45. Mapping DNA sequences in rice using in situ hybridization

Perry GUSTAFSON1 and John E. DILLE2

1) USDA-ARS, Curtis Fall, University of Missouri, Columbia, Missouri 65211, USA

2) Winthrop-Biology, Oakland Ave., Rock Hill, South Carolina 29733, USA

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. 1990 a 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small and/or metacentric chromosomes, such as found in rice, are involved (Gustafson et al. 1990b; Gustafson and Dille, unpublished data).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 1.0 kb in size using fluorochromes.


Bhatt, B., J. Burns, D. Flannery, and J.O.D. McGee, 1988. Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic In situ hybridization. Nucleic Acids Res. 16: 3951-3961.

Dooner, H. K., E. Weck, S. Adams, E. Ralston, M. Favreau and J. English, 1985. A molecular genetic analysis of insertions in the bronze locus in maize. Mol. Gen. Genet, 200: 240-246.

Dooner, H. K., 1986. Genetic fine structure of the bronze locux in maize. Genetics 113: 1021-1038.

Gaul, J. G. and M. I. Pardue, 1969. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Scie. USA 63: 378-383.

Gustafson, J. P., E. Butler and C. L. McIntyre, 1990a. Physical mapping of low copy DNA sequences in rye, Secale cereale L. Proc. Nat. Acad. Sci., USA 87: 1899-1092.

Gustafson, J. P., C. L. Mclntyre and J. E. Dille, 1990b. Physical mapping rice restriction fragment length polymorphisms. Proc. 2nd Intl. Rice Genetics Symposium. Manila, Philippines. In press.

Lapitan, N.L.V., R. G. Sears, A. L. Rayburn and B. S. Gill, 1986. Wheat-rye translocations. J. Hered. 77: 415-419.

Lawrence, J. B., C. A. Villnave and R. H. Singer, 1988. Sensitive, high resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBU in a lymphoma line. Cell 52: 51-61.

Le, H. T., K. C. Armstrong and B. Miki, 1989. Detection of rye DNA in wheat-rye hybrids and wheat translocation stocks using total genomic DNA as a probe. Mol. Biol. Reporter, 7: 150-158.

Lichter, P., T. Cremer, J. Borden, L. Manuelidis and D. C. Ward, 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-234.

Meagher, R. B., M. D. McLean and J. Arnold, 1988. Recombination within a subclass of restriction fragment length polymorphisms may help link classical and molecular genetics. Genetics 120: 809-818.

McCouch, S. R., G. Kochert, Z. H. Yu, Z.Y. Wang, G. S. Khush, W. R. Coffman and S. D. Tanksley, 1988. Molecular mapping of rice chromosomes. Theor. Appl. Genet. 76: 815-829.

Mouras, A., M.W. Saul, S. Essad and I. E. Potrykus, 1987. Localization of in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer. Mol. Gen. Genet. 207: 204-209.

Moyzis, R. K., J. M. Buckingham, L. S. Cram, M. Dani, L. L. Deaven, M. D. Johns, J. Meyne, R. L. Ratliff and J. R. Wu, 1988. A highly conserved repetitious DNA sequence, (TTAGGG)n, present in telomeres of human chromosomes. Proc. Natl. Acad. Sci., USA 85: 6622-6626.

Pinkel, D. T., T. Straume and J.W. Gray, 1986. Cytogenetic analysis using quantitative, high sensitivity, fluorescene hybridization. Proc. Natl. Acad. Sci., USA 85: 2934-2938.

Rayburn, A. L. and B. S. Gill, 1985. Use of biotin-labelled probes to map specific DNA sequence on wheat chromosomes. J. Hered. 76: 78-81.

Riethman, H. C., R. K. Moyzis, J. Meyne, D. T. Burke and M.V. Olsen, 1989. Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using yeast artiflcial chromosome vector. Proc. Natl. Acad. Sci., USA 86: 6240-6244.

Sharp, P. J., S. Chao, S. Desai and M. D. Gale, 1989. The isolation, characterization and application in the Triticaese of a set of wheat RFLP probes identifying each homoelogous chromosome arm. Theor. Appl. Genet. 78: 342-348.

Shin, J. S., S. Chao, L. Corpuz and T. Blake, 1990. A partial map of the barley genome incorporating restriction fragment length polymorphism, polymerase chain reaction, isozyme, and morphological marker loci. Genome 33: 803-810.

Singh, N. K. and K.W. Shepherd, 1984. Mapping of the genes by controlling high molecular weight glutelin subunits in rye on the long arm of chromosome 1R. Genet. Res. 44: 117-123.

Spadoro, J. P., H. Payne, Y. Lee and J. J. Resenstraus, 1990. Single copies of HIV proviral DNA detected by fluorescent in situ hybridization. Biotechniques 9: 186-195.

Tanksley, S. D., J. Miller, A. Paterson and R. Bernastsky, 1987. Molecular mapping of plant chromosomes. In : Chromosome Structure and Function. Eds. J. P. Gustafson and R. Appels. Plenum Press, N.Y. pp. 157-173.