A Database for Triticeae and Avena
The barley-wheat chromosome addition lines (AD-lines) were obtained
by Islam et al. (1981). Each of them contains one barley chromosome.
However, we know of only a few cases when they were used for the
localization of barley genes (see, for example, Bohlmann et al., 1988;
Belostotsky et al., 1989). This fact can be explained by the
difficulties in the growing and cytological control of AD-lines and some
technical problems. However, molecular markers are now widely used in
genome studies, and AD-lines are proving to be extremely useful for
Here we report the mapping of barley genes coding for ribosomal 5S-
RNA using AD-lines. These lines were kindly sent to us by Dr. Islam.
They were grown, with constant monitoring of karyotype and
reasonable fertility, at relatively high temperature and moderate
photoperiod. DNA was isolated from 7-8 days old shoots using 50 mM
Tris-Oh, 0.2 M EDTA, pH 9.0, 0.5% SDS, 200 microg/ml protease at 55 °C and
further purified using routine protocol. Barley genes coding for
ribosomal 5S-RNA were cloned earlier in our lab (Khvyrleva et al., 1988).
DNA was digested with BamHI or MvaI according to manufacturer's
instructions ("Ferment", Vilnius), purified, concentrated, and applied on
non-denaturing 5% polyacrylamide gel (Tris-borate buffer). After
electrophoresis DNA was transferred on the membrane "Hybond" (Amersham)
using the electro-blotting device Bio-Rad. DN was cross-linked to the
membrane by UV-treatment and hybridized with P32 -labelled probe. In
order to prepare probes, cloned fragments were cut out from the vector,
isolated by electrophoresis, and 50-100 ng were labelled using the random
primer procedure (Feinberg and Vogelstein, 1983).
The result of this experiment is shown on Fig. 1. We used here
the restriction enzyme MvaI which cuts the 5S RNA genes of barley and
most of 5S genes of wheat once per repeat and is not sensitive to
methylation. DNA from different barley cultivars gives a known pattern
(Khvyrleva et al., 1988); two major repeats, 301 and 450 bp long, are
seen in the lower part of the lanes. Their relative content in studied
cultivars varies significantly. Upper bands correspond to the repeats
which deviate from canonical sequence, and the shortest of them is 900
The wheat cultivar 'Chinese Spring' is a parental strain for AD-lines.
It gives two sets of bands corresponding to unit repeats grouping around
410 and 500 bp (Gerlach and Dyer, 1980). This fine heterogeneity of
wheat 5S repeat could not be shown earlier by agarose electrophoresis.
Fig. 1 shows clearly that only the AD-2 line contains the bands
301 and 900 bp corresponding to barley 5S genes. The 450 bp band of the
barley moved too close to wheat bands and is not seen at this resolution.
To check the identity of our AD-lines, the radioactive probe was
washed away from the filter and it was reprobed with a P32 labeled
fragment of the repeat coding for the major ribosomal RNAs cloned
earlier in our lab (Fig. 2) (Khvyrleva et al., 1987). Though it shows
some cross-hybridization with wheat DNA, there are barley-specific bands
seen only in the AD-lines 6 and 7. This experiment agrees well with the
known localization of barley ribosomal genes on the chromosomes 6 and 7
(Appels et al., 1980) and with the known fact of different organization
of ribosomal clusters in two nucleolar organizers (Appels and Moran,
Another way to show the presence of barley chromosomes in the AD-
lines is to hybridize their DNA with a barley-specific repeat kindly
donated to us by Dr. Metzlaff (Junghans and Metzlaff, 1988). The result
of this experiment is shown on Fig. 3. It proves the conservation of
barley chromosome in our stocks of AD-lines.
We conclude that the cluster of 5S genes is located on chromosome 2
of barley and that 5S genes are absent from chromosomes 1, 3, 4, 6, and
7. No statement can be made now concerning chromosome 5, because the
corresponding AD-line was not produced.
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1980. Molecular and chromosomal organization of DNA sequences coding
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Bohlmann, H., S. Clausen, S. Behnke, C. Giese, U. Reimann-Philipp, G.
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repeat units in the nucleus of wheat which contain 5S rRNA genes.
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