Genomic in situ hybridization (GISH) involves use of total genomic DNA as a probe in hybridization with the target chromosomes on the glass slide. It is a powerful tool to characterize parental chromosomes in interspecific hybrids, detect homoeologous pairing and alien segment introgression into the genome of cultivated species. Fukui et al. (1997) used GISH to identify the genome constitution in Oryza. The technique has been used to discriminate genomes and detect autosyndetic and allosyndetic pairing in an F₁ hybrid between O. sativa (AA) and O. australiensis (EE) (Abbasi et al. 1999) and visualize O. eichingeri (CC) chromosomes in O. sativa x O. eichingeri F₁ (Yan et al. 1999). However, GISH has not been used on characterization of meiotic chromosomes in hybrids of O. sativa (2n = 24, AA) x O. ridleyi (2n = 48, HHJJ) and their derivatives.

We used GISH to identify the O. ridleyi chromosomes in monosomic alien addition lines (MAALs) (2n+1) and double monosomic alien addition lines (2n+1+1) of rice. Total genomic DNA of O. ridleyi was extracted from the young leaves following the CTAB method. The DNA was labeled with biotin-14-dATP by nick translation. Meiotic chromosome preparations were made from anther squashes in 45% glacial acetic acid. Slides were kept in -80C for 2 hours or until use. The cover slip was removed, and the slide was air dried and passed through an aqueous ethanol series (70%, 95%, and 100% ethanol). Later, slides were treated with an enzyme mixture (0.75% Cellulase Onuzuka RS, 0.5% Pectolyase Y 23) at 37C for 30 min., washed with water and again passed through an ethanol series (5 min each step) and stored at 4C until use. The chromosome spreads were fixed in 1% formaldehyde in 1X PBS

prior to GISH.

We followed the in situ hybridization protocol as described by Fukui et al (1994) with minor modifications. These modifications included using 120 ng of O. ridleyi biotin-labeled DNA as probe for each slide in a hybridization mixture containing 50% formamide, 10% dextran sulfate, 2X SSC and 1X PBS. Slides were incubated overnight with 13 micro l of hybridization mixture. Post-hybridization involved stringent washing of slides with 10% formamide/0.1 X SSC and 0.1 X SSC at 42C for 15 min. each. The labeled probes were detected with 1% Avidin-Fluorescein in 4X SSC (Vector Lab.). Propidium iodide was used for counterstaining of chromosomes in combination with Vectashield (Vector Lab.)

The labeled total genomic DNA of O. ridleyi was used as probe in hybridization with meiotic chromosomes of MAALs (2n = 25) and double monosomic alien addition lines (2n = 26). GISH analysis showed that the line WHD IS 2706-3 has 2n = 25 chromosomes with 12 bivalents and 1 univalent at pachytene, diakinesis and metaphase stages (Fig. 1A and 1B). The labeled chromosome detected with Avidin-Fluorescein was yellow, while the unlabeled chromosomes were red due to counterstaining with propidium iodide. The extra chromosome (yellow) from O. ridleyi could be clearly distinguished from non-labeled rice chromosomes. The ridleyi chromatin could also be identified at interphase where it showed as a yellow signal. Another line WHD IS 2423-11 showed 12 bivalents and 2 univalents (2n = 26) at pachytene and at diakinesis and two separate signals in interphase cells. The two univalents fluorescing yellow originated from O. ridleyi (Fig. 1C and 1D). It was interesting to note that the labeled chromosomes of O. ridleyi could be identified easily without the use of blocking DNA of the recurrent rice parent, indicating strong divergence between A and HJ genomes. We are extending this technique to identify homoeologous pairing among different genomes of Oryza using GISH and centromere specific clones.

Acknowledgement

The financial support from the Rockefeller Foundation is gratefully acknowledged.

References

Abbasi, F.M., D.S. Brar, A.L. Carpena, K. Fukui and G.S. Khush, 1999. Detection of autosyndetic and allosyndetic pairing among A and E genomes of Oryza through genomic in situ hybridization. Rice Genet. Newsl. 16: 24-25.

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.

Fukui, K., R. Shishido, T. Kinoshita, 1997. Identification of the rice D-genome chromosomes by genomic in situ hybridization. Theor. Appl. Genet. 95: 1239-1245.

Yan, H., S. Min and L. Zhu, 1999. Visualization of Oryza eichingeri chromosomes in intergenomic hybrid plants from O. sativa x O. eichingeri via fluorescence in situ hybridization. Genome 42: 48-51.