Barley Genetics Newsletter (2005)  35:27-35

Adaptive methylation pattern of ribosomal DNA in wild barley from Israel

Shailendra Sharma¹, Harindra Singh Balyan²and Pushpendra Kumar Gupta^2*

¹Dept. of Fingerprinting, College of Biotechnology, SVBP University of Agriculture and   Technology, Meerut-250 110, India; ²Dept. 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, BamHI and HpaII, which are, methylation sensitive restriction enzymes and MspI, 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. HpaII did not cleave ribosomal DNA, while MspI gave a pattern typical of ribosomal DNA. In contrast, a total of 23 BamHI 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 (Rrn1 on chromosome 6H and Rrn2 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 SacI, BamHI has been used to study polymorphism in rDNA repeat length unit (Molnar et al., 1989; Gupta, 1996; Gupta et al., 2002). Molnar et al., (1989) reported five BamHI 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 BamHI, HpaII and MspI 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 HpaII (sensitive to CG methylation) and MspI (insensitive to CG methylation), a isochizomeric pair, and BamHI, a methylation sensitive enzyme. Both HpaII and MspI cleave the DNA sequence CCGG. However, HpaII fails to cleave the rDNA, if the internal cytosine residue (CG) at the restriction site (CCGG) is methylated. In each of the above accessions, MspI digestion and Southern hybridization with rDNA probe pTA71, showed many bands of varying sizes whereas similar experiments using HpaII showed only uncut DNA in the autoradiogram (Fig. 1).  This means that in wild barley the CCGG restriction site of HpaII/MspI in rDNA and consequently the rDNA repeat units are heavily methylated.

The restriction enzyme BamHI that cleaves the rDNA at restriction site GGATC is also sensitive to cytosine methylation. In an individual rDNA repeat unit of barley, the BamHI 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 BamHI digestion only were utilized for the study of methylation pattern of rDNA in above 44

accessions of wild barley. The digestion of DNA with BamHI 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 BamHI 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, SacI 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 BamHI 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 BamHI 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 BamHI 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 BamHI fragments of varying sizes. The availability of the varying number of BamHI fragments suggested heterogeneity for BamHI sites in rDNA repeat unit(s) of the individual wild barley accessions examined during the present study. As earlier reported, the observed heterogeneity for BamHI sites may be attributed to the alteration in sequences/methylation of BamHI sites located in IGS region, partial methylation of BamHI sites and the methylation of the BamHI 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 BamHI phenotypes, 21 (91.31%) phenotypes showed microsite specific distribution and only 2 (8.69%) BamHI 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 BamHI phenotypes is not random, and has a definite relationship with the climatic/edaphic factors at the two microsites/microniches. Since a particular BamHI phenotype is dependent on the alteration of sequences/methylation of BamHI 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 studieson 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 BamHI 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 BamHI 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 theirresponse 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.

References

Appels, R., Gerlach, W.L., Dennis, E.S., Swift, H. and Peacock, W.J. (1980) Molecular and chrosomal organization of DNA sequences coding for the ribosomal RNAs in cereals. Chromosoma 78:293-311.

Balyan, H.S., Jana, S. and Selvaraj, G. (1996) Polymorphism, ecogeographical differentiation and adaptation of ribosomal DNA (rDNA) alleles in wild and cultivated barley. In: Chopra VL, Sharma RP, Swaminathan MS (eds) Agricultural biotechnology, 2nd Asia Pacific conference. Oxford & IBH Publishing Co Pvt Ltd, New Delhi, India, pp 39-49.

Brown, S.E., Stephens, J.L., Lapitan, N.L.V. and Knudson, D.L. (1999) FISH landmarks for barley chromosomes (Hordeum vulgare L.). Genome 42:274-281.

Burn, J.E.D., Bagnali, J.D., Metzger, E.S., Dennis and Peacock W.J (1993) DNA meth-ylation, vernalization and the initiation of flowering. Proc. Natl. Acad. Sci. USA 90:287-291.

Cedar, H. (1988) DNA methylation and gene activity. Cell 53:3-4.

Conconi, A., Widmer, R..M., Koller, T. and Sogo, J.M. (1989). Two different chromatin structures coexist in ribosomal genes throughout the cell cycle. Cell 57:753-761.

Dammann, R., Lucchini, R., Koller, T. and Sogo, J.M. (1993). Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae. Nucleic Acids Res. 21:2331-2338.

Doerfler, W. (1983) DNA methylation and gene activity. Annu Rev Biochem 52:93-124.

Flavell, R.B., O’Dell, M., Sharp, P., Nevo, E. and Beiles, A. (1986) Variation in the intergenic spacer of ribosomal DNA of wild wheat, Triticum dicoccoides, in Israel. Mol. Biol. Evol.3:547-558.

Flavell, R.B., O’Dell, M. and Thompson, W.F. (1988) Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat. J. Mol. Biol. 204:523-534.

Gupta, P.K. (1996) Use of molecular probes for the study of DNA polymorphism in the genera Hordeum and Avena. In: Chopra VL, Sharma RP, Swaminathan MS (eds) Agricultural biotechnology, 2nd Asia Pacific conference. Oxford & IBH Publishing Co Pvt Ltd, New Delhi, India, pp 27-37.

Gupta, P.K., Sharma, P.K., Balyan, H.S., Roy, J.K., Sharma, S., Beharav, A. and Nevo, E. (2002) Polymorphism at rDNA loci in barley and its relation with climatic variables. Theor. Appl. Genet. 104:473-481.

Jahoor, A. and Fischbeck, G. (1987) Genetical studies of resistance of powdery mildew in barley lines derived from Hordeum spontaneum collected from Israel. Plant Breeding 99:265-273.

Jorgensen, R.A. and Cluster, P.D. (1988) Modes and tempos in the evolution of nuclear ribosomal DNA: new characters for evolutionary studies and new markers for genetic and population studies. Ann. Miss. Bot. Gard. 75:1238-1247.

Long, E.O. and Dawid, I.B. (1980) Repeated genes in eukaryotes. Ann. Rev. Biochem. 49:727-764.

Molnar, S.J., Gupta, P.K., Fedak, G. and Wheatcroft, R. (1989) Ribosomal DNA repeat unit polymorphism in 25 Hordeum species. Theor. Appl. Genet. 78:387-392.

Nevo, E., Baum, B., Beiles A. and Johnson, D.A. (1998) Ecological correlates of RAPD DNA diversity of wild barley, Hordeum spontaneum, in the Fertile Crescent. Genet. Resour. Crop Evol. 45:151-159.

Owuor, E.D., Fahima, T., Beiles, A., Korol, A, and Nevo, E. (1997) Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum. Mol. Ecol. 6:1177-1187.

Powell, W., Thomas, W.T.B., Thompson, D.M. and Swanston, J.S. (1991) Associations between rDNA  alleles and qunatitative traits in doubled haploid populations of barley. Genetics 130:187-194.

Riddle, N.C. and Richards, E.J. (2002) The Control of Natural Variation in Cytosine Methylation in Arabidopsis. Genetics 162:355-363.

Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A. and Allard, R.W. (1984) Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81:8014-8018.

Saghai-Maroof, M.A., Allard, R.W. and Zhang, Q. (1990) Genetic diversity and ecogeographical differentiation among ribosomal DNA alleles in wild and cultivated barley. Proc. Natl. Acad. Sci. USA 87:8486-8490.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a laboratory manual, 2nd edition. Cold Spring  Harbor Laboratory Press, Plainview, New York, USA.

Sardana, R., Michael, O’Dell. and Flavell, R.B. (1993) Correlation between the size of the intergenic regulatory region, the status of cytosine methylation of rRNA genes and nucleolar expression in wheat. Mol. Gen. Genet. 236:155-162.

Schmitt, F.E., Oakeley, J. and Jost, J.P. (1997) Antibiotic induces genome- wide hypermethylation in cultured Nicotiana tabacum plants. J. Biol. Chem. 272:534-1540.

Sharma, S., Beharav, A., Balyan, H.S., Nevo, E. and Gupta, P.K. (2004) Ribosomal DNA polymorphism and its association with geographical and climatic variables in 27 wild barley populations from Jordan.  Plant Sci. 166:467-477.

Fig. 1 Representative autoradiogram showing digestion pattern obtained using isochizomeric pair of MspI and HpaII restriction enzymes. Lane A: MspI showed large scale digestion depicted by number of bands from top to bottom of lane. Lane B: HpaII gave only single dense band at the top of lane depicting undigested DNA. M represents λ DNA/EcoRI+HindIII marker.

Fig. 2 Representative autoradiogram showing 11 different phenotypes obtained after BamHI digestion.

+ indicates common phenotypes at microsites and microniches