A simplified, cost- and time-effective procedure for genotyping pearl millet in resource-limited laboratories

Abstract

Procedures for DNA extraction and genotyping of large plant populations are cumbersome and expensive for resource-limited laboratories. Through eliminating or changing several steps used in DNA extraction, PCR amplification and PAGE electrophoresis in pearl millet [Pennisetum glaucum (L.) R. Br.], we developed a modified procedure that reduced the cost of consumables and required less time without compromising data quality. In the revised procedure, DNA was extracted by incubating 0.5 - 0.7 g ground young leaf tissue in 2% CTAB/-mercaptoethanol followed by refrigerated differential centrifugations with phenol:chloroform:isoamylalcohol. Steps such as additional  phenol/chloroform treatments, DNA pellet drying followed by RNase treatments and incubation were eliminated, reducing use of costly and corrosive chemicals and saving time. DNA produced from 174 genotypes exhibited an average concentration of 640 ng/ìL and average optical density ratio of 1.9. PCR amplification of SSR markers with this DNA produced clear and scorable bands following ethidium bromide stained agarose and silver stained polyacrylamide gel eletcrophoresis. Post PCR duplexing of two or more microsatellites based on different lengths of base pairs reduced the time and cost per unit data generation by up to half as compared to single marker per PAGE. Cluster analysis performed on the marker data generated 11 SSR primers following these procedures formed two main groups from genotypes of the U.S. origin. In summary, the procedures reported are simplified, shortened and economical and well suited for resource limited laboratories engaged in molecular breeding requiring large volume of genotyping.