Detection and analysis systems for microsatellite markers in wheat

This paper describes and discusses strategies for screening microsatellites for use in plant genetic research and illustrates how a subset of useful m icrosatellites can be optimised for implementation on breeding and research using a range of techniques. Beginning with the initial screening of micro satellites for potential polymorphisms in a core set of potential parental lines, through to scaling up for mapping or breeding purposes, we present a time- and cost-effective approach to microsatellite analysis in wheat line s of interest. Each stage of this process benefits from a fresh examination of the techniques applied in order to increase the efficiency with which k ey markers can be identified and implemented. For the primary screening we use primers without modification to prime PCRs in the presence of f-dNTP (fluorescently labelled nucleotide) to provide t he basis for high resolution screening for polymorphisms. As markers are de fined for use in a breeding program, the focus changes to a smaller set of primer pairs that will be used to screen large numbers of DNA samples eithe r from the analysis of progeny from a cross or the routine checking of cult ivar identity in the industry. We then examine appropriate analysis platfor ms and refinement of PCR primers and conditions in order to identify proced ures that can be implemented widely, not just in specialised well-equipped laboratories. In many cases we are able to use lower cost agarose analysis for identified polymorphisms. Where this is not feasable we examine primers for potential redesign to optimise their application either by altering th e sequence of the primer itself, based on available sequence information, o r by adding tails to the primers. The latter is shown to alter the 'stutter ' pattern that is commonly observed with wheat microsatellites so that a si ngle band is prominent and thus allows size polymorphisms to be more readil y scored. The addition of a generic 5' tail also provides a method of using a generic fluorescent primer that can be applied to multiple tagged marker s in a cost-effective fashion. The potential of alternative analytical syst ems and further refinement of primers to show plus/minus reactions with whe at lines in order to produce simple tests for use in breeding programs are also discussed.