Isolation and characterization of microsatellite markers for analysis of molecular variation in the medicinal plant Madagascar periwinkle (Catharanthus roseus (L.) G. Don) Bhumika Shokeen, Niroj Kumar Sethy, Sushil Kumar, Sabhyata Bhatia * National Center for Plant Genome Research, Aruna Asaf Ali Marg, Post Box No. 10531, Jawaharlal Nehru University Campus, New Delhi 110067, India Received 23 July 2006; received in revised form 17 October 2006; accepted 17 October 2006 Available online 27 November 2006 Abstract Catharanthus roseus (L.) G. Don, commonly known as Madagascar periwinkle, is a plant of great medicinal value. In this study microsatellite markers were developed and utilized for analysis of genetic diversity in C. roseus. In order to isolate microsatellite sequences, two small insert genomic libraries of C. roseus cv. Nirmal were constructed and screened with CA, CT, GC and GCG oligonucleotide repeats. Sixty-five microsatellite motifs were identified, from which 38 functional STMS primer pairs were designed and validated. Out of these, 24 STMS markers were used to evaluate the genetic polymorphism in 37 genotypes, which comprised of 32 accessions of C. roseus, a single accession each of two related species (C. trichophyllus and C. pusillus) and one accession each of three related genera (Vinca minor, Thevetia peruviana and Nerium indicum). The 24 STMS markers detected 26 loci with two markers amplifying more than one locus. A total of 124 alleles were amplified in the 37 genotypes ranging from 2 to 10 alleles with an average of 4.76 alleles per locus. The high average expected heterozygosity (H E ) value of 0.56 and observed heterozygosity (H O ) value of 0.52 established the efficiency of the STMS markers for discriminating the C. roseus genotypes. Nei and Li’s similarity coefficients were calculated and a UPGMA-based dendrogram was constructed which clearly distinguished all genotypes except two pairs. Sequence analysis of the length variant alleles at three STMS loci revealed that the variation in the copy number of repeat motifs was the major source of length polymorphism within C. roseus. However, isolated point mutations and indels in the microsatellite flanking regions (MFRs) of homologus loci from other species also contributed to size homoplasy and allelic size variation. This study is the first report of microsatellite development and utilization in C. roseus, providing significant insights into its genome structure and organization. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Catharanthus roseus; Microsatellites; Genetic diversity; Madagascar periwinkle; STMS 1. Introduction Catharanthus roseus (L.) G. Don (Madagascar periwinkle) of the Apocynaceae family is widely grown both for its ornamental and more recently for its medicinal importance. The plant possesses an unsurpassed spectrum of chemodiver- sity in the form of 130 mono-terpenoid indole alkaloids including the antileukemic vincristine and vinblastine, and antihypertensive ajmalicine and serpentine [1]. However, the major factor limiting the extensive use of these alkaloids for medicinal applications is their extremely low yields in vivo coupled with the difficulties in their isolation and purification. Different conventional and biotechnological approaches are being used for increasing the pharmaceutically important alkaloids [2]. In this direction major success has been achieved in dissecting the highly complex alkaloid biosynthetic pathway. A number of enzymes along with several genes have been isolated and are available for construction of transgenic cells and plants with modified alkaloid profiles [3]. Large-scale efforts are on for the metabolic engineering of the alkaloid biosynthetic pathway. Hence, due to the large research efforts targeted towards achieving better yields of the pharmaceuti- cally important alkaloids, C. roseus has become an important model medicinal plant system for biotechnology and secondary metabolism studies [4]. The genus Catharanthus consists of eight species, of which seven are endemic to Madagascar and one species (C. pusillus) is endemic to India [5]. C. roseus (2n =2x = 16) is the most www.elsevier.com/locate/plantsci Plant Science 172 (2007) 441–451 * Corresponding author. Tel.: +91 11 26735159; fax: +91 11 26716658. E-mail address: sabhyatab@yahoo.com (S. Bhatia). 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.10.010