Evaluation and implementation of DNA-based diagnostic methodology to distinguish wheat genotypes

[摘要] The aim of this study was to develop a DNA-based diagnostic system that can be used todistinguish between genotypes in the wheat breeding program at the University ofStellenbosch. Known marker systems were investigated and the chosen marker system wouldthen be implemented to determine its utility in the breeding program.Three marker systems were considered, i.e. microsatellites, Amplified Fragment LengthPolymorphisms (AFLPs) and various retrotransposon-based markers. Each system is basedon polymerase chain reaction (PCR) amplification from specific primer pairs. The multitudeof primer options was narrowed down during a review of published literature regarding wheatmolecular markers. Thirty nine microsatellite primer pairs and nine AFLP primercombinations were chosen for the initial genotype evaluation. Four different retrotransposonbasedtechniques 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 fivebreeding programmes. The genotypes were chosen as representative of the respectivebreeding populations and were used in the initial testing of the marker systems. Eighteenmicrosatellites were evaluated using the panel of twenty genotypes. From this, six primerpairs (Xgwm190, Xgwm437, Xgwm539, Xwmc11, Xwmc59 and Xwmc177) were chosen to testthe semi-automated DNA sequencer detection system. A single band/peak in eachmicrosatellite profile was used for genotyping. Four of the primer pairs were labelled withdifferent fluorochromes which enabled them to be multiplexed. The differences inamplification products of the six microsatellites meant that all six could be detected in oneelectrophoresis run.The banding pattern produced by microsatellite Xwmc177 was complex and highlypolymorphic and was therefore also analysed in the same way as the AFLP patterns. Whenanalyzed in this manner it proved to be more informative than the combination of sixmicrosatellites (with a single prominent band scored in each). Three AFLP primer combinations could also be multiplexed and visualised together. Thethree EcoRI selective primers were labelled with different dyes and used with one MseIselective primer. The SSAP system also used fluorescently labelled primers and proved to bethe most useful of the retrotransposon-based methods. However, this system produced such alarge amount of data that it made analysis too time consuming. Therefore the sixmicrosatellites and three AFLP primer combinations (MseI-CTC and EcoRI-ACA, -AAC, -AGG) were selected for routine genotyping. Due to the numerous highly polymorphic bandsproduced by the SSAP system it could be very useful to differentiate very closely relatedgenotypes 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 usingthe SAS cluster analysis function. The clusters showed that most of the lines could bedistinguished from each other. The MseI-CTC and EcoRI-AGG primer combination was themost informative. It produced the largest number of clusters (53) and could thereforediscriminate between more of the lines than any other method.The dendrograms and clusters allowed sixteen of the breeding lines to be selected to test theoptimal number of seeds to represent an entire population (variety/breeding line) as one seedwas not sufficient. It was decided that eight seeds could provide a good representation of theintra-line variability.

[发布机构] Stellenbosch University