|Wilson, Jeffrey - Jeff|
Submitted to: African Journal of Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/28/2009
Publication Date: 5/17/2010
Citation: Gulia, S.K., Singh, B., Wilson, J.P. 2010. A simplified, cost- and time-effective procedure for genotyping pearl millet in resource-limited laboratories. African Journal of Biotechnology. 9:2851-2859.
Interpretive Summary: Pearl millet is predominantly grown as a staple food crop in the semit-arid regions of Africa, the Indian subcontinent, and in Brazil. Abiotic and biotic stresses limit yield of grain and biomass. Understanding the genetic inheritance of these traits would be facilitated by application of DNA marker technology. In the regions where it is most widely grown, use of DNA technology can be limited because of costs. This research developed a modified pearl millet DNA extraction procedure, which differed from earlier ones in terms of time, simplicity, and resource and technology requirements, while reducing use of costly enzymes and corrosive and hazardous chemicals. The protocols developed in this study should result in a more economical application of DNA marker technology in resource-limited laboratories.
Technical 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 0.5-0.7g ground young leaf tissue in 2% CTAB/ß-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 640ng/µL 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 electrophoresis. 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. In summary, the procedure reported is an intermediate between maxi- and mini-prep DNA extractions suited for resource limited laboratories engaged in molecular breeding requiring large volume of genotyping.