Identification and Sequence-Based Validation of the EST-SSR Markers from Calotropis procera

Calotropis procera (Aiton) Dryand is a medicinally and economically important woody shrub used to cure various diseases. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from C. procera and studied the cross-species transferability of the selected markers. We have identified 34,355 potential EST-SSRs from C. procera and classified them based on the type of repeat motifs and Gene Ontology (GO) annotation. Virtual primers for 17,036 EST-SSRs were designed, and 55 primer pairs representing di-, tri-, tetra-, penta-, and hexa-nucleotide repeats were used for the validation of the EST-SSRs. A total of 48 out of 55 EST-SSR primers showed clear amplification in Calotropis procera . To validate EST-SSRs at the nucleotide level, four randomly selected EST-SSR primer sets were amplified and sequenced. Sequence alignment confirmed the presence of SSRs in the amplified primers. The polymorphisms of the 16 primer pairs were studied, and it was observed that 15 primer sets produced polymorphic bands. Out of these 15 pairs of primers, we identified 141 alleles with an average of 2.56 alleles per locus. The allelic diversity was estimated for 15 EST-SSR markers using three accessions of Calotropis . The study has succeeded in the development of novel EST-SSR markers from Calotropis procera and establishment of the molecular marker resources that can serve as a useful genomic tool for further development of germplasm resources and effective marker-assisted breeding and domestication programs for C alotropis procera .