|Wilson, Jeffrey - Jeff|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/18/2009
Publication Date: 3/28/2009
Citation: Gulia, S.K., Singh, B.P., Wilson, J.P., Ma, X. 2009. Successful application of new cost-effective procedures for genotyping pearl millets for genetic diversity and linkage mapping. 15th Biennial Agricultural Research Director's Symposium, Atlanta, Georgia. March 28 - April 1, 2009. p. 118.
Interpretive Summary: not required
Technical Abstract: In spite of technology advancement, procedures of DNA extraction and genotyping of large plant populations are cumbersome and expensive. Therefore, in order to genotype large mapping populations for studying genetic diversity, and linkage/QTL mapping for disease and pest resistance in pearl millet (Pennisetum glaucum), we have engaged in developing time saving and economical procedures for DNA extraction, PCR amplification and PAGE electrophoresis. Plant materials in this study included germplasm, landraces, advanced breeding lines and cultivars collected from India, Africa and the U.S. for genetic diversity studies and a RIL mapping population based on cross Tift 454 × Tift 99B for linkage mapping. In newly modified procedure, DNA was extracted by incubating 0.5-0.7g ground young leaf tissues in 2% CTAB/ß-mercaptoethanol followed by refrigerated differential centrifugation with phenol:chloroform:isoamylalcohol and again with chloroform:isoamylalcohol. Steps such as additional phenol/chloroform treatments, DNA pellet drying followed RNase treatments and incubation were eliminated, reducing use of costly and corrosive chemicals, and saving time over the old two-day DNA extraction procedure. DNA produced from 185 RILs exhibited average concentration of 640ng/µL and average optical density ratio of 1.9. Upon PCR amplification, this DNA produced clear and scorable bands following ethidium bromide stained agarose and silver stained polyacrylamide gels. Post PCR multiplexing of two or more microsatellites based on different lengths of base pairs reduced the time and cost of per unit data generation up to half. Microsatellite screening of RIL parental lines recorded more than 25% polymorphism that will be used for fingerprinting of RILs and other genotypes.