0. saliva L. x 0. australiensis Domin through genomic in situ hybridization

EM. ABBASI’, D.S. Brar1, A.L. CARPENA2, K. FUKUI3 and G.S. KHUSH’

In situ hybridization is useful technique to characterize parental genomes in wide hybrids and to detect introgressed segments and chromosomal rearrangements. Total genomic DNA has been used as a probe in in situ hybridization to detect the rye chromosomes in barley x rye hybrids and in discriminating closely related Triticeae species (Anamthawat-Jonsson et a!. 1990). We used genomic in situ hybridization (GISH) to characterize parental chromosomes in anther culture derived plant from the F1 of hybrid (0. sativa and 0. australiensis). This plant with 27 chromosomes may have originated from polyploid cells of the callus. Total genomic DNA of 0. australiensis was extracted from the young leaves according to the method of Dellaporta et a!. (1983). The DNA was digested with EcoRI and labelled with biotin-14-dATP. The protocol followed for in situ hybridization was that of Rayburn and Gill (1985) and Fukui et al. (1994) with minor modifications.

Genomic DNA of 0. australiensis was used as a probe in in situ hybridization of the somatic chromosomes of anther culture derived plant. The probe produced uniform labelling pattern over the entire length of all the 14 austra!iensis chromosomes (bluish- green) whereas 13 chromosomes of sative appeared light blue after Giemsa staining (Fig.

1). The australiensis chromosomes showing the hybridization signal appeared bluish- green under blue light excitation allowing the identification of all australiensis chromosomes whereas the sativa chromosomes appeared blue due to counterstaining with 4’-6diamidino-2-phenylindole (DAPI) (Fig. 2). The same cell showed green fluorescence of fluorescein isothiocyanate (FITC) only on the 14 chromosomes of 0. australiensis (Fig.

These results showed that the parental chromosomes of 0. sativa and 0. australiensis can be distinguished using GISH. The technique is being extended to detect the introgressed segments from 0. australiensis into the sativa genome.

References

Anamthawat-Jonsson, K., T. Schwarzacher, A.R. Leitch, M.D. Bennett and J.S. Heslop-Harrison, 1990. Discrimination between closely related triticeae species using genomic DNA as a probe. Theor. AppI. Genet.