Plant Molecular Biology 20: 165-166, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
165
Nucleotide sequence of the internal transcribed spacer region of rDNA in barley, Hordeum vulgate L. (Gramineae) N.J. Chatterton, C. Hsiao, K.H. Asay, K.B. Jensen and R.R-C. Wang USDA Agricultural Research Service, Forage and Range Laboratory, Utah State University, Logan, Utah 84322-6300, USA Received 10 March 1992; accepted 12 March 1992
Key words: rRNA, PCR, ITS, DNA sequence, nucleotide, Hordeum vuIgare, barley
The internal transcribed spacer (ITS) region (598 bp) between the 18S and 26S rRNA genes of barley (Hordeum vulgare L.) was amplified by the polymerase chain reaction (PCR) and sequenced by Sanger's dideoxy method [1]. Primers for PCR and sequencing, ITS5(F)-5' GGAAGTAAAAGTCGTAACAAGG 3' and ITS4(R)-5'-TCCTCCGCTTATTGATATGC 3' as designed by White et al. [2] were used for amplification. The region was sequenced in opposite directions twice with the amplification primers and two internal primers, ITS2(R)-5'-GCTGCGTTCTTCATCGATGC 3' and ITS3(F)-5' G C A T C G A T G A A G A A C G C A G C 3'. The
junctions of the coding and spacer regions were determined by their homology with that of rice [3], and the 5.8S region of wheat [4, 5]. The ITS 1, 5.8S and ITS 2 regions in barley are 218, 163, and 217 bp in length, respectively. The 5.8S rRNA gene of barley has complete homology with that of wheat (Triticum aestivum L.), mountain rye (Secale montanum Guss), (Chatterton, et al., submitted) and rice [3]. However, barley has different nucleotides at a significant number of locations in the ITS 1 and ITS 2 regions, compared to wheat. The AT and GC contents, 41 and 59~o respectively, of the entire ITS region in barley are only slightly different from 39~o ATand 61 ~o GC
1TCGTGACCCT GACCAAAACAGACCGTGCTCGCGTCATCCAATCCTCCGAC 51 GATGGCATTGTTCGTCGTTC GGCCAATTCCTCGACCGCCTCCACTCCTAG 101 GAGCGGGGGCTCGTGGTAAAAGAACCCACG GCGCCGAAGGCGTCAAGGAA ITS I 151 CACTGTGCCTAACCCGGGGAGATGGCTAGCTTGCTGGTCGTCACCTGTGT 201TGCAAATATA TTTAATCCAC ACGACTCTCGGCAACGGATATCTCGGCTCT 251 CGCATCGATGAAGAACGTAGCGAAATGCGATACCTGGTGTGAATTGCAGA 5.8S 301ATCCCGCGAA CCATCGAGTCTTTGAACGCAAGTTGCGCCC GAGGCCACTC 351GGCCGAGGGC ACGCCTGCCT GGGCGTCACGCCAAAACACGCTCCCAACCA 401 CCCTCTTCGG GAATTGGGAT GCGGCATATG GTCCCTCGTC CTGCAAGGGG 451CGGTGGGCCG AAGATCGGGCTGCCGGCGTA CCGCGTCGGACACAGCGCAT ITS 2 501GGTGGGCGTC CTTGCTTTAT CAATGCAGTG CATCCGACGCGTAGACGGCA 551TCATGGCCTC GAAACGACCCATCGAACGAAGTGCACGTCG CTTCGACC Fig. 1. Nucleotide sequences of the ITS 1, 5.8S and ITS 2 region in Hordeum vulgate. The 5.8S region is underlined.
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Zl1759.
166 in wheat. This suggests that DNA sequences of the ITS region are useful in quantifying species relatedness.
Acknowledgements We thank Utah Agricultural Experiment Station for their excellent cooperation and support.
References 1. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 (1977).
2. White TJ, Bruns T, Lee S, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfan DH, Sninsky JJ, White TJ (eds) PCR protocols: A Guide to Methods and Applications, pp. 315-322. Academic Press, San Diego (1990). 3. Takaiwa F, Oono K, Sugiura M: Nucleotide sequence of the 17S-25S spacer regions from rice rDNA. Plant Mol Biol 4:355-364 (1985). 4. MacKay RM, Spencer DF, Doolittle WF, Gray MW: Nucleotide sequences of wheat-embryo cytosol 5-S and 5.8S ribosomal ribonucleic acids. Eur J Biochem 112: 561576 (1980). 5. Wildeman AG, Nazar RN: Studies on the secondary structure of wheat 5.8 S rRNA: conformational changes in the A + U-rich stem during ribosome assembly. Eur J Biochem 121:357-363 (1982).
165
Nucleotide sequence of the internal transcribed spacer region of rDNA in barley, Hordeum vulgate L. (Gramineae) N.J. Chatterton, C. Hsiao, K.H. Asay, K.B. Jensen and R.R-C. Wang USDA Agricultural Research Service, Forage and Range Laboratory, Utah State University, Logan, Utah 84322-6300, USA Received 10 March 1992; accepted 12 March 1992
Key words: rRNA, PCR, ITS, DNA sequence, nucleotide, Hordeum vuIgare, barley
The internal transcribed spacer (ITS) region (598 bp) between the 18S and 26S rRNA genes of barley (Hordeum vulgare L.) was amplified by the polymerase chain reaction (PCR) and sequenced by Sanger's dideoxy method [1]. Primers for PCR and sequencing, ITS5(F)-5' GGAAGTAAAAGTCGTAACAAGG 3' and ITS4(R)-5'-TCCTCCGCTTATTGATATGC 3' as designed by White et al. [2] were used for amplification. The region was sequenced in opposite directions twice with the amplification primers and two internal primers, ITS2(R)-5'-GCTGCGTTCTTCATCGATGC 3' and ITS3(F)-5' G C A T C G A T G A A G A A C G C A G C 3'. The
junctions of the coding and spacer regions were determined by their homology with that of rice [3], and the 5.8S region of wheat [4, 5]. The ITS 1, 5.8S and ITS 2 regions in barley are 218, 163, and 217 bp in length, respectively. The 5.8S rRNA gene of barley has complete homology with that of wheat (Triticum aestivum L.), mountain rye (Secale montanum Guss), (Chatterton, et al., submitted) and rice [3]. However, barley has different nucleotides at a significant number of locations in the ITS 1 and ITS 2 regions, compared to wheat. The AT and GC contents, 41 and 59~o respectively, of the entire ITS region in barley are only slightly different from 39~o ATand 61 ~o GC
1TCGTGACCCT GACCAAAACAGACCGTGCTCGCGTCATCCAATCCTCCGAC 51 GATGGCATTGTTCGTCGTTC GGCCAATTCCTCGACCGCCTCCACTCCTAG 101 GAGCGGGGGCTCGTGGTAAAAGAACCCACG GCGCCGAAGGCGTCAAGGAA ITS I 151 CACTGTGCCTAACCCGGGGAGATGGCTAGCTTGCTGGTCGTCACCTGTGT 201TGCAAATATA TTTAATCCAC ACGACTCTCGGCAACGGATATCTCGGCTCT 251 CGCATCGATGAAGAACGTAGCGAAATGCGATACCTGGTGTGAATTGCAGA 5.8S 301ATCCCGCGAA CCATCGAGTCTTTGAACGCAAGTTGCGCCC GAGGCCACTC 351GGCCGAGGGC ACGCCTGCCT GGGCGTCACGCCAAAACACGCTCCCAACCA 401 CCCTCTTCGG GAATTGGGAT GCGGCATATG GTCCCTCGTC CTGCAAGGGG 451CGGTGGGCCG AAGATCGGGCTGCCGGCGTA CCGCGTCGGACACAGCGCAT ITS 2 501GGTGGGCGTC CTTGCTTTAT CAATGCAGTG CATCCGACGCGTAGACGGCA 551TCATGGCCTC GAAACGACCCATCGAACGAAGTGCACGTCG CTTCGACC Fig. 1. Nucleotide sequences of the ITS 1, 5.8S and ITS 2 region in Hordeum vulgate. The 5.8S region is underlined.
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Zl1759.
166 in wheat. This suggests that DNA sequences of the ITS region are useful in quantifying species relatedness.
Acknowledgements We thank Utah Agricultural Experiment Station for their excellent cooperation and support.
References 1. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 (1977).
2. White TJ, Bruns T, Lee S, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfan DH, Sninsky JJ, White TJ (eds) PCR protocols: A Guide to Methods and Applications, pp. 315-322. Academic Press, San Diego (1990). 3. Takaiwa F, Oono K, Sugiura M: Nucleotide sequence of the 17S-25S spacer regions from rice rDNA. Plant Mol Biol 4:355-364 (1985). 4. MacKay RM, Spencer DF, Doolittle WF, Gray MW: Nucleotide sequences of wheat-embryo cytosol 5-S and 5.8S ribosomal ribonucleic acids. Eur J Biochem 112: 561576 (1980). 5. Wildeman AG, Nazar RN: Studies on the secondary structure of wheat 5.8 S rRNA: conformational changes in the A + U-rich stem during ribosome assembly. Eur J Biochem 121:357-363 (1982).
