A Database for Triticeae and Avena
H. bulbosum has been used extensively in crosses with cultivated
barley in order to either produce haploids for barley breeding (Kasha
and Kao, 1970) or to transfer some useful variations such as disease
resistance and winter hardiness into barley (Snape, et al., 1988).
Based upon cytological observations, several researchers have suggested
that suppression of nucleolus-organizing region (NOR) activity of H.
bulbosum chromosomes in the interspecific hybrids could be related to
chromosome elimination of H. bulbosum, giving rise to haploid
production. Recently, Xu and Snape (1988) have confirmed that the H.
bulbosum NOR is located on short arm of chromosome 6, based upon its
homoeologous pairing with chromosome 6 of H. vulgare. They suggested
that H. bulbosum chromosome 6 appears to be the first chromosome to be
eliminated in the interspecific hybrids. Thus, it would be beneficial
to obtain detailed information on NOR activity in H. bulbosum using in
In this study, a tetraploid H. bulbosum clone (GBC141, 2n=4x=28)
was used. The root tips were taken from plants and pretreated in ice-
water for 10 hours before being fixed in acetic alcohol (1:3) for 24
hours. A 1% aceto-carmine solution was used to stain root tips before
the preparations were made. pHbR26, a 4.3 kb region of the tandemly
repeated ribosomal genes isolated from tetraploid H. bulbosum DNA by
restriction with BAM H1 and cloned in the vector pBR322 (Kasha,
unpublished), was used as the probe for in situ hybridization
experiments (Fig. 1). The biotin labelling technique as described by
Gustafson et al. (1988) was followed with the modification that
incubation of preparations in a complex strapavidin-horseradish
peroxidase (SA-HRP) and in diaminobenzidine tetrahydrochloride (DAB) was
prolonged to 40-50 minutes and 3-4 hours, respectively.
The hybridization sites with the biotinylated probe were detected
clearly as dark spots against light green chromosome background (Fig. 2).
Non-homologous hybridization sites, which occur invariably with
radioactive labelling methods, were not observed. Therefore, the DNA
sequence could be physically mapped on the homologous chromosome region
with high precision. The increase of incubation time in SA-HRP and DAB
solution tended to enhance the hybridization signal without causing
Tetraploid H. bulbosum has 28 chromosomes, four of which normally
exhibit satellites in metaphase cells. We examined 20 metaphase cells
stained with aceto-carmine for the secondary constrictions (gaps between
the satellites and short arm of chromosome 6, Table 1). Although the
number of visible gaps varied from I to 4, which was most likely due
to the squashing procedures, most of cells (50%) showed 4 gaps as
expected. This suggests that ribosomal genes in the 4 NORs are active.
Strong in situ hybridization signals were observed at the NOR
region of all 4 satellite chromosomes as expected (Fig. 2). However,
the observation that no more than 2 of the 4 chromosomes exhibited the
gap in the biotin label of cells was unexpected. Ten cells with well-
spread metaphase chromosomes were analyzed for a gap in the label (Table
1). In eight cells, only two of four satellite chromosomes showed clear
gaps in the label. In the remaining two cells, only one of four
satellite chromosomes exhibited the gap. Gustafson et al. (1988)
interpreted the gaps expressed when using in situ hybridization as due
to the active state of the ribosomal genes, whereas no gap meant an
inactive state of the ribosomal genes, whereas no gap meant an inactive
state in rye and wheat. This interpretation does not appear to be
consistent with our observations. When stained with aceto-carmine, the
gaps are present on all four of the NOR chromosomes of the H. bulbosum
clone, indicating activity of rDNA genes. With in situ hybridization,
no more than 2 of the 4 chromosomes exhibit a gap in the label. Rather
than based upon activity or inactivity or NOR regions, we suggest an
alternative hypothesis to explain the in situ observations. The NOR
region consists of a large number (perhaps 2000-3000 in barley) of
tandemly repeated genes for ribosomal RNA. Only a portion of these
genes are normally expressed. If the portion being expressed which
leads to the gap was in the middle of the tandemly repeated gene
sequence, you would observe label on both sides of the gap. If the
portion being expressed was a terminal section of the repeated sequence,
you would not observe a gap in the in situ label. Our observations
suggest that the regional expressed may be predetermined or possibly
genetically controlled since the expression can be different for
different chromosomes within the same plant.
It has been reported in cereals (Thompson and Flavell, 1988) that
the ribosomal genes with a larger intergenic spacer region are
preferentially transcribed. While such variation could also exist
within a single plant of a cross-pollinated, self-incompatible species
like H. bulbosum, the observation of gaps on all four chromosomes when
stained with aceto-carmine makes this a less plausible explanation.
The authors wish to express their thanks to Miss Rosalinda Oro for
her kind assistance during the course of this study and to Miss Anne
Dera for providing biotin labelling procedures and helpful discussions.
Financial support from NSERC and OMAF are gratefully acknowledged.
Gustafson, T.P., A.R. Dera, and S. Petrovic. 1988. Expression of
modified rye ribosomal RNA gene in wheat. Proc. Natl. Acad. Sci. USA
Kasha, K.J., and K.N. Kao. 1970. High frequency haploid production in
barley (H. vulgare L.) Nature (Lond.) 225:874-876.
Snape, J.W., J. Xu, and B.B. Parker. 1988. Wide crossing and alien
gene transfer. In Annual Report 1988, AFRC Institute of Plant Science
Research and John Innes Institute. pp. 2-3.
Thompson, W.F., and R.B. Flavell. 1988. DNAse I sensitivity of
ribosomal RNA genes in chromatin and nucleolar dominance in wheat. J.
Mol. Biol. 204:535-548.
Xu, J., and J.W. Snape. 1988. The cytology of hybrids between H.
vulgare and H. bulbosum revisited. Genome 30:486-494.
Xu, J., and J.W. Snape. 1989. The resistance of H. bulbosum and its
hybrids with H. vulgare to common fungal pathogens. Euphytica (in