marker-assisted selection in wheat - ictsd

Chapter 3 – Molecular markers for use in plant molecular breeding and germplasm evaluation 35Table 1SNP technologiesAllele discrimination• Hybridization• Primer extension• Ligation• Invasive cleavageDetection methods• Gel separation• Arrays• Mass spectrometry• Plate readersrequires obtaining an initial DNA sequencein a reference individual followed by someform of re-sequencing in other varietiesto find variable base pairs. In addition todirect sequencing, SNPs can be discoveredthrough ecotilling with the CEL I enzyme(Comai et al., 2004) or by denaturing highpressure liquid chromatography (DHPLC)to measure small conformational differenceswhen PCR amplified sequences arehybridized to a reference sequence (Kwok,2001). In addition to SNP discovery, bothDHPLC and ecotilling are viable technologiesfor SNP detection. There is a myriad ofother SNP assay technologies in developmentand to date no single method standsout as superior to the others. Table 1 listssome examples of SNP allele discriminationmethods and detection systems that can becombined in various ways (see reviews byKwok, 2001 and Gut, 2001). The benefits ofSNP assays include increased speed of genotyping,lower cost and the parallel assay ofmultiple SNP.Single feature polymorphisms andmicroarray-based genotypingIndel polymorphisms, also known as singlefeature polymorphisms (SFPs), are particularlyamenable to microarray-basedgenotyping. These assays are done by labellinggenomic DNA (target) and hybridizingto arrayed oligonucleotide probes that arecomplementary to indel loci. Each SFPis scored by the presence or absence of ahybridization signal with its correspondingoligonucleotide probe on the array. Bothspotted oligonucleotides (Barrett et al.,2004) and Affymetrix-type arrays (Borevitzet al., 2003) have been used in these assays.The SFPs can be discovered throughsequence alignments or by hybridizationof genomic DNA with whole genomemicroarrays. The advantage of microarrayplatforms for genotyping is that they arehighly parallel, and they are well suited forapplications such as quantitative trait loci(QTL) analysis, where whole genome coveragewith many markers is desirable.Special considerations fordiversity studies and germplasmevaluationThe interpretation of molecular marker datafor germplasm classification and diversitycan be confounded by uncertainty about theunderlying sources of the polymorphismsand by homoplasy (false homology). ForRFLPs in rice, indels can account for asmuch or more of the polymorphism aschanges in the restriction sites themselves(Edwards, Lee and McCouch, 2004). AFLPsand RAPDs can also be sensitive to bothindels and base changes. The ratio of indelsto base changes is important for diversitystudies because, when molecular markersare used to estimate nucleotide divergence,the divergence will be overestimated ifindel-derived polymorphisms are common(Upholt, 1977; Nei and Miller, 1990; Innanet al., 1999). The greatest certainty of theunderlying polymorphism comes fromSNP technologies that directly assay forsingle base changes.For SSR markers among closely relatedindividuals, most polymorphism shouldbe caused by expansion or contractionof the number of repeat units. However,as genetic distance between the varietiesincreases, there is an increasing chancethat indel events will cause additional size