Figure 1.
Figure 1.

Genomic organization of the P34 gene for Williams 82 and low P34 soybean accessions PI 567476 and PI 603570A. A. Four exons (open boxes) and three introns (lines separating exons) were assembled from trace archive sequence reads and amplification and sequencing of Williams 82 genomic DNA in the P34 gene region. Short sequences of 5′ and 3′ untranslated regions were also amplified and sequenced as represented by dashed lines. At the start codon of the P34 allele of the low P34 accessions, a four-basepair insertion results in a direct sequence repeat (TATGTATG). The position of the insertion is represented by a solid triangle. B. Wild-type sequence of the P34 gene in the start codon region. 5′ untranslated nucleotides are in lowercase letters, the start codon ATG is in italics, and the translated nucleotides are in uppercase letters, with each codon separated by a space. Only the first ten codons are represented. Below the nucleotide sequence is a translation of the first ten amino acids of the wild-type P34 protein. C. Mutant sequence of the P34 allele in the start codon region. One possible effect of the P34 four-basepair insertion allele (the direct repeat is underlined) is translation initiation at the first ATG (italics) and a frameshift producing a 17 amino acid peptide. The second ATG is listed in bold, and the sequence only represents the first ten codons. Below the nucleotide sequence is the frameshifted peptide that would result from translation of the mutant P34 allele. D. Mutant sequence of the P34 allele in the start codon region. Another possible effect of the P34 four-basepair insertion is a reduction or elimination of translation initiation at the second ATG (bold) due to sequence changes just upstream from the start codon, including an additional ATG (italics).

 


Figure 2.
Figure 2.

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.

 


Figure 3.
Figure 3.

Association of the low P34 phenotype with the PI 567476 and PI 603570A P34 four-basepair insertion alleles. A. Coomassie Blue stained total seed proteins extracted from ground bulk seed samples and separated by SDS-PAGE from Population 1 and the parental lines PI 567476 and Elite 1. The allele bulks consisted of mixed ground seed from the indicated genotype class listed and each lane represents the proteins extracted from 50 μg of dried seed corrected for dilution. MW indicates protein molecular weight standards, and the individual proteins are labeled with their apparent molecular mass. B. Western blot with anti-P34 monoclonal antibodies on duplicate gel, as in panel A. C. Coomassie Blue stained total seed proteins extracted from ground bulk seed samples and separated by SDS-PAGE from Population 2 and the parental line PI 603570A as well as the wild-type P34 cultivar Williams 82. Each lane is labeled similar to those in panel A. D. Western blot with anti-P34 monoclonal antibodies on duplicate gel, as in panel C.

 


Figure 4.
Figure 4.

Reduction of P34 protein level in four-basepair-insertion P34 lines. A. Coomassie Blue stained total seed proteins extracted from ground bulk seed samples representing 50 μg dry ground seed and separated by SDS-PAGE from the PI 603570A and the mutant P34 allele bulk sample from Population 2. The cultivar Williams 82 and the wild-type P34 allele bulk samples were diluted an additional eightfold (representing 6.25 μg dried seed). The allele bulks consisted of mixed ground seed from the indicated genotype class. MW indicates protein molecular weight standards, and the individual proteins are labeled with their apparent molecular mass. B. Western blot with anti-P34 monoclonal antibodies on duplicate gel, as in panel A.