Abstract
In the present study, two DNA extraction methods, namely, promega kit and CTAB method were utilized to optimize Polymerase chain reaction (PCR) protocol to amplify microsatellite of Coffea arabica based on a standardized PCR experiment. The experiment was conducted using explant of a newly released variety of coffee named Aba buna and tissue culture derived lines propagated from Aba buna. Primarily, the hybrid coffee, Aba buna was studied using 7 primer pairs that amplified microsatellite with genbank accession number AJ308755, AJ308779, AJ308782, CFGA91, CFGA92, AJ250251 and AJ250253. And for further experiments, three individuals of the coffee explant and the other two, tissue culture derived individuals micropropagated from Aba buna were studied at molecular level using 11 primer pairs, which amplified 11 microsatellites known to be with genbank accession number AJ308755, AJ308779, AJ308782, AJ308790, AJ308837, CFGA91, CFGA92, CFGA69, CFGA100, CFGA465 and CFGA502. The PCR amplicons were analyzed in horizontal agarose gel electrophoresis. Microsatellite allele separation was investigated using vertical polyacryl amide gel electrophoresis. PCR amplification failed using a highly colourful template DNA extracted by the promega kit. However, the PCR experiments resulted in high yield of microsatellite amplicon when a template DNA extracted by the Doyle & Doyle CTAB method was used. The present finding suggests that diverse secondary metabolites occurring in coffee leaf might have acted as PCR inhibitor though the exact mechanism of inhibition is unclear. The present research gave insight in promotion of the Doyle & Doyle CTAB method for isolating coffee DNA and recommends the need of modifying the promega kit to extract a coffee genomic DNA free of impurities. In a nutshell, the Doyle & Doyle CTAB method seems the most promising method to purify clean DNA, which is an ideal template to amplify SSR of C.arabica for genotyping coffee cultivars.
