal genes in organisms. A balanced lethal stock is one which carries a lethal gene in one of the homologues and its wild allele on the other homologue.
Homozygous individuals will die, so what survives is always heterozygous. Balanced lethals can be studied by using a specific strain of Drosophila melanogaster called the curly lobe plum.
In this, a stock of flies carry on one of the autosomes two dominant genes C (curly wing) and L (lobed eye) and a recessive lethal gene.
An individual with two CYL chromosomes will not survive because the recessive lethals will be in double dose and its homologue carried the Pm (plum colored eye) gene which is also dominant
When two individuals with the genotype {(CYL/Pm) X (CyllpM)} are crossed, the heterozygous individuals only would survive, as homozygous for CYLJ would carry lethal genes in double dose, because CYL chromosome the gene CY to the chromsome carrying Pm gene.
A female CYL/Pm is crossed to another normal male which is irradiated.
Four types of progeny are produced in the F, These are:
(a) Curly lobe (with an irradiated autosome A)
(b) Curly lobe (with another irradiated autosome B)
(c) Plum (with an irradiated autosome A)
(d) Plum (with another irradiated autosome B)
One of these flies is selected (because each one has one irradiated chromosome) and back crossed to curly lobe plum. The progenies will be of the following types.
(a) Homozygous curly lobe (dies)
(b) Curly lobe plum.
(c) Curly lobe with an irradiated chromosome.
(d) Plum with an irradiated chromosome. Among the above progeny, (c) flies are crossed among themselves to obtain –
(a) Homozygous curly lobe (dies)
(b) Heterozygous curly lobe (with an irradiated chromosome)
(c) Same as b.
(d) A fly with both irradiated chromosomes.
It is in the (d) category that any mutation induced would appear. If the induced mutation is lethal, it also dies and only the curly lobed individuals survive. If the mutation induced is not lethal, but a recessive trait, that would be expressed, since it is homozygous.
The same test can be done by selecting the plum with an irradiated chromosome and crossing it with a curly lobe plum with an irradiated chromosome.
The significant aspect of this cross is to bring together the same irradiated chromosome in the homozygous condition to detect all the recessive mutations.
4. Detection of Induced Mutations in Plants:
L.J. Stadler of the University of Missouri and Ralph Singleton of the University of Virginia have worked on the methods to detect mutations in maize and barley.
Stadler (1942) has made a detailed study of spontaneous mutations in maize. The following are the important steps.
(a) A genetic stock dominant for a number of genes is grown as the female parent. The tassels are removed before pollen discharge (to avoid self pollination).
(b) A multiple recessive plant in every fifth row is used as the pollen providing parent for (a) plants.
(c) Seeds were observed in large numbers for endosperm characters. Most of them show dominant characters. If any recessive character (of the male parent) appeared it is due to mutation in the female gametes.
Singleton (1962) used a technique similar to that of Stadler do detect both spontaneous and induced mutations in the pollen of maize.
His method involves collecting pollen from plants dominant for many genes and cultivated in a field irradiated with cobalt 60 and transferring them (pollen) on to the silks of plants with all recessive traits and cultivated in a normal field.
The seeds of the progeny would normally show dominant characters. If any mutation has been induced on a dominant gene, the recessive trait would appear in the progeny.
In this way Geton studied four genes Su -su, on chromosome 4, Pr – pr on chromosome -y on chromosome 6 and Sh -sh on chromosome 9.
The phenotypes of these red normal endosperm, su-sugary endosperm; .Pr- Purple aleurone, pr-aeurone; Y- yellow endosperm, y – white endosperm and Sh-full endosperm, shrunken endosperm.
