Detection and analysis systems for microsatellite markers in wheat

Abstract

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 ‘stutter’ pattern that is commonly observed with wheat microsatellites so that a single band is prominent and thus allows size polymorphisms to be more readily 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 markers in a costeffective fashion. The potential of alternative analytical systems and further refinement of primers to show plus/minus reactions with wheat lines in order to produce simple tests for use in breeding programs are also discussed.

An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts

“…Due to the abundance of single nucleotide polymorphisms (SNPs) and development of sophisticated high-throughput SNP detection systems, it has recently been proposed that SNP markers will have a great influence on future mapping research studies and MAS (Rafalski, 2002;Koebner & Summers, 2003). At present, methods for detection and analysis of widely-used markers are becoming faster and more sophisticated, and many of these methods are automated Hori et al, 2003;Rampling et al, 2001;Warburton et al, 2002). One example of an improvement in the efficiency of marker analysis is multiplex PCR, which enables multiple marker loci to be tested simultaneously Donini et al, 1998;Masi et al, 2003).…”

Molecular discrimination of Bx7 alleles demonstrates that a highly expressed high-molecular-weight glutenin allele has a major impact on wheat flour dough strength

“…Genescan analysis of PCR products of the whole DH population, was carried out with the addition of the 0.04 mlL/PCR reaction dUTP-labelled with the fluorescent dye, R110 (Applied Biosystems). The PCR product was run on an ABI 377 sequencer and the data subsequently processed using Genescan 3.1 software (Rampling et al 2001). …”

“…Amplification conditions for the PCR reaction were an initial cycle at 95C for 5 min, followed by 38 cycles of; 95C for 30 s, 58C for 30 s and 72C for 1 min, followed by a final extension at 72C for 5.25 min. PCR products were visualised as described by Rampling et al (2001). Genescan analysis of PCR products of the whole DH population, was carried out with the addition of the 0.04 mlL/PCR reaction dUTP-labelled with the fluorescent dye, R110 (Applied Biosystems).…”

Fine mapping of qGW1, a major QTL for grain weight in sorghum

“…PCR reactions were carried out using an M13-tagged forward primer (Rampling et al 2001) as previously described (Li et al 2009). The reaction mixture (10 μl) contained 20 ng template DNA, 1 pmol forward primer, 5 pmol reverse primer, 5 pmol M13 (−29) primer, 1 μl of 10 × PCR buffer, 0.8 μl of 2.5 mM dNTPs, and 0.5 U Taq polymerase.…”