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This paper describes and discusses strategies for screening microsatellites for use in plant genetic research and illustrates how a subset of useful microsatellites can be optimised for implementation on breeding and research using a range of techniques. Beginning with the initial screening of microsatellites 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 lines of interest. Each stage of this process benefits from a fresh examination of the techniques applied in order to increase the efficiency with which key 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 the basis for high resolution screening for polymorphisms. As markers are defined 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 either from the analysis of progeny from a cross or the routine checking of cultivar identity in the industry. We then examine appropriate analysis platforms and refinement of PCR primers and conditions in order to identify procedures 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 the sequence of the primer itself, based on available sequence information, or by adding tails to the primers. The latter is shown to alter the |
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