Internal transcribed spacer (ITS) sequences of ribosomal DNA of wild barley
and their comparison with ITS sequences in common wheat

S. Sharma, S. Rustgi, H.S. Balyan and P.K. Gupta

Molecular Biology Laboratory, Department of Agricultural Botany

Ch. Charan Singh University, Meerut-250 004, India

 

Hard-copy edition pages 38 - 45.

Abstract

ITS (Internal Transcribed Spacer) region of ribosomal DNA was studied in 10 wild barley (Hordeum spontaneum) and six common wheat (Triticum aestivum) accessions. Alignment of sequences suggested the presence of both insertions/deletions (indels) and substitutions (transitions and transversions) in both ITS1 and ITS2 regions. The indels were responsible for length variation. Barley accessions, collected from narrow geographical range were less diverse in comparison to wheat accessions which were collected from six countries belonging to three different continents. Presence of sequence and length variation in the ITS region suggests that it can be used for assaying genetic diversity in crops like barley and wheat at the intraspecific level.  

 

Introduction

Eukaryotic ribosomal RNA genes (known as ribosomal DNA or rDNA) are found as parts of repeat units that are arranged in tandem arrays, located at the chromosomal sites known as nucleolar organizing regions (NORs). Each repeat unit consists of a transcribed region (having genes for 18S, 5.8S and 26S rRNAs and the external transcribed spacers i.e. ETS1 and ETS2) and a non-transcribed spacer (NTS) region. In the transcribed region, internal transcribed spacers (ITS) are found on either side of 5.8S rRNA gene and are described as ITS1 and ITS2.

The length and sequences of ITS regions of rDNA repeats are believed to be fast evolving and therefore may vary. Universal PCR primers designed from highly conserved regions flanking the ITS and its relatively small size (600-700 bp) enable easy amplification of ITS region due to high copy number (up to-30000 per cell, Dubouzet and Shinoda 1999) of rDNA repeats. This makes the ITS region an interesting subject for evolutionary/phylogenetic investigations (Baldwin et al. 1995; Hershkovitz et.al. 1996, 1999) as well as biogeographic investigations (Baldwin 1993; Suh et al. 1993; Hsiao et al. 1994; Dubouzet and Shinoda 1999). The sequence data of the ITS region has also been studied earlier to assess genetic diversity in cultivated barley (Petersen and Seberg 1996). In this communication, the results on the variation in length and sequence of  ITS region of rDNA in wild barley, Hordeum spontaneum and common wheat, Triticum aestivum L., are presented and discussed in relation to their utility in assessing genetic diversity at intraspecific level in these two species.

 

Material and Methods

Plant material Ten accessions of wild barley, Hordeum spontaneu, belonging to 10 different populations from Jordan and six accessions of common wheat, Triticum aestivum, from six different countries belonging to four continents were used in the present study. The wild barley material was supplied by Prof. E. Nevo, University of Haifa, Israel and common  wheat material was procured from Directorate of Wheat Research (DWR), Karnal, India. All the accessions were grown at the research farm of Ch. Charan Singh University, Meerut, India. Barley accessions, collected from different locations of Jordan, included the following: 1-4 (Shuni N); 3-9 (Irbid E Technion); 4-12 (Mafrak W); 5-3 (Mafrak); 7-25 (Amman); 9-10 (Mount Nebo); 11-14 (Wadi Arnon Mujeib); 14-30 (Waddi Hassa); 15-30 (Waddi Hassa S); 19-50 (Karak Dead Sea). Similarly, the Wheat accessions included the following: E-965 (Yugoslavia); E-1000 (Cyprus); E-680 (Argentina); E-3275 (The Netherlands); E-4813 (Kenya); E-2055 (Poland).

 

DNA extraction, ITS amplification, cloning and sequencing (a) DNA extraction and purification: Total cellular DNA was isolated from two to three plants (one month old) per accession, using  modified CTAB method of Saghai-Maroof et al. (1984). The isolated DNA was purified by RNaseA treatment and phenol: chloroform: isoamyl alcohol extraction. The quality and quantity of DNA samples was checked on agarose gel using lambda DNA as marker.

(b) ITS amplification: ITS1-5.8S- ITS2 rDNA region was amplified using the following primer pair (White et al. 1990):  ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’)

 ITS-5 (5’- GGAAGTAAAAGTCGTAACAAGG-3’)

Amplifications were carried out in 50 µl reaction mixture containing 35.7µl sterile water , 5µl of 10x PCR buffer, 3µl of 25mM MgCl2, 2µl of 10mM dNTPs, 1µl of each primer (0.7µM ), 0.3µl  (1.5 U)  of  Taq polymerase (Promega Corp., USA) and 2 µl (50 ng) template  DNA. Perkin Elmer DNA thermal cycler was used with the following PCR profile: an initial denaturation for 5 min at 95o C, 35 thermal cycles (1 min at 95o C, 1min at 50o C and 1 min at 72o C), and a final 5 min extension at 72o C. The amplified DNA was purified using QIAGEN QIA QUICK PCR purification kit following manufacturers instructions (QIAGEN, Germany). 

(c) Cloning and sequencing: Purified DNA was ligated in pGEMâ-T Easy vector (Promega Corp., USA) overnight at 16o C. The ligated DNA was transformed in DH5a competent cells.  The recombinant clones were identified through blue/white colour selection and the presence of insert  in the recombinant clones (white colonies) was confirmed following colony PCR. For sequencing, plasmid DNA was  isolated following alkali lysis method (Sambrook et al. 1989). The insert DNA was sequenced on contract using automated sequencing facility at IISc, Bangalore, India.

(d) Sequence alignment: The sequences of  ITS1-5.8S-ITS2 regions were manually  aligned with the corresponding sequences of  Hordeum vulgare (Chatterton et  al. 1992a) and  Triticum aestivum L. (Chatterton et  al. 1992b) that were already available in the database. 

(e) Sequence submission: Sequences of clones were submitted directly to GenBank through Bankit (a world wide web sequence submission server available at NCBI home page). The sequences are available on line (http://www.ncbi.nlm.nih.gov) and can be located by accession numbers or GI numbers AF438186 – AF438197 and AF440676 – AF440679.

 

Results and discussion

Length, GC content and variation in sequence of entire ITS  The total length of the entire ITS of rDNA and its GC content (%) in wild barley and wheat genotypes studied during the present study were variable but were in agreement with the results of earlier studies on wild and cultivated barley and with those on bread wheat (Hsiao et al. 1994; Petersen and Seberg 1996). However, the variation in the total length of the entire ITS of  wild barley (595 to 598 bp) was lower than that in wheat (597 to 605 bp). The alignment of sequences of entire ITS showed differences among sequences of clones of wild barley genotypes and also among those of wheat genotypes. These sequences of ITS also differed from corresponding published sequences each for cultivated barley and bread wheat (Hsiao et al. 1994; Chatterton et  al. 1992a, b). The sequence data showed that the total variable sites in wild barley (7.4%) were comparable with the total variable sites in wheat (6.90%) (Table 1). These variable sites included both the substituions which arise due to point mutation at specific sites, and indels that presumably result from slippage replication (Dover 1986; Stephan 1989) and short mispairing during replication (Jobst et al. 1998). The substitutions and indels were equally frequent both in wild barley and wheat. However, the frequency of such sites in the present study was higher than those reported earlier in the ITS sequences of different species of Triticeae. For instance, in a study involving ten genotypes of cultivated barley, H. vulgare, only 0.84% substitution sites were available while the indels were completely absent (Petersen and Seberg, 1996). In another study involving different species of Triticeae including wild and cultivated barley, no substitutions or indels were detected in a study of two accessions of each species procured from widely separated geographical regions (see Hsiao et al. 1995). In the present study, among the substitutions, transitions were more frequent than transversions, and among the indels, deletions were more frequent than insertions. In both wild barley and wheat, always only single base deletions were observed except in a solitary wheat genotype (E-965) where besides single base deletions, a single deletion of 2 bases was also detected. Insertions of single base were also noticed in barley and wheat sequences. Over all, in the entire ITS region, 40 (6.62%) autapomorphic sites (variation at a same site in one sequence) in barley and 33 (5.40%) autapomorphic sites in wheat were detected. Similarly, 5(0.82%) synapomorphic sites in barley and 9 (1.50%) synapomorphic sites (variation at the same site in more than one sequence) in wheat were detected.

 

Intraspecific sequence divergence in ITS1, ITS2 and 5.8S regions Of the three regions of the entire ITS, the sequence of 5.8S rRNA coding region, as expected, is conserved both in wild barley and wheat and this region exhibited only three substitutions (including two transitions and one transversion) and a single deletion in one wheat genotype (Table 1). This suggested similar level of intraspecific sequence divergence in 5.8S region in the two species. Substitutions and indels of the same order in 5.8S region were also reported in earlier studies on Zea and several species of Triticeae (Hsiao et al. 1995, Buckler 1996). The sequence divergence of the ITS1 in wild barley due to substitutions ranged from 0.0 to 2.71% and that due to substitutions plus indels ranged from and 0.0 to 5.42% which were lower than those observed in wheat (substitutions; 0.0 to 3.12% and substitutions plus indels; 0.44 to 7.0% ) (Tables 1). The sequence divergence of the ITS2 in wild barley due to substitutions ranged from 0.0-2.28% and that due to substitutions plus indels ranged from 0.0 to 5.0% while in wheat substitutions ranged from 0.45 to 2.26% and substitutions plus indels, ranged from  0.45 to 4.07% (Table1). Thus both in wild barley and wheat sequences divergence was greater in the ITS1 than in the ITS2 region.

 

The higher level of divergence in ITS1, observed during the present study is in conformity with earlier reports in a variety of plant species (Kollipara et.al. 1997, Baldwin 1993, Moller and Cronk 1997). The deletions within ITS1 and ITS2 are believed to interfere with rRNA processing. For instance, in vivo mutational studies in yeast (Saccharomyces cerevisiae) indicated that deletions of certain regions within ITS1 inhibited production of mature small and large subunit rRNAs (Musters et al. 1990; Nues et al. 1994), whereas certain deletions and point mutations in ITS2 prevented or reduced processing of large subunit rRNA (Sande et al. 1992).

A characteristic conserved sequence GGCG- (4 to 7n) -GYGYCAAGGAA (where Y=C or T), was also available in the ITS1 of both wild barley and wheat. In previous studies on many flowering plants this characterstic sequence has been reported in the middle of ITS1 and this sequence is presumed as a recognition site for processing of a primary transcript into the structural rRNA (see Liu and Schardl 1994. In three wild barley accessions and one wheat genotype, a single base insertion was also noticed in the above characteristic sequence.

 

Diversity among wild barley and wheat genotypes due to substitutions in the entire ITS

The pairwise comparison between possible pairs of 10 wild barley and six wheat genotypes was done to work out sequence divergence based on substitutions (Tables 2 and 3). The sequence divergence among pairs of wild barley genotypes with a mean of 0.85% and a range of 0.16-1.70% was lower than the divergence among pairs of wheat genotypes having a mean of 1.30% and a range of 0.65-2.13%. This clearly indicated that on the basis of sequence divergence due to substitutions in the ITS region, the barley genotypes were less diverse than the wheat genotypes. It was not surprising, bacause wheat genotypes were sampled from six countries belonging to three different continents while the wild barley genotypes were sampled from limited geographical area of Jordan. The wheat genotypes studied here actually belong to an elite germplasm collection and might have been bred for specific purposes utilizing very diverse ancestrally unrelated parents and hence are more diverse. The sequence divergence data further suggested that wheat genotypes E-680 (from Argentina) and E 3275 (from The Netherlands) were most divergent (2.13%) as compared to other pair of genotypes. In wild barley, the maximum divergence (1.70) was noted between three different pairs of genotypes, namely 4-12 (from Mafrak W) and 15-30 (from Waddi Hassa S); 9-10 (from Mount Nevo) and 19-50 (from Karak Dead Sea); and 14-30 (from Waddi Hassa) and 19-50 (from Karak Dead Sea). These genotypes were collected from independently evolving wild populations that may be adapted to specific climatic and edaphic conditions and hence may be genetically more diverse.

 

Conclusion

The data on length and sequence of ITS may be a useful parameter for the assessment of genetic diversity at the intraspecific level in species like barley and wheat, although the level of diversity detected using ITS data at interspecific level is much higher in different groups of plants.

    

Acknowledgements

During the course of study Prof. P. K. Gupta, was CSIR-Emeritus Scientist. Thanks are due to the Council of Scientific and Industrial Research (CSIR), New Delhi, India and National Agricultural Technology  Project (NATP) Programme for financial assistance. Thanks are also due to Prof. E. Nevo of University of Haifa, Israel for supply of barley material and to the Directorate of Wheat Research (DWR), Karnal, India for supply of wheat material.

 


 

 

 

 

 

 

 

Table 1. Sequence characteristics of ITS region of barley and wheat accessions. 

 

S.No

Parameter

ITS1

5.8S

ITS2

Entire sequence

1

Length range (nt)

214-217

217-222

164

162-163

215-217

216-220

595-598

597-605

2

Length mean (nt)

216.09

220.14

164.0

163.0

216.50

217.28

596.5

600.28

3

Aligned length(nt)

221

224

164

163

 

219

221

 

604

608

 

4

G+C content, range (%)

54.29-55.65

58.03-62.0

59.14-59.75

58.00-59.00

60.7-62.10

59.22-60.63

57.94-58.77

59.04-60.52

5

G+C content, mean (%)

54.84

660.66

59.53

58.4

61.59

60.37

58.49

60.00

6

Sequence divergence, range (%)

based on substitutions only

0-2.71

0-3.12

0-1.20

0-1.22

0-2.28

0.45-2.26

0.16-1.70

0.65-2.13

7

Sequence divergence, range (%)

based on substitutions plus indels

0-5.42

0.44-7.0

0-1.21

0-1.84

0-5.0

0.45-4.07

0.50-3.0

1.0-4.0

8

No. of indels

13

15

0

1

9

6

22

22

9

No. of variable sites  (%)

24 (10.85)

23 (10.26)

4 (2.43)

5 (3.06)

17 (7.76)

14 (6.33)

45(7.4)

42 (6.90)

10

No. of constant sites (%)

197 (89.10)

201(89.73)

160 (97.56)

158(96.93)

202 (92.23)

207 (94.0)

559 (92.54)

566 (93.09)

11

No. of synapomorphic sites (%)

5 (2.26)

6 (3.00)

0 (0.0)

0 (0.0)

0 (0)

3 (1.36)

5 (0.82)

9 (1.50)

12

No. of autapomorphic sites (%)

19 (8.59)

17 (8.00)

4 (2.43)

5 (3.06)

17 (7.76)

11(5.00)

40 (6.62)

33 (5.4)

13

Transitions®

0-4

0-4

0-2

0-2

0-2

0-3

0-7

1-8

14

Transversions®

0-2

0-3

0-1

0-1

0-3

0-2

0-5

1-6

Note: In each box, upper value is for barley and lower values is for wheat.

®= Based on pairwise comparisons

 

 

 

 

Table 2. Pairwise nucleotide sequence divergence (%), based on substituions, among 10 accessions of wild barley (Hordeum spontaneum).   

 

Acc.

No

1-4

3-9

4-12

5-3

7-25

9-10

11-14

14-30

15-30

19-50

1-4

-

0.33

0.33

 0.70

0.16

0.82

0.50

0.82

0.70

1.15

3-9

1/1

-

0.50

0.70

0.33

0.82

0.50

0.82

0.70

1.15

4-12

2/1

3/0

-

0.50

0.50

0.82

0.33

0.82

1.70

1.32

5-3

2/2

3/1

2/1

-

0.33

1.00

0.50

1.00

0.82

1.50

7-25

1/0

2/0

3/0

1/1

-

0.82

0.50

0.85

0.70

1.15

9-10

1/4

2/3

2/3

2/4

2/3

-

0.82

1.32

1.15

1.70

11-14

2/1

3/0

2/0

2/1

3/0

2/3

-

0.82

0.70

1.32

14-30

4/1

5/0

5/0

5/1

5/0

5/3

5/0

-

1.15

1.70

15-30

3/1

4/0

4/0

4/1

4/0

4/3

4/0

6/1

-

1.32

19-50

4/3

5/2

6/2

6/3

5/2

5/5

6/2

7/3

6/2

-

    Note: Percentage of sequence divergence distance is shown above the diagonal.

        Direct counts of transitions/transversions are shown below the diagonal.

 

 

 

Table 3. Pairwise nucleotide sequence divergence (%), based on substituions, among six accessions of common wheat (Triticum aestivium).

 

Acc.No

E-965

E-1000

E-680

E-3275

E-4813

E-2055

E-965

    -

0.65

1.15

1.31

0.98

1.31

E-1000

2/2

     -

1.48

1.64