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Home >>Molecular Maps of Plant Genome >>Physical Maps Using In Situ Hybridization (ISH)
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Physical Maps Using In Situ Hybridization (ISH) - Utilizing molecular probes derived from cDNA or gDNA (even if they were not derived from the same species i.e. heterologous probes), In situ hybridization could be successfully utilized for mapping genes with multiple copies on specific chromosomes.
For instance cDNA copies of wheat rDNA were used for in situ hybridization to mitotic metaphase chromosomes of bread wheat cv. Chinese Spring. It was found that 90% of the ribosomal genes were located on chromosomes 18 and 6B and the remaining repeat units located on 50 (Miller et al., 1980, Peacock et al., 1981).
This had suggested that the diploid genomes like A genome, which had two chromosomal sites for rRNA genes (Gerlach et al., 1980) had undergone a change after incorporation into hexaploid (6x) bread wheat. Similarly Appels et al. (1988) sucessfully collected information on physical location of the 5S rRNA loci in wheat, rye and barley.

Using in situ hybridization (ISH), they found that 5S rRNA loci are located distal to the secondary constrictions on chromosome IB of wheat and 1R of rye and on non-nucleolar chromosome in barley. Recently another additional 5S rRNA locus in rye was located on 5R (Reddy and Appels, 1989)

In another recent report, physical mapping of the 5S rRNA multigene families in bread wheat was achieved by combining the technique of in situ hybridization (ISH) with deletion mapping (Mukai et al., 1990).

Terminal deletion stocks produced by using monosomic addition of a gametocidal chromosome from Aegilops cylindrica, were utilized for detection and location of twelve 5S rRNA loci on short arms of chromosomes of homoeologous groups 1 (1AS, IBS and 10S) and 5 (5AS, 5BS and 5DS).
Specific chromosome arms and regions of the arms associated with only five different loci were determined using ditelocentrics (DT) and deletion stocks. The position of ISH sites on a telocentric chromosome was determined as a fraction of the total telosome length from centromere.
The post ion of five loci of 5S rRNA designated as 5S-Rrna AI, 5S-Rma D1, 55- Rrna A2, 55-Rrna B2 and 5S-Rrnll D2 were 0.77, 0.96, 0.76, 0.63 and 0.64 on arms lAS, 1DS, 5AS, 5BS and 5DS respectively. The 5S rRNA locus on lAS was discovered for the first time during this study.

Recently (Sept., 1992), in situ hybridization has also been used for physically locating the molecular markes earlier mapped in rice. It has been shown that distances on linkage maps (in cM) are not proportionate to physical distances on chromosomes.

Techniques have also been described, where each of two or three DNA sequences can be labelled with different non radioactive haptens, each of which can be visualized with an independent detection system. It is, therefore, possible to detect two or three DNA sequences simultaneously on animal or plant chromosomes, in presence of each other, for physical mapping.

For instance, in rye chromosomes, the location of pTa71 (rDNA sequence from wheat) and pSc1l9.2 (a repeat sequence from rye) could be detected by labelling one with biotin and the other with digoxigenin and then using a mixture of the two for in situ hybridization (Leitch et al., 1991)
 

 

 
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