TECHNICAL NOTE Development of micro satellite markers for a critically endangered species, Ceropegia fantastica from the Western Ghats, India R. C. Sumangala Æ L. Naveen Kumar Æ B. T. Ramesha Æ R. Uma Shaanker Æ K. N. Ganeshaiah Æ G. Ravikanth Received: 9 January 2009 / Accepted: 19 January 2009 / Published online: 1 February 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Ceropegia fantastica L. (Asclepiadaceae) is a highly endemic and endangered species in the Western Ghats of India. Fourteen microsatellite markers were developed for C. fantastica. Eight microsatellite primers screened had 2–5 alleles per locus and the observed and expected heterozygosity ranged from 0.48 to 0.83 and 0.48 to 0.62, respectively. The primers were also evaluated for their cross amplification against two related species Ce- ropegia hirsuta and Ceropegia oculata. The microsatellites developed for this species could be used for addressing population genetics of this endemic and critically endan- gered species. Keywords Microsatellites Á Ceropegia fantastica Á Western Ghats Á Asclepiadaceae Á Cross amplification Á Endangered species Ceropegia L. (Apocynaceae) is an old world tropical genus containing about 200 species (Bruyns 1985). There are about 48 species of Ceropegia in India, with many of them being endemic (Huber 1957). Ceropegia fantastica is an endangered species mostly confined to the northern regions of Western Ghats, a mega diversity hotspot in south India. The species is sparsely distributed with no more than 50 individuals at any given population (Yadav et al. 2006). In this paper, we report the development of microsatellite markers and discuss the utility of these markers in addressing questions related to the population genetics of this species. The plant material of C. fantastica and its closely related species, Ceropegia hirsuta and Ceropegia oculata were collected from north Western Ghats (N 16°06 0 041 00 E074°08 0 8 00 ) in south India. Genomic DNA was extracted from all the individuals of each species using CTAB method (Doyle and Doyle 1987). The extracted DNA was purified and digested with RsaI and ligated to linker oligonucleotides SNX-F and SNX-R (5 0 -GTTTAAGGCCTAGCTAGCAGAATC-3 0 ;5 0 -GATTC TGCTAGCTAGGCCTTAAACAAAA-3 0 ) using the rapid DNA ligation kit (Fermentas International). These DNA fragments were hybridized to biotinylated oligos (micro- satellite probes) and captured by using dynabeads. Beads and attached probes were separated magnetically from the supernatant. Following stringent washes, the bound DNA (pure gold) was recovered using Fermentas TA cloning kit. The pure gold DNA was incorporated into a pTZ57R plasmid (NEB) vector. Ligation and transformation was performed as per Fermentas TA cloning kit. The positive clones were picked and subjected for colony PCR using the universal M13 forward and M13 reverse primers. The purified PCR products were sequenced using Cycle sequencer (ABI PRISM 3100 Genetic Analyzer, Applied R. C. Sumangala Á L. Naveen Kumar Á R. Uma Shaanker Á K. N. Ganeshaiah Á G. Ravikanth (&) Conservation Genetics Laboratory, Ashoka Trust for Research in Ecology and the Environment, #659, 5th A Main, Hebbal, Bangalore 560024, India e-mail: gravikanth@gmail.com B. T. Ramesha Á R. Uma Shaanker Á K. N. Ganeshaiah Á G. Ravikanth School of Ecology and Conservation, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India R. Uma Shaanker Department of Crop Physiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India K. N. Ganeshaiah Department of Forestry and Environmental Sciences, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India 123 Conserv Genet (2009) 10:1825–1827 DOI 10.1007/s10592-009-9825-0