Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR

Abstract

The Pi-ta gene from indica introgressed into japonica rice has been used to control the blast disease caused by the fungal pathogen Magnaporthe grisea (Herbert) Barr. (anamorph Pyricularia oryzae Cav.) worldwide. A single nucleotide length polymorphism (SNLP) was identified at the intron region of the Pi-ta gene to develop a codominant Pi-ta gene marker suitable for genotyping with an automated machine. The DNA primer specific to the resistant Pi-taallele was labeled with blue dye (FAM, 6-carboxyfluorescein) as a forward primer, the DNA primer specific to the susceptible pi-ta allele was labeled with green dye (HEX, 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) as another forward primer and the DNA primer identical to both Pi-ta/pi-ta alleles was unlabeled as the reverse primer for polymerase chain reaction (PCR). Using these three primers, a 181-bp blue peak in homozygous resistant and a green peak of 182–183 bp in homozygous susceptible, and both peaks in heterozygous plants were produced by PCR. The utility of marker was verified using a segregating F₂ population, inbred cultivated lines, dominant markers and pathogenicity testing. A codominant Pi-ta marker was thus developed for effective Pi-ta assisted selection for crop improvement. Using highly homologous competitive primers for allele detection by PCR can benefit the study of genome organization of the complex locus.