Journal of Genetics

, 98:101 | Cite as

Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability

  • Sazda Abdi
  • Anuj Dwivedi
  • Shashi
  • Suresh Kumar
  • Vishnu BhatEmail author
Research Article


Most of the grasses of the genus Cenchrus (20–25 species) and Pennisetum (80–140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus–Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus–Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus–Pennisetum complex, and one (CcSSR_47) with Bothriochloa–Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a ‘bristle clade’. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.


microsatellite marker Cenchrus Pennisetum apomixis agamic complex allelic sequence divergence. 



Sazda Abdi was supported by INSPIRE fellowship from DST, New Delhi, India. The authors gratefully acknowledge the financial support from University Grants Commission, India (F.No.41-402/2012(SR)) towards transcriptome analysis and the University of Delhi R&D grant provided to VB, to undertake this research work. We are grateful to the Director, ICAR-IGFRI, Jhansi for providing some of the research material for this study. The authors duly acknowledge USDA, ARS, PGRCU, 1109, Experiment Street, Griffin, Georgia 30223-1797 for providing plant samples used in this study.


  1. Abdi S., Shashi, Dwivedi A. and Bhat V. 2016 Harnessing apomixis for heterosis breeding in crop improvement. In Molecular breeding for sustainable crop improvement (ed. V. R. Rajpal, S. R. Rao and S. N. Raina), pp. 79–99. Springer International Publishing, Switzerland.CrossRefGoogle Scholar
  2. Akiyama Y., Goel S., Conner J. A., Hanna W. W., Yamada-Akiyama H. and Ozias-Akins P. 2011 Evolution of the apomixis transmitting chromosome in Pennisetum. BMC Evol. Biol. 11, 289.PubMedPubMedCentralCrossRefGoogle Scholar
  3. Ambreen H., Kumar S., Murali T. V., Joshi G., Bali S., Agarwal M. et al. 2015 Development of a novel set of genomic microsatellite markers in Carthamus tinctorius L. using next generation sequencing and assessment of their utility for diversity analysis and cross species transferability. PLoS One 10, e0135443.Google Scholar
  4. Bashaw E. C. and Hignight K. W. 1990 Gene transfer in apomictic buffelgrass through fertilization of an unreduced egg. Crop Sci. 30, 571–575.CrossRefGoogle Scholar
  5. Bashaw E. C., Hussey M. A. and Hignight K. W. 1992 Hybridization (N+N and 2N+N) of facultative apomictic species in the Pennisetum agamic complex. Int. J. Plant Sci. 153, 466–470.CrossRefGoogle Scholar
  6. Chabane K., Ablett G. A., Cordeiro G. M., Valkoun J. and Henry R. J. 2005 EST versus Genomic derived microsatellite markers for genotyping wild and cultivated barley. Genet. Resour. Crop EV. 52, 903–909.CrossRefGoogle Scholar
  7. Chase A. 1921 The Linnaean concept of pearl millet. Am. J. Bot. 8, 41–49.CrossRefGoogle Scholar
  8. Chemisquy M. A., Giussani L. M., Scataglini M. A., Kellog E. A. and Morrone O. 2010 Phylogenetic studies favour the unification of Pennisetum, Cenchrus and Odontelytrum (Poaceae): a combined nuclear, plastid and morphological analysis, and nomenclatural combinations in Cenchrus. Ann. Bot. 106, 107–130.PubMedPubMedCentralCrossRefGoogle Scholar
  9. Chen S. L. and Phillips S. M. 2006 Cenchrus. In Flora of China, Poaceae (ed. Z. Y. Wu and P. H. Raven), vol. 22, pp. 552–553. Science Press, Beijing.Google Scholar
  10. Clayton W. D. and Renvoize S. A. 1986 Genera Graminum: grasses of the world. Her Majesty’s Stationery Office, London.Google Scholar
  11. Clayton W. D. 1989 Gramineae (Paniceae, Isachneae and Arundinelleae). In Flora Zambesiaca (ed. E. Launert and G. V. Pope), vol. 10, pp. 1–231. Whitstable Litho Printers Ltd.Google Scholar
  12. Conner J. A., Goel S., Gunawan G., Cordonnier-Pratt M, Johnson V. E., Liang C. et al. 2008 Sequence analysis of BAC clones from the apospory-specific genomic region (ASGR) of Pennisetum and Cenchrus. Plant Physiol. 147, 1396–1411.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Crins W. J. 1991 The genera of Paniceae (Gramineae: Panicoideae) in the southeastern United States. J. Arnold Arbor. 1 (suppl), 171–312.Google Scholar
  14. Daniell H., Lin C. S., Yu M. and Chang J. W. 2016 Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 17, 134.PubMedPubMedCentralCrossRefGoogle Scholar
  15. de Wet J. M. J. and Harlan J. R. 1970a Bothriochloa intermedia: a taxonomic dilemma. Taxon 19, 339– 340.CrossRefGoogle Scholar
  16. de Wet J. M. J., Harlan J. R. 1970b Apomixis, polyploidy and speciation in Dichanthium. Evolution 24, 270–277.PubMedCrossRefGoogle Scholar
  17. DeLisle D. G. 1963 Taxonomy and distribution of the genus Cenchrus. Iowa State Coll. J. Sci. 37, 259–351.Google Scholar
  18. Donadío S., Giussani L. M., Kellogg E. A., Zuloaga F. O. and Morrone O. 2009 A preliminary molecular phylogeny of Pennisetum and Cenchrus (Poaceae-Paniceae) based on the trnL-F, rpl16 chloroplast markers. Taxon 58, 392–404.CrossRefGoogle Scholar
  19. Doust A. N. and Kellogg E. A. 2002 Inflorescence diversification in the panicoid “bristle grass” clade (Paniceae, Poaceae): evidence from molecular phylogenies and developmental morphology. Am. J. Bot. 89, 1203–1222.PubMedCrossRefGoogle Scholar
  20. Doust A. N., Penly A. M., Jacobs S. W. L. and Kellogg E. A. 2007 Congruence, conflict, and polyploidization shown by nuclear and chloroplast markers in the monophyletic “Bristle clade” (Paniceae, Panicoideae, Poaceae). Syst. Bot. 32, 531–544.CrossRefGoogle Scholar
  21. Eujayl I, Sorrells M. E., Wolters P., Baum M. and Powell W. 2002 Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theor. Appl. Genet. 104, 399–407.PubMedCrossRefGoogle Scholar
  22. Fisher W. D., Bashaw E. C. and Holt E. C. 1954 Evidence of apomixis in Pennisetum ciliare and Cenchrus setigerus. Agron. J. 46, 401–404.CrossRefGoogle Scholar
  23. Giordano A., Cogan I. O. N., Drayton M., Mouradov A., Panter S., Schrauf E. G. et al. 2014 Gene discovery and molecular marker development, based on high-throughput transcript sequencing of Paspalum dilatatum Poir. PLoS One, 10, e85050.CrossRefGoogle Scholar
  24. Giussani L. M., Nchez J. H. C-S, Zuloaga F. O. and Kellogg E. A. 2001 A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C4 photosynthesis. Am. J. Bot. 88, 1993–2012.PubMedCrossRefGoogle Scholar
  25. Gómez-Martínez R. and Culham A. 2000 Phylogeny of the subfamily Panicoideae with emphasis on the tribe Paniceae: evidence from the trnLF cpDNA region. In Grasses: systematics and evolution (ed. S. W. L. Jacobs and J. E. Everett), pp. 136–140. CSIRO Publishing, Collingwood, Australia.Google Scholar
  26. Gupta S., Kumari K., Sahu P. P., Vidapu S. and Prasad M. 2012 Sequence based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet (Setaria italica (L.) P. Beauv.). Plant Cell Rep. 31, 323–337.PubMedCrossRefGoogle Scholar
  27. Hojsgaard D. and Hörandl E. 2015 A little bit of sex matters for genome evolution in asexual plants. Front. Plant Sci. 6, 82.PubMedPubMedCentralCrossRefGoogle Scholar
  28. Hojsgaard D., Pellino M., Sharbel T. and Horandl E. 2015 Resolving genome evolution pattern in asexual plants. In next-generation sequencing in plant systematics, pp. 119–153 (ed. E. Hörandl and M. S. Appelhans). Ruggell: Gantner Verlag, IAPT, Slovakia.Google Scholar
  29. Jauhar P. P. 1981 Cytogenetics and breeding of pearl millet and related species, pp. xix + 289. Alan R. Liss, New York.Google Scholar
  30. Jungmann L., Vigna B. B., Boldrini K. R., Sousa A. C., Valle C. B., Resende R. M. et al. 2010 Genetic diversity and population structure analysis of the tropical pasture grass Brachiariahumidicola based on microsatellites, cytogenetics, morphological traits, and geographical origin. Genome 53, 698–709.PubMedCrossRefGoogle Scholar
  31. Kalia R. K., Rai M. K., Kalia S., Singh R. and Dhawan A. K. 2011 Microsatellites markers: an overview of the recent progress in plants. Euphytica 177, 309–334.CrossRefGoogle Scholar
  32. Kharrat-Souissi A., Baumel A., Torre F., Juin M., Yakovlev S. S., Roig A. et al. 2011 New insights into the polyploid complex Cenchrus ciliaris L. (Poaceae) show its capacity for gene flow and recombination process despite its apomictic nature. Aust. J. Bot. 59, 543–553.CrossRefGoogle Scholar
  33. Kumar S. and Saxena S. 2016 Sequence characterized amplified regions linked with apomictic mode of reproduction in four different apomictic Cenchrus species. Mol. Plant Breed. 7, 1–14.Google Scholar
  34. Kumar S., Saxena S., Rai A., Radhakrishna A. and Kaushal P. 2019 Ecological, genetic, and reproductive features of Cenchrus species indicate evolutionary superiority of apomixis under environmental stresses. Ecol. Indic. 105, 126–136.CrossRefGoogle Scholar
  35. Kumari K., Muthamilarasan M., Misra G., Gupta S., Subramanian A., Parida S. K. et al. 2013 Development of eSSR-markers in Setaria italica and their applicability in studying genetic diversity, cross transferability and comparative mapping in millet and non-millet species, PLoS One 8, e67742.PubMedPubMedCentralCrossRefGoogle Scholar
  36. Martel E., De Nay D., Siljak-Yakovlev S., Brown S. and Sarr A. 1997 Genome size variation and basic chromosome number in pearl millet and fourteen related Pennisetum species. J. Hered. 88, 139–143.CrossRefGoogle Scholar
  37. Martel E., Poncet V., Lamy F., Siljak-Yakovlev S., Lejeune B. and Sarr A. 2004 Chromosome evolution of Pennisetum species (Poaceae): implications of ITS phylogeny. Plant Syst. Evol. 249, 139–149.CrossRefGoogle Scholar
  38. Martins W. S., Lucas D. C. S., Neves K. F. S. and Bertioli D. J. 2009 WebSat - A web software for microsatellite marker development. Bioinformation 3, 282–283.PubMedPubMedCentralCrossRefGoogle Scholar
  39. Nagy S. P., Cernák I. P., Gorji A. M., Hegedűs G. and Taller J. 2012 PIC calc: an online program to calculate polymorphic information content for molecular genetic studies. Biochem. Genet. 50, 670–672.PubMedCrossRefGoogle Scholar
  40. Oliveira A. F., Cidade W. F., Fávero A. P., Vigna B. B. Z. and Souza A. P. 2016 First microsatellite markers for Paspalum plicatulum (Poaceae) characterization and cross amplification in different Paspalum species of the Plicatula group. BMC Res. Notes 9, 511.PubMedPubMedCentralCrossRefGoogle Scholar
  41. Pernès J. 1975 Organization evolution d’un groupe agamique: la section des Maximae du genre Panicum (Gramineae), pp. 1–106. ORSTOM, Paris.Google Scholar
  42. Perrier X. and Jacquemoud-Collet J. 2006 DARwin software (
  43. Powell W., Machray G. and Provan J. 1996 Polymorphism revealed by simple sequence repeats. Trends Plant Sci. 1, 215–222.CrossRefGoogle Scholar
  44. Rozen S. and Skaletsky H. 2000 Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics methods and protocols (ed. S. Krawetz and S. Misener), pp. 365–386. Humana Press, Totowa, NJ.Google Scholar
  45. Snyder L. A., Hernandez A. R., Warmke H. E. 1955 The mechanism of apomixis in Pennisetum ciliare. Bot. Gazet. 116, 209–221.CrossRefGoogle Scholar
  46. Souza J. S., Chiari L., Simeaao R. M., Vilela M. M., Salgado L. R. 2018 Development, Validation and Characterization of Genic Microsatellite Markers in Urochloa Species. Am. J. Plant Sci. 9, 281–295.CrossRefGoogle Scholar
  47. Srivastava M. K., Yadav C. B., Bhat V. and Kumar S. 2011 Cloning and characterization of apartial cDNA encoding Xyloglucan endotransglucosylase in Pennisetum glaucum L. Afr. J. Biotechnol. 10, 9242–9252.CrossRefGoogle Scholar
  48. Temnykh S., DeClerck G., Lukashova A., Lipovich L., Cartinhour S. and McCouch S. 2001 Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res. 11, pp. 1441–1452.PubMedPubMedCentralCrossRefGoogle Scholar
  49. Thiel T., Michalek W., Varshney R. K. and Graner A. 2003 Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). TAG Theor. App. Genet. 106, 411–422.CrossRefGoogle Scholar
  50. Varshney R. K., Sigmund R., Borner A., Korzun V., Stein N., Sorrels M. E. et al. 2005 Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat, rye and rice. Plant Sci. 168, 195–202.CrossRefGoogle Scholar
  51. Wang C., Yan H., Li J., Zhou S., Liu T., Zhang X. et al. 2018 Genome survey sequencing of purple elephant grass (Pennisetum purpureum Schum ‘Zise’) and identification of its SSR markers. Mol. Breed. 38, 94.CrossRefGoogle Scholar
  52. Wipff J. K. 2003 Pennisetum Rich. In Flora of North America North of Mexico, Magnoliophyta: Commelinidae (in part): Poaceae, part 2 (ed. M. E. Barkworth, K. M. Capel, S. Long, M. B. Piep), vol. 25, pp. 515–529. Oxford University Press, New York.Google Scholar
  53. Yadav C. B., Dwivedi A., Kumar S. and Bhat V. 2019 AFLP-based genetic diversity analysis distinguishes apomictically and sexually reproducing Cenchrus species. Braz. J. Bot. 42, 361–371.CrossRefGoogle Scholar
  54. Zhou S., Wang C., Yin G., Pennerman K. K., Zhang J., Yan H. et al. 2019 Phylogenetics and diversity analysis of Pennisetum species using Hemarthria EST-SSR markers. Grassl. Sci. 65, 13–22.CrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2019

Authors and Affiliations

  • Sazda Abdi
    • 1
  • Anuj Dwivedi
    • 1
  • Shashi
    • 1
  • Suresh Kumar
    • 2
  • Vishnu Bhat
    • 1
    Email author
  1. 1.Department of BotanyUniversity of DelhiDelhiIndia
  2. 2.Division of BiochemistryICAR-Indian Agricultural Research InstituteNew DelhiIndia

Personalised recommendations