marker-assisted selection in wheat - ictsd

Chapter 3 – Molecular markers for use in plant molecular breeding and germplasm evaluation 33can be added to the primers to reduce thenumber of co-amplified bands. AFLPs havemany of the advantages of RAPDs, but havemuch better reproducibility. AFLP technologyrequires greater technical skill thanRAPDs and, because AFLPs run on polyacrylamidegels instead of agarose, they alsorequire a larger investment in equipmentthan RAPDs. Using manual gels, AFLPbands are detectable using silver stain, orby labelling of the primers with a radioactiveisotope. Alternatively, for higherthroughput, AFLPs can be detected with anautomated DNA sequencer by using fluorescentlylabelled primers.Diversity array technology (DArT) isa modification of the AFLP procedureusing a microarray platform (Jaccoud et al.,2001) that greatly increases throughput. InDArT, DNA fragments from one sampleare arrayed and used to detect polymorphismsfor the fragments in other samplesby differential hybridization (Wenzl etal., 2004).Developing molecular markerswith DNA sequence informationWhen the DNA sequence is available, it ispossible to design primers to amplify acrossa specific locus. However not all loci willbe polymorphic. Targeting highly variablesequence features increases the likelihoodof detecting polymorphism. These highlyvariable features include tandem repeatssuch as microsatellites, and dispersed complexrepeats such as transposable elements.MicrosatellitesSimple sequence length polymorphisms(SSLPs), also known as simple sequencerepeats (SSRs), or microsatellites, consist oftandemly repeated di-, tri- or tetra-nucleotidemotifs and are a common feature ofmost eukaryotic genomes. The number ofrepeats is highly variable because slippedstrand mis-pairing causes frequent gain orloss of repeat units. With their high levelof allelic diversity, microsatellites are valuableas molecular markers, particularly forstudies of closely related individuals.PCR-based markers are designed toamplify fragments that contain a microsatelliteusing primers complementary tounique sequences surrounding the repeatmotif (Weber and May, 1989). Differencesin the number of tandem repeats are readilyassayed by measuring the molecular weightof the resulting PCR fragments. As the differencesmay be as small as two base pairs,the fragments are separated by electrophoresison polyacrylamide gels or usingcapillary DNA sequencers that providesufficient resolution.Without prior sequence knowledge,microsatellites can be discovered by screeninglibraries of clones. Clones containing therepeat motif must be sequenced to findunique sites for primer design flanking therepeats. Microsatellite marker developmentfrom pre-existing sequence is far more direct.Good reviews of microsatellite markerdevelopment include those of McCouchet al. (1997) and Zane, Bargelloni andAtarnello (2002). Microsatellites discoveredin non-coding sequence often have a higherrate of polymorphism than microsatellitesdiscovered in genes. However, in somespecies such as spruce (Picea spp.) withhighly repetitive genomes, SSR markersdeveloped from gene sequences have fewerinstances of null alleles, i.e. failure of PCRamplification (Rungus et al., 2004).Microsatellite markers have severaladvantages. They are co-dominant; the heterozygousstate can be discerned from thehomozygous state. The markers are easilyautomated using florescent primers on anautomated sequencer and it is possible