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Home >>Molecular Maps of Plant Genome >>Preparation of Genetic Maps Using RFLP Loci
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Preparation of Genetic Maps Using RFLP Loci - The first step for the preparation of linkage maps based on RFLPs is to procure as many unique DNA sequence clones as possible and to determine which clones are effective in revealing polymorphisms. As discussed earlier, both cDNA clone£ (derived from mRNA) and genomic DNA (gDNA) clones have been used in this connection.
In order to determine which clones are actually useful in determining RFLPs, the clones are subjected to an initial screening. The following criteria, are used for selecting useful clones:
(i) it should yield strong hybridization signal;
(ii) it should reveal a small number of fragments and
(iii) it should reveal polymorphism among homozygous lines (as in maize) or related species (as in tomato) which will be used for crosses to yield F2 segregating population for linkage studies.

The second step is to make crosses between homozygous lines or related species showing polymorphism for the selected DNA clones. The Fi hybrids are selfed to yield segregating F2 population. Heterozygous parents like those in potato can also be used rarely for crosses to estimate recombination frequencies leading to construction of RFLP maps.

The DNA samples extracted from parents, Fi hybrids and from individual plants of F2 population can be used for collection of RFLP data using DNA clones (selected as above) as probes. A population of 50 F2 individuals has been shown lo be adequate to give information on linkage among RFLPs, if any.

Recombination frequencies can also be estimated from backcross populations and doubled haploids (DH) derived from pollen of Fl plants. In maize, recombinant inbred (RI) lines have also been produced from a number of crosses, through continuous selfing or sib mating in F2 plants.

These RI lines are comparable to RI lines used in mouse and are also comparable to doubled haploids (DH). They can be used in much the same way as the F2 plants and offer following advantages
(i) They can be maintained indefinitely and supply unlimited DNA, unlike individual F2 plants.
(ii) They are produced by multiple rounds of meioses and, therefore, the linked genes have a better chance of recombination.
The data on recombination frequencies are processed using a computer device, with the help of suitable software. One such software in common use for RFLP maps is MAPMAKER (other software’s like LINKAGE was used in earlier studies).
Once RFLP genetic and chromosome maps are available, if unmarked regions are available, chromosome walking or chromosome hopping or insertion targeting may be used, starting from a known RFLP locus. This wilt helps saturation of the unmarked region or in reaching close to a gene of interest.

The most extensive molecular maps using RFLPs, morphological markers and QTLs (quantitative trait loci; for QTLs see later) have been prepared in maize and tomato. For maize the two inbred lines used initially were H427 and 761. For tomato, however, two different species (L. esculentum and L. hirsutum or L. esculentum and L. pennellii) had to be used due to limited variability available in L. esculentum.

Segregation of codominant RFLP loci (1 : 2 : 1) could be detected in F2 generation, and linkage, if any, could be detected by deviation from the expected segregation ratio. For the linked RFLP loci, recombination frequencies were also worked out using maximum likelihood analysis (MAPMAKER, Lander et al., 1987).

In preparing potato RFLP maps at Cologne in Germany, following two modifications were used to improve the technique of RFLP mapping:
(i) only those restriction enzymes were used, whose restriction sites consisted of only four rather than six base pairs, so that the restriction sites will be more frequent and consequently the genomic DNA will be cleaved more frequently to release additional polymorphism;.
(ii) denaturing polyacrylamide sequencing gels were used instead of agarose gels to produce better resolution of DNA fragments, which differ in length by only a few base pairs.
Heterologous probes may also be used for RFLP mapping in plants. For instance a number of DNA clones from maize have also been utilized for RFLP mapping in sorghum. Similarly, DNA clones belonging to tomato could also be successfully used for potato (also for pepper) and vice versa.
The resulting RFLP maps of potato and tomato were found to be similar. Such studies also suggest that similar RFLP loci occur in related species and can also be used, therefore, for phylogenetic studies.

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