M. N. ISLAM-FARIDI, T. IsHii, V. KUMAR, L. A. SITCH and D. S. BRAR
International Rice Research institute, P. O. Box 933, Manila, Philippines
In situ hybridization is an important cytological technique to locate gene(s) on chromosomes. This technique has been used extensively in mammals using radioactively labelled DNA probes and also in some plant species. During the last few years, non-radioactive labelling of DNA has become more attractive. In non-radioactive method, the results can be obtained on the same day or following day, while the radioactive method requires a few to several weeks exposure for detection of hybridization signal (Mouras et al. 1987). In addition, the use of non-radioactive in situ hybridization method allows better resolution of the signal (Ambros et al. 1986).
Rayburn and Gill (1985) used the non-radioactive in situ hybridization technique in wheat. Since then this method has also been used in maize, barley and rye. We used this technique in rice using biotin labelled rDNA probe.
The mitotic chromosome preparations were made from root tip cells digested with cellulase and pectolyase enzymes (Islam-Faridi and Sitch 1989). The 7.8 kb rDNA probe (a kind gift from Dr. F. Takaiwa, National Institute of Agrobiological Resources, Japan) was labelled with biotin-11-dUTP (Bethesda Research Laboratories, BRL) by nick translation (BRL kit) method. The protocols for in situ hybridization and enzyme detection were same as described by Rayburn and Gill (1985).
The somatic chromosome preparations of IR36 were used in in situ hybridization using biotin labeled rDNA probe. Four of the 24 somatic chromosomes showed hybridization signals (Fig. 1). Indica rice var. IR36 has two nucleolus organizer regions, on chromosomes 9 and 10 (Khush et al. 1984). Hybridization of the rDNA probe with somatic chromosomes of primary trisomics 9 and 10 showed hybridization signal on five (2 homologous pairs and one extra chromosome) of the 25 chromosomes (Fig. 2), indicating that ribosomal RNA genes are located on chromosomes 9 and 10 of rice. The results demonstrate the usefulness of in situ hybridization for cytological location of genes on rice chromosomes.
Fig. 1. Four chromosomes of IR36 showing hybridization signal of rDNA (arrowed).
Fig. 2. Five chromosomes of IR36 (trisomic for chromosome 9) showing hybridization signal of rDNA (arrowed).
Ambros, P. F; M. A. Matzke and A.J.M. Matzke, 1986. Detection of a 17kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94: 11-18.
Istam-Faridi, M. N. and L. A. Sitch, 1989. A rapid, reliable method for preparing somatic chromosomes of rice. RGN 6: 176-177.
Khush, G. S; R. J. Singh, S. C. Sur and A. L. Librojo, 1986. Primary trisomics of rice: origin, morphology, cytology and use in linkage mapping. Genetics 107: 141-163.
Mouras, A., M. W. Saul, S. Essad and I. Potrykus, 1987. Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer. Mol. Gen. Genet. 207: 204-209.
Rayburn, A.L. and B.S. Gill, 1985. Use of biotin-labelled probes to map specific sequences on wheat chromosomes. J. Hered. 76: 78-81.