Oryza through genomic in situ hybridization

F.M. ABBASI’, D.S. Brar1, A.L. CARPENA2, K. FUKUI3 and G.S. Khush1

     Genomic in situ hybridization (GISH) is becoming an important technique to characterize parental genomes in wide hybrids and to detect introgressed segments and chromosome rearrangements. Total genomic DNA as a probe in in situ hybridization has been used to detect parental chromosomes in wheat-rye hybrids and in discriminating closely related Triliceae species (Anamthawat-Jonsson et al. 1990). in situ hybridization using genomic probes has been demonstrated to be an effective technique for the detection of meiotic pairing between wheat-rye (Begona et a!. 1995). However, GISH has not been used to characterize meiotic pairing in Oryza. Detection of autosyndetic and allosyndetic pairing in wide hybrids is difficult through conventional meiotic analysis. We used GISH to characterize meiotic pairing in F1 hybrids of 0. sativa (2n = 24 AA genome) x 0. australiensis (2n = 24 EE genome). Total genomic DNA of 0. australiensis was extracted from the young leaves according to the method of Dellaporta et at. (1983). The DNA was digested with EcoRI and labeled with biotin-14-dATP by nick translation method, in situ hybridization protocol as described by Fukui et a!. (1994) was used on meiotic chromosomes with minor modifications.

     GISH analysis showed that 12 out of 24 chromosomes fluoresced yellow (australiensis chromosomes) and other 12 chromosomes appeared red (sativa chromosomes) upon counter staining with propidium iodide (Fig. 1). Meiotic analysis using GISH was carried out on 20 metaphase cells. Two types of pairing was observed (1) between A and E genome chromosomes and (2) Within A genome chromosomes. Of the 20 cells examined through GISH, 12 cells lacked pairing, while in other cells both autosyndetic and allosyndetic pairing was observed. GISH revealed 1-2 bivalents resulting from pairing among A genome chromosomes and allosyndetic pairing involving 1 to 4 homoeologous chromosomes (A and E genomes). None of the australiensis chromosomes showed pairing among themselves. The autosyndetic and allosyndetic pairing of chromosomes resulted into rod shaped bivalents. The GISH technique used here would be extended to detect pairing among A genome and other distantly related genomes of Oryza at pachytene to precisely understand the process of alien introgression particularly of small chromosome segments.