Identification of molecular markers in wheat and maize using AFLP technology

Abstract

The optimisation and implementation of a molecular fingerprinting system and its ultimate use in marker assisted selection of identified maize and wheat genotypes for the CSIR are described in this dissertation. The Amplified Fragment Length Polymorphism (AFLP) method was optimised for the maize trait gamete sterility, and for the wheat traits eye spot resistance and cytoplasmic male fertility. Maize and wheat DNA were extracted from selected genotypes employing standardized protocols. A commercial AFLP kit was used to obtain suitable fingerprint fragments for analysis. As this kit was not designed for very large genomes, conditions for the large wheat genome required optimisation. AFLP fingerprints generated from the different genotypes of maize and wheat were evaluated for potentially trait linked markers. Polymorphic fragments were identified by their presence in genotypes containing the trait of interest, and their absence from fingerprints of genotypes that did not contain the trait of interest. These amplification products were identified and selected as possible markers. These potential markers were converted to Sequence Characterised Amplified Regions (SCARs) by cloning the selected amplification products to T-tailed pSK vector, and subsequent sequencing of the fragment. Primers were designed to recognise these amplification regions in the tested genotypes. Primers were optimally designed to include or exclude the restriction sites of the enzymes at each end of the amplified fragments and annealing temperatures normalised for each of the primer sets. The primers were validated by amplification of the tested genotypes with the primer sets at specific reaction and temperature cycling conditions. For maize, two primer sets were identified that could be developed to identify gamete sterility genotypes. In the case of wheat, one primer set for each of the traits spot resistance and cytoplasmic male fertility was identified for their potential use as markers.