Evaluation and implementation of DNA-based diagnostic methodology to distinguish wheat genotypes
Thesis (MSc)--Stellenbosch University, 2007.
The aim of this study was to develop a DNA-based diagnostic system that can be used to distinguish between genotypes in the wheat breeding program at the University of Stellenbosch. Known marker systems were investigated and the chosen marker system would then be implemented to determine its utility in the breeding program. Three marker systems were considered, i.e. microsatellites, Amplified Fragment Length Polymorphisms (AFLPs) and various retrotransposon-based markers. Each system is based on polymerase chain reaction (PCR) amplification from specific primer pairs. The multitude of primer options was narrowed down during a review of published literature regarding wheat molecular markers. Thirty nine microsatellite primer pairs and nine AFLP primer combinations were chosen for the initial genotype evaluation. Four different retrotransposonbased techniques were investigated; namely Inter-Retrotransposon Amplified Polymorphism (IRAP), REtrotransposon-Microsatellite Amplified Polymorphism (REMAP), Sequence- Specific Amplified Polymorphism (SSAP) and, a derivative of these developed in this study, Wis-2 Retrotransposon Amplification. The study started with twenty genotypes which included varieties/breeding lines from five breeding programmes. The genotypes were chosen as representative of the respective breeding populations and were used in the initial testing of the marker systems. Eighteen microsatellites were evaluated using the panel of twenty genotypes. From this, six primer pairs (Xgwm190, Xgwm437, Xgwm539, Xwmc11, Xwmc59 and Xwmc177) were chosen to test the semi-automated DNA sequencer detection system. A single band/peak in each microsatellite profile was used for genotyping. Four of the primer pairs were labelled with different fluorochromes which enabled them to be multiplexed. The differences in amplification products of the six microsatellites meant that all six could be detected in one electrophoresis run. The banding pattern produced by microsatellite Xwmc177 was complex and highly polymorphic and was therefore also analysed in the same way as the AFLP patterns. When analyzed in this manner it proved to be more informative than the combination of six microsatellites (with a single prominent band scored in each). Three AFLP primer combinations could also be multiplexed and visualised together. The three EcoRI selective primers were labelled with different dyes and used with one MseI selective primer. The SSAP system also used fluorescently labelled primers and proved to be the most useful of the retrotransposon-based methods. However, this system produced such a large amount of data that it made analysis too time consuming. Therefore the six microsatellites and three AFLP primer combinations (MseI-CTC and EcoRI-ACA, -AAC, - AGG) were selected for routine genotyping. Due to the numerous highly polymorphic bands produced by the SSAP system it could be very useful to differentiate very closely related genotypes that cannot be distinguished with the markers proposed for routine use. A panel of 119 breeding lines were then used to implement the two chosen marker systems. The results obtained for these markers were used to produce a dendrogram of the lines using the SAS cluster analysis function. The clusters showed that most of the lines could be distinguished from each other. The MseI-CTC and EcoRI-AGG primer combination was the most informative. It produced the largest number of clusters (53) and could therefore discriminate between more of the lines than any other method. The dendrograms and clusters allowed sixteen of the breeding lines to be selected to test the optimal number of seeds to represent an entire population (variety/breeding line) as one seed was not sufficient. It was decided that eight seeds could provide a good representation of the intra-line variability.