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Strong phosphine resistance in Tribolium castaneum is due to point mutations in DNA that code for amino acid changes of P45S and/or G131S in the enzyme dihydrolipoamide dehydrogenase (DLD). One allele coding for P45S is the most common in all phosphine-resistant US populations and in one strain from Brazil (TCBR), whereas another allele, G131S, occurs only in Australian beetles. Dose-mortality studies found that the TCBR strain was more resistant to phosphine than US populations. To investigate strong resistance mutations in TCBR, we sequenced cDNA for DLD in TCBR and compared results with a US population from Kansas. The common P45S mutation was detected in both populations, but two additional mutations G131D and V167A were identified only from TCBR. We used a CAPS marker (Cleaved Amplified Polymorphic Sequence) for P45S, herein designated M1, to survey this resistance allele in TCBR. We also developed a marker for the G131D mutation, designated M2. Only two genotypes, R1R1S2S2 (homozygous for resistance at M1, but homozygous susceptible at the M2 site) and R1S1R2S2 (heterozygous for resistance at M1 and M2) existed in TCBR. However, phosphine resistance levels were similar between individuals with the two genotypes. Beetles with strong resistance in TCBR may be homozygous for either the presence of the common P45S allele, or heterozygous for two resistance alleles at a second locus, G131D. The strong resistance phenotype in TCBR may be expressed differently based on previously unknown mutations at the DLD locus, but this will require further research to resolve. [ABSTRACT FROM AUTHOR]