New Resistance Gene Analog Polymorphism Primers Designed Based On Conserved Domain Sequences of Disease Resistance Genes in Cereals.

Most previously designed resistance gene analog polymorphism (RGAP) primers were based on disease resistance genes in dicot crops and might not be very effective to identify markers linked to disease resistance genes in cereals.  Here we report the development and utilization of RGAP markers using primers designed based on cloned wheat and barley genes for disease resistance.  The DNA sequences of the conserved motifs (P-loop, Kin2, NBS-B and GLPL) of Yr10, Lr1, Lr10, Lr21, Lr35, Pm3b, and Mla1 and their start code flanking regions were used to design primers.  A total of 35 primers were designed. The lengths of the primers range from 15 to 18 nucleotides and the annealing temperatures range from 38 to 54^oC.  PCR amplicons were resolved using 5.0-6.5% polyacrylamide gels or an automatic Li-COR DNA analyzer. When various wheat genotypes were tested, the sizes of the amplicons ranged from 50 to 700 bp, and the average of the amplicon numbers was 74 (ranged from 42 to 131). Compared to previous primers designed based on resistance genes from other plant species, the bands produced by the new primers were more reproducible and robust. These primers were successfully used in mapping three wheat genes for stripe rust resistance in populations developed from wheat crosses AvS/PI 183527, AvS/PI 178759, and AvS/ PI 192252. The markers developed with these primers can be used in mapping resistance genes, determining genetic diversity, and identifying genotypes of wheat and barley; and also possibly other plants.