Molecular marker assays for wild-type or mutant P34 alleles. A. Sequence alignment encompassing all of the primers utilized in the P34 molecular marker assays. P34WT represents Williams 82 genomic DNA sequence on soybean chromosome 8; P34x represents PI 567476 and PI 603570A mutant P34 alleles; ps05 and ps08 represent the P34 pseudogenes on soybean chromosomes 5 and 8, respectively. Lower case text represents the 5′-untranslated region, and the first exon sequences are in uppercase text. Red sequences indicate bases that differ for one or both of the P34 pseudogenes. Blue bolded bases represent the direct repeat resulting from the four-basepair insertion in PI 567476 and PI 603570A alleles. Gray highlighted sequences represent the regions corresponding to the primers used for the P34 size assay and the SimpleProbe assay. The italicized bases in the P34x sequence represent the SimpleProbe sequence. Underlined sequences in the P34WT and P34x sequences represent the regions corresponding to the primers used for the GC tail assay. These assay primers also contained tails (described in Materials and Methods). B. Example of genotype output from the P34 GCtail assay. Melting curves generated from the negative first derivative SYBR Green I fluorescence disappearance over temperature. The blue peak is from a wild-type P34 sample, the red peaks are from a heterozygous P34 sample, and the green peak is from a four-basepair insertion P34 sample. C. Example of genotype output from the P34 size assay. Shown is fragment analysis output of GeneScan 600 LIZ standards (orange) with their sizes listed on the x-axis. The blue peaks represent a heterozygous P34 sample which produced two intense peaks on either side of the 160 bp standard, with the 157 bp product representing the wild-type P34 allele product, and the 161 bp product representing the four-basepair insertion P34 product. D. Example of genotype output from the P34 SimpleProbe assay. Melting curves generated from the negative first derivative of SimpleProbe fluorescence disappearance over temperature. The blue peaks are from wild-type P34 samples, the red peaks are from heterozygous P34 samples, and the green peaks are from four-basepair insertion P34 samples.