Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.)

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resis tance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusar ium wilt resistance, we developed cleaved amplified polymorphic sequences ( CAPS) and restriction fragment length polymorphisms (RFLP) markers by conve rting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectiv ely. The RAPD-PCR polymorphic fragments from the susceptible line 'Vedranta is' were cloned and sequenced in order to construct primers that would ampl ify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (de signated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible me lon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragmen ts amplified from resistant (PI 161375) and susceptible ('Vedrantais') line s and were then confirmed by electrophoresis of restriction endonuclease di gestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI , and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 11 9 F-2 individuals from crosses of 'Vedrantais' x PI 161375 and 'Ananas Yokn eam'x MR-1 respectively, and 17 families from a backcross BC1S1 population derived from the breeding line 'MD8654' as a resistance source. BclI- and M spI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resist ant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed ove r 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F-2 individuals and BC1S1 families tested. This is the first report of PCR-base d CAPS markers linked to resistance/susceptibility for Fusarium wilt in mel on. The RFLP markers resulting from probing with a clone-derived 1.05-kb SC G17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were codominant, easier to score, and more accura te and consistent in predicting the melon phenotype than the RAPD markers f rom which they were derived.