Development of a PCR marker for rapid identification of the Bt-10 gene forcommon bunt resistance in wheat

In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) i s effective against all the known races of common bunt caused by Tilletia t ritici and T. laevis. The genotypes of 199 F-2 plants, originated from a cr oss between BW553 containing Bt-10 and the susceptible spring wheat cultiva r 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bu nt reaction, indicating that Bt-10 was expressed in a partially dominant fa shion. A polymorphic DNA fragment, amplified using RAPD, and previously sho wn to be linked to Bt-10 was sequenced and SCAR (sequence characterized amp lified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a pol ymorphic fragment of 490 bp resulting from a single base pair difference be tween lines possessing Bt-10 and those lacking the gene. As per the base pa ir difference, FSD and RSA primers were designed to generate a 275-bp polym orphic DNA fragment. Both 275- and 490-bp polymorphic fragments were presen t in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cul tivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F-2 population. Using Southern analysis, the 490-bp f ragment was effective in differentiating homozygous resistant plants from t hose heterozygous for Bt-10, based on its presence and the hybridization si gnal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was als o observed for the molecular marker and corresponded to 88% of the phenotyp es deduced from the original F-2 population. The molecular marker was estim ated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, b ased on the segregation analysis. Segregation analyses of Bt-10 and the 275 -bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3:1 for the molecular mark er in two of the populations. These results demonstrated that the PCR marke r system using the FSD and RSA primer pair permitted a rapid and reliable i dentification of individual lines carrying the Bt-10 gene for resistance to common bunt.