European Journal of Biotechnology and Bioscience  ~ 7 ~  Volume: 3, Issue: 4, 7-9 May 2015 www.biosciencejournals.com ISSN: 2321-9122 Impact Factor: 3.742 T. Amutha Priya Lab of Developmental Biology and Plant Biotechnology SPK Centre for Environmental Sciences Manonmaniam Sundaranar University, Tamil Nadu, India V. Manimekalai Lab of Developmental Biology and Plant Biotechnology SPK Centre for Environmental Sciences Manonmaniam Sundaranar University, Tamil Nadu, India P. Ravichandran Lab of Developmental Biology and Plant Biotechnology SPK Centre for Environmental Sciences Manonmaniam Sundaranar University, Tamil Nadu, India Correspondence: T. Amutha Priya Lab of Developmental Biology and Plant Biotechnology SPK Centre for Environmental Sciences Manonmaniam Sundaranar University, Tamil Nadu, India Intra Specific Genetic Diversity Studies on Calotropis gigantea (L) R. Br. - Using RAPD Markers T. Amutha Priya, V. Manimekalai, P. Ravichandran Abstract Calotropis gigantea (L.) (Asclepiadaceae) is an important medicinal plant which exhibits variations in flower color, morphology and anatomy. Taxonomists treat these variants as two different forms due to contrasting floral features. Hence, a molecular marker based study using random amplified polymorphic DNA (RAPD) was carried out to ascertain the origin of morphological variations as well as to check whether the floral variation is due to phenotypic or genotypic factor. Further the study was aimed to assess the genetic diversity of these two forms collected from ten different accessions. Genomic DNA was isolated and purified from flower petals using the procedure described by Jobes et al., with minor modifications. The DNA content was quantified and purity was checked using Spectrophotometer. Twenty decamer random operon primers were used to amplify the DNA and RAPD-PCR was done. High level of genetic similarity was observed between the white and pink form accessions. Ten random primers, each with 10 bases generated a total of 16 polymorphic bands out of 209 total bands. In addition to the morphological variations a significant level (16 %) of polymorphism of was observed. Overall genetic similarity based on 10 random primers was 84 %. Cluster analysis was carried out based on UPGMA Jaccard coefficient. The results reveal relatively a rich source of genetic diversity of the selected variants. The observed variation and polymorphism among the morphological forms deemed to be worth for conservation of C. gigantea. The molecular variations and data obtained from these two forms can be of taxonomic importance. Further analysis involving ISSR or mini/micro satellite markers is required to confirm the results obtained. Keywords: Genetic diversity, Calotropis gigantea, RAPD-PCR, primers, ISSR Introduction Calotropis gigantea is a common xerophytic perennial shrub. This plant is a soft-wooded, evergreen, perennial shrub. C. gigantea ranges from 8 to 10 feet in height, and grows in various climates and soils, and sometimes in habitats where nothing grows. Consequently, it is a common wasteland weeding [1]. Calotropis is used many diseases such as fevers, rheumatism, indigestion, cough, cold, eczema etc [2]. Calotropis gigantea yields a durable fibre (commercially known as bowstring of India) useful for roots, carpets, fishing nets, and sewing thread. Molecular techniques have found to be more useful and accurate for determination of both intra and inter specific variations in plants. Randomly amplified polymorphic DNA (RAPD) markers, in particular, have been successfully employed for determination of intra-specific genetic diversity in several species. These include date palm [3]. Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Molecular marker based study using random amplified polymorphic DNA (RAPD) was carried out to ascertain the origin of morphological variations as well as to check whether the floral variation is due to phenotypic or genotypic factor. Materials and Methods Calotropis gigantea plants were collected from Ambur, Alwarkurichi, Tenkasi, and Papanasam, along the road sides and wastelands of Tirunelveli district, South India. The plants were maintained in the centre’s nursery for tissue culture and further genetic diversity studies. Plant Material and DNA extraction Calotropis gigantea two forms collected genomic DNA was isolated from flower petals by [4] with minor modifications.