A Database for Triticeae and Avena
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
(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.
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.
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.
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.
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.
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 |
1.31 |
1.64 |
E-680 |
4/3 |
4/5 |
- |
2.13 |
0.82 |
1.15 |
E-3275 |
6/2 |
6/4 |
8/5 |
- |
1.64 |
1.64 |
E-4813 |
3/3 |
3/5 |
3/2 |
7/3 |
- |
0.65 |
E-2055 |
4/4 |
4/6 |
4/3 |
6/4 |
3/1 |
- |
Note: Percentage of sequence divergence
distance is shown above the diagonal.
Direct counts of transitions/transversions are shown below
the diagonal.
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