marker-assisted selection in wheat

Chapter 7 – Marker-assisted selection in common beans and cassava 85Figure 1Examples of gel multiplexing for MAS of A) BGYMV and B) BCMV resistance genesLoad 1S alleleR alleleLoad 2S alleleR alleleLoad 3S alleleR alleleA) bgm-1 geneRS SCAR (Urrea et al., 1996/CIAT)Controls530bp 570bpGel 1Load 2 Load 1 Gel 2Load 3 Load 2 Load 1 Gel 2Load 3Load 2Load 1B) bc-3 geneROC11 SCAR (Johnson et al., 1997)intermediate level of resistance, while otherminor genes must be recovered throughconventional phenotypic selection to assurehigher resistance.Scaling up of MAS required the developmentof simple operational procedures inboth the field (tagging, tissue collection) andthe laboratory (DNA extraction, markerevaluation). For gamete selection strategiesin the field, individual, evenly-spaced plantsfrom segregating populations were markedwith numbered tags that were coated withparaffin to protect them until seed harvest.Leaf disks were sampled from young vegetativetissue with a paper hole puncherand placed directly into pre-numbered cellsof microtitre 96-well plates stored on ice,ready for grinding and extraction in thelaboratory. The implementation of MASfor bgm-1 and subsequently for SW12.700in the laboratory required substantial adaptationof standard protocols to establishhigh-throughput procedures. Grinding ofsamples in microtitre plates was accomplishedwith a block of 96 pegs that fitinto each well. Alkaline DNA extraction(Klimyuk et al., 1993) was employed withsuccess for both markers, and eventually itwas possible to multiplex the markers inboth the amplification and gel phases usingmultiple primer PCR and multiple loadingper gel wells (Figure 1A). With experienceand improved procedures, efficiency morethan doubled over a two-year period. MASwas often carried out before flowering todecide on a plant’s status as a carrier of theresistant allele for further use in crossing.Two small red seeded lines developed inthe Panamerican School using MAS havereached the stage of validation in Honduras(J.C. Rosas, personal communication) andshown resistance to the BGYMV strainsprevalent there. Resistance to BGYMV ofdrought tolerant lines selected at CIAT wasmaintained using MAS for one or moregenes, followed by field selection in CentralAmerica. Similarly, red mottled lines developedin CIAT with the aid of MAS showedfield resistance in the Caribbean and one ofthese lines from the red mottled advancedline for the Caribbean (RMC) series hasbeen released (Blair et al., 2006). MAS hasalso been an important element of maintainingBGYMV viral resistance in CIAT’sprogramme as other breeding objectivessuch as nutritional value have beenassumed, necessitating the inclusion of susceptibleparents in crosses with resistant