Genomic DNA extraction, PCR amplification and sequencing

The genomic DNA from the sterilized plant material was extracted using the method suggested by Doyle and Doyle⁵⁸. Extracted DNA was quantified employing the method of Sambrook et al.⁵⁹. Purity of the DNA was further determined by electrophoresis in agarose gel (0.8%). PCR amplification of three chloroplast DNA (cpDNA) markers (trnL-F, rbcL and psbA-trnH) was carried out using the extracted DNA as template. A single PCR protocol was recruited in respect of all three chloroplast regions. In the process, 20 μL reaction mixture containing extracted genomic DNA template (2 μL) (1:10 dilution of the extracted DNA), forward primer (1 μL), reverse primer (1 μL), 1 × final concentration of ReadyMix Taq PCR reaction mix (SIGMA-ALDRICH) (10 μL) and nuclease free water (6 μL). The PCR was carried out in Thermal cycler (BIO-RAD iCycler). PCR program settings included: 94 °C for 4 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1.30 min, and a final elongation step at 72 °C for 10 min. Amplified chloroplast markers (trnL-F, rbcL and psbA-trnH) were visualized on 1% agarose gel under UV light by staining with ethidium bromide. Amplified PCR products were purified using GenElute PCR Clean-up kit (SIGMA-ALDRICH) and sequenced at Barcode Biosciences, Bangalore.