45. Mapping DNA sequences in rice using in situ hybridization

In situ hybridization (ISH), as defined by Gaul and Pardue (1969), involves the cytological location of labelled DNA to chromosomal sites. It is seen such as a powerful and useful tool for the molecular cytogeneticist. ISH can be used to physically map both repetitive, low-copy, and unique DNA sequences in plant chromosomes. It would enable the molecular biologist, cytogeneticist, and breeder to locate and track alien gene and gene complexes that have been inserted into host plants by transformation (Mouras et al. 1987; Le et al. 1989; Rayburn and Gill 1985; Lapitan et al. 1986).

Genetic (recombinational) maps in many species including wheat (Sharp et al. 1989), corn (Hoisington et al. 1990), barley (Shin et al. 1990), rice (McCouch et al. 1988), and tomato (Tanksley et al. 1987) are being developed and expanded. Improved ISH techniques now make it possible to map genes, restriction fragment length polymorphisms (RFLPS) and other small DNA sequences to establish the relationship(s) between genetic (recombinational) distances and physical distances. There are indications that significant differences occur between physical and genetic distances as determined by recombinational map units (Dooner et al. 1985; Dooner 1986; Singh and Shepherd 1984; Meagher et al. 1988; Gustafson et al. 1990a and b). The implications of these findings are at present unknown.

Analysis of several rice RFLP linkage groups have shown that a limited portion of the chromosomes' physical length is involved (Gustafson et al. 1990b). It is of considerable interest to geneticists and breeders to know how much of the physical length of a genome is covered by the presently existing linkage maps. As previously mentioned, recent in situ hybridization studies utilizing root-tip protoplasts have determined the physical location of linked RFLP probes in rice. However the physical mapping becomes extremely difficult when small and/or metacentric chromosomes, such as found in rice, are involved (Gustafson et al. 1990b; Gustafson and Dille, unpublished data).

Double in situ hybridizations are required when mapping a linkage group to metacentric chromosomes. One probe is mapped and the second probe is mapped relative to the first one. This time consuming process requires two separate in situ hybridizations, i.e., a single hybridization followed by a double hybridization. At present, the technique for mapping two probes at the same time is available for mapping from highly repeated to unique sequence probes.

Recently, fluorescence techniques have been developed that can detect low-copy hybridization sites (Lawrence et al. 1988; Spadoro et al. 1990). These techniques are capable of detecting DNA probes 5.3 kb and smaller with the use of image enhancement (Pinkel et al. 1986; Bhatt et al. 1988; Spadoro et al. 1990). However, fluorescence isothiocyanate (FITC) fluorescence techniques have not been very successful when detecting low-copy and unique-sequence probes in plants. We have adapted a technique from Lichter et al. (1988) which works well with highly and moderately repeated DNA probes from Secale. At the present time, the technique has worked only on mitotic chromosome preparations using highly- and moderately-repeated probes. The recent use of fluorochromes in labeling needs to be further explored. With this technique the possibility of labeling two probes at the same time with different colored fluorochromes holds promise for rapidly producing physical maps of DNA markers where two markers can be mapped one relative to the other simultaneously.

This approach would greatly increase the accuracy of physical mapping. At present, fluorochromes used to detect human DNA sequences work with probes that are either large in size, from 7.0 kb to cosmid-sized probes (Riethman et al. 1989) or with biotinylated deoxynucleotide oligomers (Moyzis et al. 1988; Meyne et al. 1990). Most probes of around 7 kb in size need to be image enhanced before they can be seen by the naked eye. We are not aware that anyone has detected a unique sequence DNA probe below 1.O kb in size using fluorochromes.

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