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
The financial support from the Rockefeller Foundation is gratefully acknowledged.
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: