Skip to main content

Intra-individual and intra-species heterogeneity in nuclear rDNA ITS region of Vigna species from subgenus Ceratotropis


The extent of intra-individual and intra-species heterogeneity in the nuclear rDNA internal transcribed spacer (ITS) was investigated among the ‘Asiatic Vigna’ species (subgenus Ceratotropis). High intra- and inter-individual ITS polymorphism was observed among Vigna radiata accessions, where multiple ITS length variants ranging from ~700 to ~770 bp were detected on PCR amplification. Subsequent analysis revealed that the variants are ‘heteroduplex ITS fragments’ generated during the PCR process. Analysis of ITS from wild and cultivated forms of ten Vigna species from subgenus Ceratotropis revealed substantial intra-species divergence in four species: Vigna umbellata, Vigna trilobata, V. radiata and Vigna minima. However, no other species analysed showed intra-individual ITS heterogeneity as observed in V. radiata. The results demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. radiata. Evidence indicates that intra-species hybridization and a slow ‘molecular drive’ are responsible for this phenomenon. Sequence analysis of 5·8S, ITS1 and ITS2 and secondary-structure analysis of ITS regions indicate that the ITS variants do not belong to pseudogenic rDNA repeat units. Further, reverse transcriptase-PCR (RT-PCR) analysis showed that rDNA repeat units harbouring certain intra-individual ITS variants were transcriptionally inactive, indicating the regulation of these loci by epigenetic gene silencing. The V. radiata ITS variants, when analysed together, did not cause any phylogenetic errors at the species level.

Corresponding author
*Corresponding author. Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Tel: +91-22-25595078. Fax. +91-22-25505151. e-mail:
Hide All
Ajibade, S. R., Weeden, N. F. & Chite, S. M. (2000). Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica 111, 4755.
Álvarez, I. & Wendel, J. F. (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29, 417434.
Bailey, C. D., Timothy, G. C., Stephen, A. H. & Colin, E. H. (2003). Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes. Molecular Phylogenetics and Evolution 29, 435455.
Baldwin, B. G., Sanderson, M. J., Porter, J. M., Wojeiechowski, M. F., Campbell, C. S. & Donoghue, M. J. (1995). The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82, 247277.
Baudoin, J. P. & Maréchal, R. (1988). Taxonomy and evolution of the genus Vigna. In Proceedings of the Second International Mungbean Symposium, pp. 212. Shanhua, Taiwan: Asian Vegetable Research and Development Center.
Bjourson, A. J. & Cooper, J. E. (1992). Band-stab PCR: a simple technique for the purification of individual PCR products. Nucleic Acids Research 20, 4675.
Campbell, C. S., Wojciechowski, M. F., Baldwin, B. G., Alice, L. A. & Donoghue, M. J. (1997). Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae). Molecular Biology and Evolution 14, 8190.
Chandel, K. P. S. (1981). Wild Vigna species in the Himalayas. Plant Genetic Resources Newsletter 45, 1719.
Chandel, K. P. S., Lester, R. N. & Starling, R. J. (1984). The wild ancestors of urd and mungbeans (Vigna mungo (L.) Hepper and V. radiata (L.) Wilcz.). Botanical Journal of the Linnean Society 89, 925937.
Dadejová, M., Lim, K. Y., Soucková-Skalická, K., Matyášek, R., Grandbastien, M.-A., Leitch, A. & Kovařík, A. (2007). Transcription activity of rRNA genes correlates with a tendency towards intergenomic homogenization in Nicotiana allotetraploids. New Phytologist 174, 658668.
Delgado-Salinas, A., Turley, T., Richman, A. & Lavin, M. (1999). Phylogenetic analysis of the cultivated and wild species of Phaseolus (Fabaceae). Systematic Botany 24, 438460.
Doi, K., Kaga, A., Tomooka, N. & Vaughan, D. A. (2002). Molecular phylogeny of genus Vigna subgenus Ceratotropis based on rDNA ITS and atpB-rbcL intergenic spacer of cpDNA sequences. Genetica 114, 129145.
Dover, G. A. (1986). Molecular drive in multigene families: how biological novelties arise, spread and are assimilated. Trends in Genetics 6, 159165.
Egawa, Y., Chotechuen, S., Tomooka, N., Ngampongsai, S. & Kitbamroong, C. (1996). Cross-Compatibility among the Subgenus Ceratotropis Species of the Genus Vigna. In Phylogenetic Differentiation of Mungbean Germplasm (Subgenus Ceratotropis of the Genus Vigna) and Evaluation for Breeding Program, pp. 1930. Under the collaborative research program between JIRCAS (Japan) and DOA (Thailand).
Egawa, Y., Nakagahra, M. & Fernandez, G. C. J. (1988). Cytogenetical analysis of tetraploid Vigna glabrescens by interspecific hybridization involving diploid Asian Vigna species. In Proceedings of the Second International Mungbean Symposium, pp. 200204. Shanhua, Taiwan: Asian Vegetable Research and Development Center.
Elder, J. F. J. & Turner, B. J. (1995). Concerted evolution of repetitive DNA sequences in eukaryotes. Quarterly Review of Biology 70, 297320.
Fatokun, C. A., Danesh, D., Young, N. D. & Stewert, E. L. (1993). Molecular taxonomic relationship in the genus Vigna based on the RFLP analysis. Theoretical and Applied Genetics 86, 97104.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using bootstrap. Evolution 39, 783791.
Fitch, W. M. (1971). Towards defining the course of evolution: minimum change for a specified tree topology. Systematic Zoology 20, 406416.
Fuertes, A. J., Roselló, J. A. & Nieto, F. G. (1999). Nuclear ribosomal DNA (nrDNA) concerted evolution in natural and artificial hybrids of Armeria (Plumba-ginaceae). Molecular Ecology 8, 13411346.
Fujii, K. & Miyazaki, S. (1987). Infestation resistance of wild legumes (Vigna sublobata) to azuki bean weevil Callosobruchus chinensis (L.) (Coleoptera: Bruchidae) and its relationship with cytogenetics classification. Applied Entomology and Zoology 22, 319322.
Galasso, I., Schmidt, T., Oignone, D. & Heslop-Harrison, J. S. (1995). The molecular genetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18S–5·8S–25S rRNA genes, 5S rRNA genes, telomere-like sequences and family of centromeric repetitive DNA sequences. Theoretical and Applied Genetics 91, 928935.
Gerstner, J., Schiebel, K., von Waldburg, G. & Hemleben, V. (1988). Complex organisation of the length heterogeneous 5′external spacer of mungbean (Vigna radiata) ribosomal DNA. Genome 30, 723733.
Goel, S., Raina, S. N. & Ogihara, Y. (2002). Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of nuclear ribosomal DNA in the Phaseolus–Vigna complex. Molecular Phylogenetics and Evolution 22, 119.
Graf von der Schulenburg, J. H., Hancock, J. M., Pagnamenta, A., Sloggett, J. J., Majerus, M. E. N. & Hurst, G. D. D. (2001). Extreme length and length variation in the first ribosomal internal transcribed spacer of ladybird beetles (Coleoptera: Coccinellidae). Molecular Biology and Evolution 18, 648660.
Hartmann, S., Nason, J. D. & Bhattacharya, D. (2001). Extensive ribosomal genic variation in the columnar cactus Laphocereus. Journal of Molecular Evolution 53, 124134.
Hemleben, V., Ganal, M., Gerstner, J., Schiebel, K. & Torres, R. A. (1988). Organization and length heterogeneity of plant ribosomal RNA genes. In Architecture of Eukaryotic Genes (ed. Kahl, G.), pp. 371383. Weinheim: VCH Verlagsgesellschaft.
Jaaska, V. & Jaaska, V. (1990). Isozyme variation in Asian beans. Botanica Acta 103, 281290.
Jobst, J., King, K. & Hemleben, V. (1998). Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae. Molecular Phylogenetics and Evolution 9, 204209.
Kaga, A., Tomooka, N., Egawa, Y., Hosaka, K. & Kamijima, O. (1996). Species relationship in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis. Euphytica 88, 1724.
Karvonen, P. & Savolainen, O. (1993). Variation and inheritance of ribosomal DNA in Pinus sylvestris L. (Scots pine). Heredity 71, 614622.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111120.
Krishna, T. G. & Jawali, N. (1997). DNA isolation from single or half seeds suitable for random amplified polymorphic DNA analysis. Analytical Biochemistry 250, 125127.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology 288, 911940.
Miyazaki, S. (1982). Classification and phylogenetic relationships of the Vigna radiata–mungo–sublobata complex. Bulletin of the National Institute of Agricultural Sciences 33, 161.
Nei, M. & Kumar, S. (2000). Molecular Evolution and Phylogenetics. New York: Oxford University Press.
Nicholas, K. B., Nicholas, H. B. Jr, & Deerfield, D. W. II, (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4, 14.
Niyodham, C. (1992). Note on Thai and Indo-Chinese Phaseolae (Leguminosae-Papilionoideae). Nordic Journal of Botany 12, 339346.
Ohta, T. & Dover, G. A. (1983). Population genetics of multiple gene families that are dispersed into two or more chromosomes. Proceedings of the National Academy of Sciences of the USA 80, 40794083.
Prasad, S., Reddy, K. S. & Jawali, N. (1999). Abundance of random amplified hybridizing microsatellites in mungbean (Vigna radiata L. Wilzeck). Asia Pacific Journal of Molecular Biology and Biotechnology 7, 173177.
Razafimandimbison, S. G., Kellogg, E. A. & Bremer, B. (2004). Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: a case study from Naucleeae (Rubiaceae). Systematic Biology 53, 177192.
Saini, A., Krishna, T. G., Reddy, K. S. & Jawali, N. (2000 a). The BamH1 site in the internal transcribed spacer region of mungbean ribosomal RNA gene is partially methylated. Euphytica 114, 5559.
Saini, A., Reddy, K. S. & Jawali, N. (2000 b). Variation of the internal transcribed spacer region of ribosomal DNA in mungbean cultivars. In Proceedings of the DAE-BRNS Symposium, on The Use of Nuclear and Molecular Techniques in Crop Improvement, pp. 348356. Bhabha Atomic Research Centre, Mumbai, India.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406425.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory.
Sang, T., Crawford, D. J. & Stuessy, T. F. (1995). Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proceedings of the National Academy of Sciences of the USA 92, 68136817.
Schiebel, K. A. & Hemleben, V. (1989). Nucleotide sequence of the 18S–25S spacer region from rDNA of mungbean. Nucleic Acids Research 17, 28522856.
Schiebel, K., von Waldburg, G., Gerstner, J. & Hemleben, V. (1989). Termination of transcription of ribosomal RNA genes of mung bean occurs within a 175-bp repetitive element of the spacer region. Molecular Genetics and Genomics 218, 302307.
Schlötterer, S. & Tautz, D. (1994). Chromosomal heterogeneity of Drosophila ribosomal DNA arrays suggests intrachromosomal exchanges drives concerted evolution. Current Biology 4, 777783.
Souframanien, J., Joshi, A. & Gopalakrishna, T. (2003). Intraspecific variation in the internal transcribed spacer region of rDNA in black gram (Vigna mungo (L.) Hepper). Current Science 85(6), 798802.
Suh, Y., Thien, L. B., Reeve, H. E. & Zimmer, E. A. (1993). Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of ribosomal DNA in Winteraceae. American Journal of Botany 80, 1942–1055.
Tateishi, Y. & Maxted, N. (2002). New Species and combinations in Vigna subgenus Ceratotropis (Piper) Verdc. (Leguminosae, Phaseoleae). Kew Bulletin 57(3), 625633.
Thulin, M., Lavin, M., Pasquet, R. & Delgado-Salinas, A. (2004). Phylogeny and biogeography of Wajira (Leguminosae): A monophyletic segregate of Vigna centered in the Horn of Africa region. Systematic Botany 29, 903920.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 48764882.
Tomooka, N., Maxted, N., Thavarasook, C. & Jayasurya, A. H. M. (2002). AFLP analysis of diploid Vigna subgenus Ceratotropis species. Genetic Resources and Crop Evolution 49, 521530.
Tomooka, N., Vaughan, D. A., Moss, H. & Maxted, N. (2003). The Asian Vigna: Genus Vigna Subgenus Ceratotropis Genetic Resources. Dordrecht, The Netherlands: Kluwer Academic Publishers.
Unfried, K., Schiebel, K. & Hemleben, V. (1991). Subrepeats of rDNA intergenic spacer present as prominent independent satellite DNA in Vigna radiata but not in Vigna angularis. Gene 99, 6368.
Vaughan, D. A., Tomooka, N., Kaga, A., Doi, K. & Yoon, M. S. (2000). Research on Vigna subgenus Ceratotropis genetic resources that provide insights into in situ conservation strategies. In Proceedings of the MAFF International Workshop on Genetic Resources. Wild Legumes, pp. 129150. Tsukuba, Japan: MAFF, NIAR.
Volkov, R. A., Komarova, N. Y., Zentgraf, U. & Hemleben, V. (2006). Molecular cell biology: epigenetic gene silencing in plants. In Progress in Botany (ed. Esser, K., Lüttge, U., Beyschlag, W. & Hellig, F.) vol. 67, pp. 101133. Berlin, Heidelberg and New York: Springer-Verlag.
Wallace, A. J. (2001). Combined single stranded conformation polymorphism and heteroduplex analysis. In Laboratory Methods for Detection of Mutations and Polymorphism in DNA (ed. Taylor, G. R.), pp. 7994. Boca Raton, FL: CRC Press.
Wei, X.-X., Wang, X.-Q. & Hong, D.-Y. (2003). Marked intragenomic heterogeneity and geographical differentiation of nrDNA ITS in Larix potaninii (Pinaceae). Journal of Molecular Evolution 57, 623635.
Wendel, J. F. (2000). Genome evolution in polyploids. Plant Molecular Biology 42, 225249.
Yamaguchi, H. (1992). Wild and weed azuki beans in Japan. Economic Botany 46, 384394.
Yasuda, K. & Yamaguchi, H. (1996). Phylogenetic analysis of the subgenus Ceratotropis (genus Vigna) and an assumption of the progenitor of azuki bean using isozymes variation. Breeding Science 46, 337342.
Yoon, M. S., Doi, K., Kaga, A., Tomooka, N. & Vaughan, D. A. (2000). Analysis of genetic diversity in the Vigna minima complex and related species in East Asia. Journal of Plant Research 113, 375386.
Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31, 34063415.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Genetics Research
  • ISSN: 0016-6723
  • EISSN: 1469-5073
  • URL: /core/journals/genetics-research
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
Type Description Title
Supplementary materials

Saini et al. supplementary material
Supplementary Table S1 and Figs S1 S2 S3A and S3B

 Word (4.8 MB)
4.8 MB
Supplementary materials

Saini et al. supplementary material
Supplementary Table S2 and Figs S4 and S5

 Word (1.5 MB)
1.5 MB


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed