marker-assisted selection in wheat - ictsd

32Marker-assisted selection – Current status and future perspectives in crops, livestock, forestry and fishunsequenced RFLP markers amongresearchers requires an infrastructure forthe maintenance and distribution of clonedprobes for use by multiple researchers.However, if end-sequence or full-clonesequence information is available, the probescan be amplified readily from genomicDNA via the polymerase chain reaction(PCR), and the cumbersome aspects ofclone maintenance and distribution areavoided. The polymorphisms detected byRFLPs may result from single base changescausing a loss of restriction sites or a gain ofnew restriction sites, or from insertions anddeletions (indels) between restriction sites(McCouch et al., 1988; Edwards, Lee andMcCouch, 2004).PCR-based markersMany advances in molecular marker technologyhave come through applications ofthe PCR method (Mullis et al., 1986). InPCR, a thermo-stable DNA polymeraseenzyme makes copies of a target sequencebeginning from two small pieces of syntheticallyproduced DNA (primers) thatare complementary to sequences bracketingthe target. Through iterations of theprocess with heating to separate the doublestranded DNA molecules and cooling toallow the primers to re-anneal, the targetsequence is exponentially amplified.Polymerase chain reaction-based markersrequire much less DNA per assay thanRFLPs and are more compatible with automatedhigh-throughput genotyping (i.e. theability to process large numbers of samplesquickly and efficiently).Randomly amplified polymorphic DNAmarkersRandomly amplified polymorphic DNAmarkers (RAPDs) use PCR to amplifystretches of DNA between single primersof arbitrary sequence (Williams et al., 1990;Welsh and McClelland, 1990). Amplificationoccurs only where sequences complementaryto the primers are in close enoughproximity for successful PCR. The typicaloligonucleotide used for RAPDs isten bases long and will amplify many locisimultaneously, allowing multiple markersto be assayed in a single PCR reactionand a single lane on an agarose gel. As theprimers are arbitrary, RAPD technologycan be applied directly to any species withno prior sequence knowledge. This technologyis particularly useful when thereis a need to assay loci across the entiregenome. The polymorphisms are detectedonly as the presence or absence of a bandof a particular molecular weight, and itis not possible to differentiate betweenhomozygous and heterozygous markers.RAPDs are notoriously unreliable because,aside from sequence differences, the amplificationor failure of amplification of anyband may be sensitive to any number offactors, including DNA template quality,PCR conditions, reagents and equipment.Amplified fragment lengthpolymorphismsAmplified fragment length polymorphisms(AFLPs) are molecular markers derivedfrom the selective amplification of restrictionfragments (Vos et al., 1995). GenomicDNA is digested with a pair of restrictionenzymes and oligonucleotide adaptors areligated to the ends of each restriction fragment.The fragments are amplified usingprimers that anneal to the adaptor sequenceand extend into the restriction fragment.Only a portion of restriction fragmentswill be within the range of sizes than can beamplified by PCR and visualized on polyacylamidegels (between 50 and 350 bp). Forlarge genomes, additional selective bases