Primers that amplify inserts in a multicloning site also hybridize to Sorghum bicolor DNA

One or both members of a pair of primers developed to permit polymerase chain reaction amplification of sorghum DNA fragments cloned into the PstI site of pUC18 were shown to hybridize to sorghum DNA. The presence of the same primer sequences on the ends of amplified inserts posed a problem in using the amplified inserts as hybridization probes because the high signal level of the primer-detected DNA fragments often obscured the segregation patterns of the restriction fragments detected by the insert DNA. Conditions that favor annealing of the insert rather than the primers were experimentally defined, however, so that directly amplified DNA sequences could be used as RFLP probes. Cosegregation analysis of 51 F2 individuals from a cross between BTx 623 and IS 3620C established a linkage group containing the Pd1 locus. Alleles at the locus are revealed as codominant bands on Southern blots of heterozygotes, but the segregation ratio among the F2 progeny deviated significantly from the expected 1:2:1. The distortion favored the allele from parent BTx 623.