DNA-barcoding, SCoT and SRAP Based Somaclonal Variation in Micropropagated Withania somnifera Plantlets

Ashwagandha (Withania somnifera) is one of the recognized plant species that considered of most traditional natural supplements. Tissue culture is an efficient method as fast and affordable in plant propagation. Few studies have discussed the genetic impact of such method on ashwagandha plant. The aim of this research was to identify the genetic stability of micropropagated plantlets and to assess the impact of in vitro-propagation on somaclonal variability in ashwagandha using start codon-targeted (SCoT), sequence-related amplified polymorphism (SRAP) and DNA-barcoding assays. SCoT marker assay produced a total number of 132 bands with an average of 11 bands per primer, where scorable PCR fragments were generated from all primers. The phylogenetic tree constructed using SCoT binary data, revealed genetic variability among studied plant samples. SRAP primer combinations showed a total of 78 bands by an average of 11.1 bands / combination, in which all combinations produced scored PCR fragments. Over SRAP assay, one specific band was obtained that was present in different ashwagandha micropropagated plant samples compared to the control (mother plant). This PCR fragments were obtained using me1F/em1R primer combination (287 bp). The phylogenetic tree constructed using SRAP data was successful to differentiate between micro-propagated plants and the control. The DNA- barcoding analysis using chloroplast gene RNA polymerasel (rpoCl) gene was used to detect the soma- clonal variation between control and one micro-propagated plant of ashwagandha. The phylogenetic tree constructed using DNA-barcoding sequences was successful to differentiate between the two samples, where control and micropropagated plantlets were grouped in two different groups. This study suggests the valuableness of using SRAP and DNA-barcoding in detecting soma-clonal variation among micropropagated plantlest of ashwagandha.