Physical Maps Using in Situ Hybridization (ISH), Heterologous probes, Mitotic metaphase, Chromosomes, Hybridization

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Physical maps using insituhybridization (ISH)
Utilizing molecular probes derived from cDNA or gDNA (even if they were Got derived from the same species i.e. heterologous probes), in situhybridization could be successfully utilized for mapping genes with multiple copies on specific chromosomes. For instance cDNA copies of wheat rDNA were used for in situhybridization to mitotic metaphase chromosomes of bread wheat cv. Chinese Spring. It was found that 90% of the ribosomal genes were located on chromosomes, lB and 6B and the remaining repeat units located on 5D. 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, information was collected on physical location of the 5S rRNA loci in wheat, rye and barley. Using in situhybridization (ISH), it was found that 5S rRNA loci are located distal to the secondary constrictions on chromosome IB of wheat and IR of rye and on non-nucleolar chromosome in barley. An additional 5S rRNA locus in rye was located on 5R.

Physical mapping of the 5S rRNA multigene families in bread wheat was also achieved by combining the technique of in situhybridization (ISH) with deletion mapping. Terminal deletion stocks produced by using monosomic addition of a gametocidal chromosome from Aegilopscylindrica, ere utilized for detection and location of twelve 5S rRNA loci on short arms of chromosomes of homoeologous groups I (1AS, 1BS and 1DS) 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 centromeres. The positions of five loci of 5S rRNA, which were designated as 5S-Rrna AI, 5S-Rrna DI, 55-Rrna A2, 5S-Rrna B2 and 5S-Rrna D2were 0.77, 0.96, 0.76, 0.63, and 0.64, on arms of lAS, IDS, 5AS, 5BS and 5DS respectively. The 5S rRNA locus on IAS was discovered for the first time during this study.

Terminal Detection for 5DS wheat chromosomes
(a) Metaphase showing 40 normal, 2 telocentric chromosomes	(b) Cell showing ISH with 12 sites of hybridization

In situhybridization has also been used for physically locating the molecular markers 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 animals 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 pSc119.2 (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 situhybridization.

In most of the above approacles involving ISH, individual DNA sequences (e.g. ribosomal RNA, or a specific repeat sequence) were physically localized and no real large physical mapping at the level of whole genome or a whole chromosome was undertaken. However, during 1995-2002, high resolution physical mapping of BACs anchored by genetic markers was undertaken using interphase and pachytene chromosomes. Fibre FISH technique was also used for physical mapping. The interphase and pachytene chromosomes being elongated give a better resolution in the physical map. Orientation of BACs with respect to telomeres and the centromere was also possible in pachytene chromosomes.

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