# Barley Genetics Newsletter (2005) 35:27-35

![](media/image1.jpeg){width="4.5625in" height="0.4270833333333333in"}

**Adaptive methylation pattern of ribosomal DNA in wild barley from
Israel**

> Shailendra Sharma^1^, Harindra Singh Balyan^2^ and Pushpendra Kumar
> Gupta^2\*^

^1^Dept. of Fingerprinting, College of Biotechnology, SVBP University of
Agriculture and Technology, Meerut-250 110, India; ^2^Dept. of Genetics
and Plant Breeding, Charan Singh University, Meerut-250 004, India;
\*corresponding author, e-mail: pkgupta36@ vsnl.com, Telefax:
+91-121-2768195

**Abstract**

Forty four accessions of wild barley, *H. spontaneum* were assayed to
study the methylation status of ribosomal DNA repeat units. For this
purpose, *Bam*HI and *Hpa*II, which are, methylation sensitive
restriction enzymes and *Msp*I, which is methyl insensitive enzyme, were
used for restriction digestion. Southern blots were hybridized with
pTa71 probe, which represented a complete ribosomal DNA repeat unit from
bread wheat. Wild barley material studied belonged to two ecologically
diverse climatic and edaphic microsites (the "Evolution Canyon" at Lower
Nahal Oren, Mount Carmel, and Tabigha, Eastern Upper Galilee Mountains),
from Israel, each having two different ecogeographcally contrasting
microniches. *Hpa*II did not cleave ribosomal DNA, while *Msp*I gave a
pattern typical of ribosomal DNA. In contrast, a total of 23 *Bam*HI
phenotypes were observed due to methylation. Same rDNA repeat length
units exhibited different methylation status at different microsites and
miocroniches, as inferred from RFLPs observed. Differential methylation
status of same rDNA repeat unit seems to be associated with ecological
conditions dominating at a particular microsite or microniche suggesting
a role of natural selection in determining methylation patterns.

**Keywords:** *Hordeum spontaneum* - Intergenic Spacer - Methylation -
Natural selection - rDNA

# Introduction 

Eukaryotic ribosomal RNA genes (known as ribosomal DNA or rDNA), that
encode 18S, 5.8S and 26S ribosomal RNAs (rRNAs), are found as parts of
repeat units that are arranged as tandem arrays, located at the
chromosomal sites known as nucleolar organizing regions (NORs) (Long and
Dawid, 1980; Jorgensen and Cluster, 1988). In barley, there are two
pairs of satellited chromosomes (6 or 6H and 7 or 5H), each carrying one
rDNA locus (*Rrn*1 on chromosome 6H and *Rrn*2 on chromosome 5H) that is
associated with the corresponding NOR (Saghai-Maroof *et al*., 1984;
Brown *et al*., 1999). Each rDNA repeat unit consists of a highly
conserved coding region (for 18S, 5.8S and 26S rRNAs) and a variable
non-coding intergenic spacer (IGS) region. IGS itself consists of a
non-transcribed spacer (NTS) region, which contains motifs referred to
as subrepeats, and is itself flanked by external transcribed spacers
(ETS) at its two ends. In the coding region also, on either side of 5.8S
rRNA gene, are found internal transcribed spacers (ITS), described as
ITS1 and ITS2.

Several studies suggest that natural selection is the major force that
directs differentiation at the level of rDNA repeat unit (Flavell *et
al*., 1986; Saghai Maroof *et al*., 1984, 1990; Gupta *et al*., 2002;
Sharma *et al*., 2004). This differentiation may provide genotypes with
variable ecological adaptations. Wild barley, *Hordeum spontaneum*, from
fertile crescent region has been shown to be rich in genetic diversity
and this genetic diversity is adaptive in nature (Balyan *et al*., 1996;
Nevo *et al*., 1998; Gupta *et al*., 2002; Owuor *et al*., 1997; Sharma
*et al*., 2004). In *Hordeum* species, besides restriction enzymes like
*Sac*I, *Bam*HI has been used to study polymorphism in rDNA repeat
length unit (Molnar *et al*., 1989; Gupta, 1996; Gupta *et al*., 2002).
Molnar *et a*l., (1989) reported five *Bam*HI restriction site maps for
25 different *Hordeum* species and suggested grouping of different
species on the basis of these maps.

The present study was undertaken to analyze the methylation patterns of
rDNA in barley, and to study the role of natural selection in
determining the current methylation status of rDNA, which could be
partly adaptive.

## 

## Materials and methods

# *Plant materials*

#  The material for the present study comprised 44 accessions of wild barley (*H. spontaneum*). Seed material was originally supplied by E. Nevo of University of Haifa, Israel and was multiplied at the Research Farm of Ch. Charan Singh University, Meerut, India. The 44 accessions of wild barley were collected from two different microsites in Israel. Twenty two (22) accessions were collected from "Evolution Canyon", out of which 9 belonged to NFS (North Facing Slope) and another 13 samples belonged to SFS (South Facing Slope). The remaining 22 accessions were collected from 'Tabigha' out of which 12 were collected from 'terra rossa' microniche and 10 were collected from 'basalt'. microniche. The two microsites are separated by 53 km. The details of two ecological microsites and those of microniches at each microsite are available elsewhere (Gupta *et al*., 2002).

# 

## *DNA extraction and purification* 

##  Total cellular DNA, from one to two plants per accession, was isolated from one month old, field-grown individual plants using the modified CTAB method of Saghai-Maroof *et al*., (1984). The isolated DNAs were further purified by RNaseA treatment and phenol: chloroform: isoamyl alcohol following Sambrook *et al*., (1989). 

## 

## *Restriction enzyme digestion, Southern blotting, hybridization and autoradiography* 

##  Appropriate amounts (\~10μg) of purified DNA samples were digested separately, with restriction enzymes *Bam*HI, *Hpa*II and *Msp*I according to manufacturer's instructions (Amersham, UK) (Figs. 1 and 2). Southern blotting, hybridization and autoradiography were performed as described earlier (Gupta *et al*., 2002).

#### 

**Results and Discussion**

*Methylation pattern in the rDNA of wild barley*

For the study of methylation patterns in rDNA, the genomic DNA from 44
accessions of wild barley, *H.* *spontaneum*, was restricted separately
with each of the three restriction enzymes including *Hpa*II (sensitive
to CG methylation) and *Msp*I (insensitive to CG methylation), a
isochizomeric pair, and *Bam*HI, a methylation sensitive enzyme. Both
*Hpa*II and *Msp*I cleave the DNA sequence CCGG. However, *Hpa*II fails
to cleave the rDNA, if the internal cytosine residue (CG) at the
restriction site (CCGG) is methylated. In each of the above accessions,
*Msp*I digestion and Southern hybridization with rDNA probe pTA71,
showed many bands of varying sizes whereas similar experiments using
*Hpa*II showed only uncut DNA in the autoradiogram (Fig. 1). This means
that in wild barley the CCGG restriction site of *Hpa*II/*Msp*I in rDNA
and consequently the rDNA repeat units are heavily methylated.

The restriction enzyme *Bam*HI that cleaves the rDNA at restriction site
GGATC is also sensitive to cytosine methylation. In an individual rDNA
repeat unit of barley, the *Bam*HI restriction enzyme has three (Molnar
*et al*., 1989, Gupta *et al*., 1996) to four (Appels *et al*., 1980)
cleavage sites. Two of the four cleavage sites are located in the IGS
region and one of these two sites show considerable heterogeneity, which
could be due to sequence alteration or to methylation. Of the remaining
two sites, one each lies in 18S and 26S coding regions (Appels *et al.*,
1980, Gupta *et al*., 1996). However, the cleavage site in 26S coding
region is presumed to be methylated (Appels *et al*., 1980, Gupta *et
al*., 1996). In the present study, results obtained due to *Bam*HI
digestion only were utilized for the study of methylation pattern of
rDNA in above 44

accessions of wild barley. The digestion of DNA with *Bam*HI followed by
Southern hybridization with rDNA probe pTA71 gave 23 different banding
patterns each of which included 5 to 9 bands of variable sizes. These 23
banding patterns were classified as 23 (I to XXIII) different *Bam*HI
phenotypes (Figure 2; Table 1).

*Methylation status of rDNA repeat units*

It may be recalled that in the same set of 44 wild barley accessions
that were used for the study of methylation, *Sac*I restriction enzyme
provided evidence of one to three rDNA repeat unit length variants at
two rDNA loci in an individual accessions (Table 1; Gupta *et al*.,
2002). Assuming four *Bam*HI cleavage sites in a single rDNA repeat unit
(Appels *et al*., 1980), a maximum of four, seven and 10 fragments are
expected following digestion of rDNA with *Bam*HI in wild barley
accessions having one, two and three rDNA repeat unit length variants
(having one common fragment in each case). However, on digestion with
*Bam*HI during the present study, the individual wild barley accessions
with one, two and three rDNA repeat units respectively gave 5 to 7, 6 to
9 and 8 to 9 *Bam*HI fragments of varying sizes. The availability of the
varying number of *Bam*HI fragments suggested heterogeneity for *Bam*HI
sites in rDNA repeat unit(s) of the individual wild barley accessions
examined during the present study. As earlier reported, the observed
heterogeneity for *Bam*HI sites may be attributed to the alteration in
sequences/methylation of *Bam*HI sites located in IGS region, partial
methylation of *Bam*HI sites and the methylation of the *Bam*HI site
located in 26S rDNA (Appels *et al*., 1980, Molnar *et al*., 1989, Gupta
*et al*., 1996).

*Possible role of ecogeographic factors in governing methylation of
rDNA*

Interestingly, out of the above 23 *Bam*HI phenotypes, 21 (91.31%)
phenotypes showed microsite specific distribution and only 2 (8.69%)
*Bam*HI phenotypes were shared by the two microsites i.e. 'Evolution
Canyon' and Tabigha, separated by 53 km. Similarly, at the 'Evolution
Canyon' microsite, out of nine exclusive phenotypes, 5 phenotypes
(55.55%) exclusively belonged to NFS (cooler more humid representing
south European and Mediterranean dense Macquis live oak forests) and
three phenotypes (33.33%) belonged to SFS (drier and much warmer
representing African Savannah) and only one (11.11%) phenotype was
common between the two microniches. At Tabigha edaphic microsite, out of
12 exclusive phenotypes, six phenotypes (50%) belonged to terra rossa
(drier and shallow soil layer) and five phenotypes (41.66%) belonged to
basalt (humid and flat soil type) microniches with one (8.33%) phenotype
common between the two microniches. This suggests that the distribution
of *Bam*HI phenotypes is not random, and has a definite relationship
with the climatic/edaphic factors at the two microsites/microniches.
Since a particular *Bam*HI phenotype is dependent on the alteration of
sequences/methylation of *Bam*HI cleavage sites, the ecogeographical
factors might have played an important role in determining
microsite/microniche specific patterns of rDNA methylation. This is in
agreement with the previous studies on several plant species (*Nicotiana
tabacum, Arabidopsis thaliana,* etc.) showing alteration of cytosine
methylation patterns due to environmental conditions/stress (Burn *et
al*., 1993; Schmitt *et al*., 1997; Riddle and Richards, 2002).

*Methylation at Nor loci and rDNA gene expression*

In most eukaryotes, rRNA genes are found in multiple copies and only a
subset of these genes is expressed in most cells (Conconi *et al.*,
1989; Dammann *et al*., 1993). These studies showed that very active
loci have a higher proportion of rRNA genes with demethylated cytosine
residues compared to less active loci (Doerfler, 1983; Cedar, 1988,
Flavell *et al*., 1988; Riddle and Richards, 2002). The length of IGS
region has also been correlated with methylation status of cytosine
residues in wheat (Sardana *et al*., 1993). For instance, longer
intergenic spacers have more unmethylated CCGG sites than shorter
intergenic spacers. Since most of the polymorphic bands contributing to
microsite specific *Bam*HI phenotypes involved major part of IGS region,
the possible role of selection at the IGS regions is speculated. It has
been noticed in studies involving both plant and animal species that IGS
region contains some regulatory sequences that play an important role in
the expression of neighboring rRNA genes (Doerfler, 1983; Cedar, 1988;
Sardana *et al*., 1993). In the present study also, microsite/microniche
specific *Bam*HI phenotypes suggests that natural selection plays an
important role in the methylation of nucleotides of the IGS and,
therefore, in the switching on or off of rDNA genes expression.
Moreover, the results indicate that the two rDNA loci differ in their
response towards methylation, which can be attributed to selection
forces regulating the sequence activity at spacer region. This
differential behavior of two loci towards methylation could be related
with one or more important traits. In the past also, polymorphism at
*Rrn2* locus was shown to be associated with water sensitivity (Powell
et al. 1991). Our earlier studies also suggested a role of
ecogeographical factors in the differentiation of rDNA in wild barley
(Gupta *et al*., 2002; Sharma *et al*., 2004). However critical
experiments, like sequence analysis of different IGS regions, are still
needed to verify this.

# 

# Acknowledgements

The work was done under the tenure of a Emeritus Scientist project of
Council of Scientific and Industrial Research (CSIR), New Delhi, India.
PKG is currently working as a Senior Scientist of Indian National
Science Academy (INSA), New Delhi. Thanks are also due to Prof. E. Nevo
of University of Haifa, Israel for supply of wild barley seed material.

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![](media/image2.png)

**Fig. 1** Representative autoradiogram showing digestion pattern
obtained using isochizomeric pair of *Msp*I and *Hpa*II restriction
enzymes. Lane A: *Msp*I showed large scale digestion depicted by number
of bands from top to bottom of lane. Lane B: *Hpa*II gave only single
dense band at the top of lane depicting undigested DNA. M represents λ
DNA/*Eco*RI+*Hin*dIII marker.

![](media/image3.png){width="5.393055555555556in"
height="4.131944444444445in"}

**Fig. 2** Representative autoradiogram showing 11 different phenotypes
obtained after *Bam*HI digestion.

<table>
<colgroup>
<col style="width: 9%" />
<col style="width: 5%" />
<col style="width: 33%" />
<col style="width: 7%" />
<col style="width: 8%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 9%" />
<col style="width: 6%" />
<col style="width: 5%" />
</colgroup>
<tbody>
<tr class="odd">
<td colspan="10"><strong>Table 1.</strong> Details of phenotypes and
number of bands with their sizes obtained after <em>Bam</em>HI
digestion</td>
</tr>
<tr class="even">
<td rowspan="2"><em>Bam</em>HI phenotype</td>
<td rowspan="2">TNB</td>
<td rowspan="2">Size of <em>Bam</em>HI bands (bp)</td>
<td colspan="4"><blockquote>
<p>No. of accessions &amp; relative frequency</p>
</blockquote></td>
<td colspan="3">Phenotypes common at:</td>
</tr>
<tr class="odd">
<td colspan="2"><blockquote>
<p>Evolution Canyon</p>
</blockquote></td>
<td colspan="2"><blockquote>
<p>Tabigha</p>
</blockquote></td>
<td>Microsites</td>
<td colspan="2">Niches</td>
</tr>
<tr class="even">
<td></td>
<td></td>
<td></td>
<td>NFS</td>
<td>SFS</td>
<td>TR</td>
<td>B</td>
<td></td>
<td>Slopes</td>
<td>Soils</td>
</tr>
<tr class="odd">
<td>I</td>
<td>5</td>
<td>9540, 7740, 5640, 3900, 1800</td>
<td>1 (0.1)</td>
<td><strong>-</strong></td>
<td>-</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>II</td>
<td>5</td>
<td>9425, 7625, 5525, 3900, 1800</td>
<td>3 (0.3)</td>
<td>1 (0.07)</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>-</td>
<td>+</td>
<td>-</td>
</tr>
<tr class="odd">
<td>III</td>
<td>7</td>
<td>9425, 7625, 5525, 3900, 3125, 2400, 1800</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>1 (0.09)</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>IV</td>
<td>7</td>
<td>9655, 7855, 5755, 3900, 3255, 2500, 1800</td>
<td>-</td>
<td>-</td>
<td>2 (0.15)</td>
<td>2 (0.18)</td>
<td>-</td>
<td>-</td>
<td>+</td>
</tr>
<tr class="odd">
<td>V</td>
<td>6</td>
<td>9310, 7510, 5410, 3990, 3610, 1800</td>
<td>-</td>
<td>3 (0.23)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>VI</td>
<td>6</td>
<td>9195, 7395, 5295, 3900, 3495, 1800</td>
<td>-</td>
<td>3 (0.23)</td>
<td>-</td>
<td>1 (0.09)</td>
<td>+</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>VII</td>
<td>6</td>
<td>9310, 7510, 5180, 3900, 3380, 1800</td>
<td>-</td>
<td>2 (0.15)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>VIII</td>
<td>6</td>
<td>9080, 7855, 5180, 3900, 3380, 1800</td>
<td>-</td>
<td>4 (0.30)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>IX</td>
<td>6</td>
<td>9310, 7510, 5755, 3955, 3900, 1800</td>
<td>1 (0.1)</td>
<td>-</td>
<td>-</td>
<td>1 (0.09)</td>
<td>+</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>X</td>
<td>7</td>
<td>9310, 7625, 5525, 5410, 3955, 3900, 1800</td>
<td>-</td>
<td>-</td>
<td>2 (0.15)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XI</td>
<td>9</td>
<td>9383, 8085, 5985, 5483, 4185, 3900, 3083, 2400, 1800</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>2 (0.18)</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>XII</td>
<td>7</td>
<td>9383, 8085, 5483, 5985, 4185, 3900, 1800</td>
<td>1 (0.1)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XIII</td>
<td>8</td>
<td>9540, 8315, 7740, 5640, 6215, 4415, 3900,1800</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>1 (0.09)</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>XIV</td>
<td>7</td>
<td>9540, 8315, 6215, 5640, 4415, 3900, 1800</td>
<td>1 (0.1)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XV</td>
<td>7</td>
<td>9425, 7625, 6215, 5525, 4415, 3900, 1800</td>
<td>1 (0.1)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>XVI</td>
<td>6</td>
<td>9383, 7583, 6100, 4300, 3900, 1800</td>
<td>1 (0.1)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XVII</td>
<td>7</td>
<td>9383, 7970, 5870, 5483, 4070, 3900, 1800</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>1 (0.07)</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>XVIII</td>
<td>7</td>
<td>9383, 7583, 5775, 3900, 3255, 2500, 1800</td>
<td>-</td>
<td>-</td>
<td>4 (0.30)</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XIX</td>
<td>7</td>
<td>9195, 7395, 5525,3900, 3025, 2500, 1800</td>
<td>-</td>
<td>-</td>
<td>-</td>
<td>1 (0.09)</td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="even">
<td>XX</td>
<td>8</td>
<td>9540,7740, 5410, 3900, 3610, 2810, 2600, 1800</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>1 (0.07)</td>
<td><strong>-</strong></td>
<td>-</td>
<td>-</td>
<td>-</td>
</tr>
<tr class="odd">
<td>XX1</td>
<td>8</td>
<td>9310, 7855, 6215, 3900, 5755, 3955, 5410, 1800</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>1 (0.07)</td>
<td><strong>-</strong></td>
<td>-</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
</tr>
<tr class="even">
<td>XXII</td>
<td>9</td>
<td>9540, 8965, 7740, 6215, 5640, 5065, 4415, 3900, 1800</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td>1 (0.09)</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
</tr>
<tr class="odd">
<td>XXIII</td>
<td>9</td>
<td>9310, 7740, 5640, 5410, 4950, 3900, 3130, 2510, 1800</td>
<td>-</td>
<td>-</td>
<td>1 (0.07)</td>
<td>-</td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
<td><strong>-</strong></td>
</tr>
</tbody>
</table>

TNB= Total number of bands obtained from *Bam*HI digestion; NFS= North
facing slope; SFS= South facing slope; TR=Terra rossa; B=Basalt;

\+ indicates common phenotypes at microsites and microniches