6. 
Molecular cytogenetic analysis of 0. saliva L. x 0. brachyantha A. Chev. et Roehr.
derivatives
EM. ABBASI’, D.S. Brar1, A.L. CARPENA2 and G.S. Khush1
1) 
International Rice Research Institute, Manila, Philippines
2) 
University of the Philippines, Los Banos, Laguna, Philippines
The genus Oryza is comprised of 24 species, having 2n = 24 or 48 chromosomes representing nine different genomes; AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ genomes. These have been classified into four species complexes sativa, officinalis, ridleyi, and meyeriana. However, 0. brachyantha, an African species is not included in any of these complexes (Vaughan 1989). Oryza brachyantha (2n = 24, FF genome) possesses useful genes for resistance to stemborer, leaffolder, bacterial blight, blast and whorl maggot. A series of hybrids have been produced through direct crosses and embryo rescue between elite breeding lines of rice and wild species, representing each of the nine genomes (Brar and Khush 1995). In addition, monosomic alien addition lines (MAALs) 2n = 25 and introgression lines have been produced from several cross-combinations of cultivated and wild species.
We used genomic in situ hybridization (GISH) to characterize parental chromosomes in the F1 hybrid of 0. sativa cv 1R56 and 0. brachyantha (accession 101232) and to identify extra chromosome in monosomic alien addition lines. The in situ hybridization protocol as given by Rayburn and Gill (1985) was followed with minor modifications. Total genomic DNA of 0. brachyantha was digested with EcoRI and labelled with biotin-l4- dATP and used as a probe. Somatic chromosome analysis showed 2n =24 in both parental species in 0. sativa and 0. brachyantha and 24 chromosomes in the F1 hybrid. Meiotic analysis showed regular formation of 12 at metaphase I in both the parents. On the other hand, the Fl hybrid showed limited chromosome pairing (0- 2 bivalents), between sativa and brachyantha chromosomes, average being 0.05 bivalents per cell. In majority of the cells, the Fl showed unpaired chromosomes as 24 univalents. At anaphase, unequal distribution of chromosomes was commonly observed.
After in situ hybridization, all the 12 chromosomes of 0. brachyantha could be identified from sativa chromosomes. The hybridization signal appeared dark brown due to probe hybridization and non labelled sativa chromosomes appeared light blue (Fig. 1-
1). In a similar experiment, two MAALs (MAAL 6 and MAAL 12) derived from 0. brachyantha were used for in situ hybridization. The extra chromosome of 0. brachyantha 
showed dark brown hybridization signal (arrow in Fig. 1-2) making it clearly distinguishable from the remaining 24 chromosomes of 0. sativa.
The results show that there is very limited homoeology between chromosomes of 0. sativa and 0. brachyantha. Aggarwal et al. (1977) also demonstrated through total gemonic DNA hybridization that the genomes of 0. sativa and 0. brachyantha are highly diverged. The chromosomes of parental species in Fl hybrid and extra chromosome in MAAL could be easily distinguished through GISH. The technique is being used to characterize introgression of alien chromosome segments from brachyantha into the sativa genome.
References
Aggarwal, R.K., D.S. Brar and G.S. Khush, 1997. Two new genomes in the Oryza complex identified on the basis of molecular divergence analysis using total genomic DNA hybridization. Mol. Gen. Genet. 252:
1-12.
Brar, D.S. and G.S. Khush, 1995. Wide hybridization for enhancing resistance to biotic and abiotic stresses in rainfed lowland rice. Pages 901-910. In: Proc. Intern. Rice Research Conference, IRRI, Manila, Philippines.
Rayburn, A.L. and B.S. Gill, 1985. Use of biotin-labeled probes to map specific DNA sequences on wheat chromosomes. J. Hered. 76: 76-81.
Vaughan, D.A., 1989. The genus Oryza L. current status of taxonomy. IRRI Research Paper Series No. 138. pp.21.