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

Abstract

In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T. laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based 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 marker in two of the populations. These results demonstrated that the PCR marker system using the FSD and RSA primer pair permitted a rapid and reliable identification of individual lines carrying the Bt-10 gene for resistance to common bunt. Key words: resistance gene, genetic segregation, molecular marker, RAPD analysis.