marker-assisted selection in wheat

Chapter 7 – Marker-assisted selection in common beans and cassava 87CIAT for the selection of BGYMV resistanceas discussed previously. Althoughmost BCMV and BCMNV resistance geneshad been tagged with SCAR markers,implementation required efforts to validateand scale up the use of the markers inapplied breeding programmes. Genotypingfor the ROC11 marker was carried out onadvanced lines given that this marker isdominant and in repulsion with the resistanceallele. In other words, the absence of aband was indicative of the presence of therecessive bc-3 allele and therefore it wasmore appropriate to evaluate after fixationof the alleles to homozygosity throughmass or pedigree selection with singleplant selections in the F 4 and F 5 generationwhen single plant rows were evaluatedfor the resistance gene marker. To determinewhether the advanced line continuedto segregate for the gene, alkaline DNAextraction was conducted on leaf discscollected from four leaflets from four individualplants per line using a hole-puncherrather than from a single plant per familyor advanced line. The presence or absenceof polymerase chain reaction (PCR) productswas evaluated for each genotype basedon scanned photographs or gel captureimagery of multiplexed gels (Figure 1B) topredict if the genotype contained the resistanceor the susceptible allele.Once optimized for parental genotypes,MAS was conducted on a large number ofprogeny rows. For example in 2003, morethan 4 000 advanced lines were evaluated forthe ROC11 marker for genotypes grown atthree sites within Colombia (CIAT-Darien,CIAT headquarters and CORPOICA-Rionegro). DNA was collected at all threesites and shipped successfully to the laboratoryin 96-well plate format as discussedabove. Both the ROC11 and SW13 markerswere single copy SCARs that did not produceextra bands and therefore were easyto multiplex. To facilitate the evaluationof markers on a large number of advancedlines, usually within two to three weeks,and increase the efficiency of MAS, severalinnovations were implemented: loading ofagarose gels (first with two and then threeloadings), increasing numbers of wells percomb (first 30-well and then 42-well combswere used), use of 384-well PCR plates andmultipipetor loading of gels. The resultingsavings decreased the time to PCR amplifyand load a gel by approximately 50 percentand increased the number of genotypes runper gel by 225 percent.The rapid increase in efficiency obtainedduring the application of the ROC11marker shows the advantages of testingnew markers in practical breeding programmes.The use and advantages of thesemolecular markers has been presented atan Organization of American States-sponsoredcourse in Colombia given in 2002and a Rockefeller Foundation-sponsoredcourse in Uganda given in 2003. Based onthis programme and the training courses,MAS for BCMV genes was initiated aspart of a recently approved Associationfor Strengthening Agricultural Research inEastern and Central Africa (ASARECA)project for three countries in eastern Africaand training of researchers from the Andeanregion has allowed more breeding linesfrom Peru to be screened (CIAT, 2004).Other examples of MAS for simplyinherited traitsSeveral pathogens, especially fungalpathogens, have co-evolved with the beanhost, and present a population structure(Andean/MesoAmerican) that mimicsthe major gene pools of bean (Pastor-Corrales, Jara and Singh, 1998). This isthe case with Phaeoisariopsis griseola, the