In-Silico Identification and Characterization of EST-SSR Markers from Catharanthus roseus for Genetic Diversity Analysis

Abstract

Catharanthus roseus is one of the most significant and important perennial plant of Apocynaceae family, globally known for its ornamental and medicinal values. Molecular markers have great importance in order to investigate composition, genetic variation and evolutionary relationships. In this study, accessions collected from five north Indian state were cultivated at Plant Conservatory, Punjabi University, Patiala and their morphological variation were analyzed. In total 22, 867 ESTs (expressed sequence tags) were downloaded from NCBI database and assembled into 2,853 sequences with an average size of 659 bp by using bioinformatic tools. Among assembled dataset a total of 427 SSRs (simple sequence repeats) were successfully detected in 358 sequences. For functional annotation all SSR containing sequences were subjected to Blast-x with E-value of 10-5 against NCBI-nr (non-redundant) database and Swiss-Prot database that resulted functional hits for 279 (75%) sequences. Furthermore, GO (Gene ontology) was predicted by comparing the sequences with Arabidopsis database (TAIR), total 268 GO ids were assigned to 259 sequences. Additionally, 141 different pathways were predicted by using KEGG database with highest enrichment of metabolic pathways category. Finally, to validate the identified SSR containing sequences, total 260 primers targeting repeat regions were designed. Further, 28 primers of class I category were selected for validation among 25 accessions of C. roseus and resulted that 17 primers amplified the corresponding repeated region. Additionally, among validated primers, 5 primers were found to be polymorphic further used for analysis of variation among accessions. These identified markers can be further used to investigate the variation among the species and can be helpful for detection of genes responsible for the survival of plant for particular stress condition.