Research Notes 
83 
II. Cytogenetics
4. 
Characterization of parental genomes in a hybrid betwen 0. sativa L. and 0.
officinalis Wall ex Watt. through fluorescence in situ hybridization
M. ASGHAR’, D.S. Brar’, J.E. Hernandez2, N. Ohmido3 and G.S. KHUSH1
1) 
International Rice Research Institute, Manila, Philippines
2) 
University of the Philippines, Los Banos, Laguna, Philippines
3) 
Hokuriku National Agricultural Experiment Station, Joetsu, 943-0154 Japan
Flourescence in situ hybridization (FISH) is a useful method for characterization of parental genomes in wide hybrids, detection of alien introgression and in phylogenetic analysis. We applied FISH to identify parental chromosomes and to locate rDNA loci in F1 hybrid of an elite breeding line of Oryza sativa and 0. officinalis (accession 100896). Somatic chromosome preparations of parents and F1 hybrid were made using enzymatic maceration method. Total genomic DNA of 0. officinalis and 45S rDNA used as probe were labeled by random primer labeling technique with biotin-16-dUTP and digoxigenin-11-dUTP respectively under supplier’s instructions. In situ hybridization protocols as described by Fukui er al. (1994) were used with minor modifications. Post- hybridization involved stringent washing to the slides with 2 x SSC at 42°C and 0.1 x SSC at 60°C.
Somatic chromosome analysis showed that the two parents, 0. sativa and 0. officinalis as well as the F1 hybrid had 24 chromosomes. Following FISH and using total genomic
 
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Rice Genetics Newsletter Vol. 15

DNA of 0. officinalis as probe, parental chromosomes of officinalis could be distinguished in the F1 hybrid from the sativa chromosomes. The bluish-green fluorescein isothiocyanate (FITC) signal appeared on officinalis chromosomes when the hybrid cell was counter- stained by 4’-6-diamidino-2-phenylindole (DAPI) (Fig. 1). This is depicted more clearly when the same cell was exposed to blue light and only officinalis chromosomes were seen as green (Fig. 2). Hybridization with 45S rDNA probe, showed reddish fluorescent signal of rhodamine on two chromosomes of 0. officinalis and on one chromosome of 0. sativa (arrows in Fig. 1). Ohmido and Fukui (1997) also used multi-color FISH to identify simultaneously A genome-specific tandem repeat sequences and telomeric nucleotide sequences on rice prometaphase chromosomes. The results show that FISH can be conveniently used to differentiate parental chromosomes of sativa and officinalis in the F1 hybrid.

References

Fukui, K., N. Ohmido and G. S. Khush, 1994. Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization. Theor. Appl. Genet. 87: 893-899.

Ohmido, N. and K. Fukui, 1997. Visual verification of close disposition between rice A genome-specific DNA sequence (TrsA) and telomere sequence. Plant Mol. Biol. 35: 963-968.