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Restriction
Fragment
Length
Polymorphism
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RFLP - These markers are based on specific restriction enzymes and probes and are based on Southern hybridization. RFLP has been described in (Appendix4.11). RFLPs have various applications, including preparation of molecular maps. An RFLP map depicts, in essence, the locations of different DNA sequences used as probes as determined by recombination data.
The DNA sequences used as probes are, of necessity, radioactive; such sequences should have the following features:
(i) each sequence must produce a strong hybridization signal,
(ii) a single sequence should produce a small number of bands, ideally, a single band, on the X-ray film following Southern hybridization, and more particularly,
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(iii) must show polymorphism (differential movement of the bands in gel) in different strains of a species (e.g., in maize, rice, etc.) or among related species, (e.g., in tomato).
The probes are used to screen different strains/related species, and a few strains/species showing polymorphism for several probes (= RFLP markers) are identified. Two selected strains (say A and B) are crossed to produce Fl and F2lback cross generations.
DNA is isolated separately from the parent strains, the Fl and 50 (or more) F2lback cross plants and used for determining RFLPs with the various probes for which they show polymorphism. DNA from each plant is digested with the appropriate restriction enzyme, the digest is subjected to electrophoresis and Southern hybridization is performed with each of the various probes.
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Ideally, a probe should reveal a single band which shows differential mobility (detected as a difference in their positions in the film) in the two parental strains. For example, probe detects a relatively slower moving band in strain A and a faster moving one in strain B.
These bands may be regarded as two alleles of a single gene, say, allele A for slow moving band and allele a for the fast moving band. Similarly, probe 2 detects a fast moving band in strain A (this may be denoted as allele b) and a slow moving one in strain B (designated as allele B). We may now write the RFLP genotypes of the strains A and B as AAbb and aaBB, respectively
The F1 hybrid between the strains A and B will show both slow and fast moving bands for each of the two probes; we may write its genotype as AaBb. Obviously, RFLP markers behave as codominant markers.
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A similar consideration will show that the back cross (F1 x strain A) population of this cross will show four types of plants when classified with the probes 1 and 2; these four types may be written as: AAbb, AaBb (both called parental types since they result from the parental type gametes produced by F1), AABb, and Aabb (the last two are recombinant types as they originate from recombinant type gametes produced by F1). The question whether the two RFLP markers are linked or not is answered by comparing the frequencies of AAbb, AaBb, AABb and Aabb with the expected frequency of 1 : 1 : I : 1 by a X2 - test.
A significant deviation from the expected frequency will show linkage between the two markers. The frequency of recombination [=(number of recombinant type progeny/total number of progeny) x 100] between the two markers can be estimated, and used as a measure of the distance between them for preparing a map, similar to a linkage map depicting their relative positions in the chromosomes.
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Ordinarily, a cross made for RFLP mapping will differ for many RFLP markers all of which are used separately to classify the back cross/F2 progeny.
The data from these classifications are analysed by a suitable computer programme, e.g., MAPMAKER, to detect linkage and estimate recombination frequencies among the RFLP loci, and to construct RFLP maps. Extensive RFLP maps have been prepared for maize, tomato, potato, rice, wheat, barley, etc. Once an RFLP map has been prepared for a species, it can be combined with its genetic map, and assigned to the specific chromosomes.
RFLP markers can be used to map quantitative trait loci (QTLs). QTLs have small individual effects, and their expression is greatly affected by the environment.
As a result, their inheritance pattern can not be followed by classical genetic techniques and they can not be mapped. But QTLs can be identified and mapped in relation to RFLP markers. Such mapping would also permit an indirect selection for QTL by selecting for RFLP marker linked with them.
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The detection of linkage between QTL and RFLP markers is done as follows. Two strains are selected, which differ, from each other for several RFLP markers and show significantly and markedly different mean values of many quantitative traits.
The two strains are crossed to produce Fit which is selfed to produce F2 population. The F2 plants are evaluated for the various RFLP markers and the concerned quantitative traits. F2 plants homozygous for the two alleles of each RFLP marker are identified and grouped into two separate classes.
The mean values of the two groups for a quantitative trait are now compared; a significant difference between the two groups will reveal a linkage between the RFLP marker and the QTL affecting the concerned quantitative trait. Computer programmes like MAPMAKER enable the detection of linkage between RFLP (and other molecular) markers an QTLs and to prepare their linkage maps.
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