Abstract Background Resistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2 , the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele. Results Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli -based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. Conclusions Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele. Nomenclature Palmer amaranth, Amaranthus palmeri S. Wats.; protoporphyrinogen oxidase, PPO; tall waterhemp, Amaranthus tuberculatus ( Moq .) J.D.Sauer

Can double PPO mutations exist in a same allele and are such mutants functional?