Indian Journal of Biotechnology Vol 13, October 2014, pp 464-472 Utilization of EST-derived SSRs in the genetic characterization of Artemisia annua L. genotypes from Ladakh, India Jitendra Kumar 1 , Prasad Bajaj 2 , Gyan P Mishra 1 , Shashi Bala Singh 1 , Harvinder Singh 2 and Pradeep K Naik 1 * 1 Defence Institute of High Altitude Research, Defence R & D Organization, Leh 194 101, Jammu & Kashmir, India 2 Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan 173 215, India Received 15 November 2012; revised 7 May 2013; accepted 19 June 2013 Artemisia annua L. is an important medicinal plant that produces substantial quantity of artemisinin, an antimalarial agent. In India, it grows wild in the Ladhak region and has harboured considerable variability over the years. EST-derived SSR markers were used to measure the genetic diversity among the A. annua germplasm collected from geographically separated Leh (11,500 ft) and Nubra (9600 ft) valleys (Ladakh, India). After analysing 68,974 non-redundant (of 3,60,906 available) ESTs of A. annua, 4,342 SSR markers were developed. On an average, one SSR was found per 8.9 kb of EST sequence with dinucleotide motifs in highest frequency (52.2%), followed by tri (42.4%), tetra (3.6%), hexa (1.2%) and pentanucleotide (0.6%) repeat types. A set of 16 primer pairs were designed by considering only the SSR-containing ESTs from the artemisinin biosynthetic pathway. In total, 38 alleles were identified from 13 polymorphic SSR loci, ranging from 1-7 alleles per locus and displayed moderate genetic diversity with an average of 0.24. It was found that the genetic diversity among individual from Nubra valley was narrower than that of Leh valley, suggesting the importance and feasibility of introducing elite genotypes from different origins for Artemisia germplasm conservation and breeding programmes. Keywords: Artemisia annua, cluster analysis, expressed sequence tag (EST), genetic diversity, simple sequence repeat (SSR) marker Introduction Artemisia annua L. is an annual herb, native to Asia, and commonly found in many countries throughout Europe, North America, Central and South America. It is one of the most important medicinal plants from cold arid regions of India, especially in the Ladakh region. It is well adapted to high altitudes of 9600-11500 ft above mean sea level (MSL) and grows well under low temperature, nutrient deficiency and environmental stress to which they are exposed. Over the years, the Artemisia populations in the Ladakh region have developed considerable variability, necessitating the study of genetic diversity and characterization. This plant synthesizes and accumulates substantial quantities of many derivatives of a cadiene skeleton, including artemisinin (endoperoxide seco sesquiterpene lactones). Artemisinin is currently the most effective agent against multidrug resistant strains of Plasmodium species, the malarial parasites 1,2 . This property of A. annua has placed it among the top ten industrial medicinal plants of the modern world. Chemical synthesis of the artemisinin is commercially nonviable, and efforts to produce artemisinin in engineered yeast cells as well as in tissue cultured cells have so far not been very fruitful 3 . Thus, the sole source of the drug is from the widely grown or cultivated plants 4 . Since the production of artemisinin varies among the genotypes, it has generated worldwide interest in studying the genetic diversity of A. annua populations, cloned variants, chemotypes, ecotypes and in the development of pure-line cultivars. For effective utilization and protection of plant genetic resources, the analysis of genetic diversity and relatedness between or within different genotypes is a prerequisite 5 . It also helps in developing DNA based molecular markers for identification of genotypes with better traits. However, so far reports are not available regarding the genetic characterization of this plant from Ladakh region and hence a detailed investigation is required. Traditionally, the genetic diversity of the plant is assessed by various DNA fingerprinting methods using specific and non-specific DNA-based markers. Microsatellites or simple sequence repeats (SSRs) based DNA markers are short tandem repeats of 1 to 6 bp in length, evenly distributed in the genomes and  *Author for correspondence: Tel: +91-1792-239227; Fax: +91-1792-245362 E-mail: pknaik1973@gmail.com