Figure 1.
Figure 1.

Sequence of soybean low phytic acid (Lpa) genes in lpa1-a SNP region and lpa1-a molecular marker assay allele discrimination. (A) Portion of the Lpa1 (Glyma03 g32500) sequence aligned with related sequences. Arrow indicates position of lpa1-a (CX1834) mutation (A3828T, relative to start codon). Black background with white text represents primers used for amplification for SimpleProbe analysis, and underline indicates region to which SimpleProbe corresponds. Gray background with black text indicates differences between Lpa1 and Lpa2. (B) Typical Lpa1 genotyping result with SimpleProbe on Lightcycler 480 II (Roche Applied Sciences). Lpa1 (W82/5601T) gives a characteristic peak of 66.5°C, and the peak for lpa1-a (CX1834) is 63°C. Heterozygotes show both peaks.

 


Figure 2.
Figure 2.

High sequence identity in a portion of the Lpa1 homologs from maize, soybean, and Arabidopsis. Alignment of a portion of the predicted protein structure for Lpa1 (Glyma03 g32500.1), Lpa2 (Glyma19 g35230.1), AtMRP5 (AT1G04120.1), and maize MRP5 (GI:162464191). Arrow and underline at position Lpa1 894 in alignment represents location of effect of the lpa1-a nonsense mutation (R894STOP). Second arrow and underline at position 1039 of Lpa2 indicates residue affected by lpa2-a and lpa2-b mutations. lpa2-a results in an R1039K change, while lpa2-b results in a R1039STOP change. Identical residues are indicated by white background with black text. Gaps and nonsimilar amino acid substitutions are indicated by black background with white text. Similar amino acid substitutions are indicated by light gray background with black text, and weakly similar substitutions are indicated by white text and dark gray background. The alignment before 701 is not shown due to space limitations, but mutant alleles do not differ from the ‘Williams 82’ gene models before residue 664 (Lpa1) or 493 (Lpa2).

 


Figure 3.
Figure 3.

Sequence of Lpa genes in lpa2-a single nucleotide polymorphism region and lpa2-a molecular marker assay allele discrimination. (A) Portion of the Lpa2 (Glyma19 g35230) sequence aligned with similar sequences. Arrow indicates position of lpa1-a (CX1834) G4866A missense mutation. Black background with white text represents primers used for SimpleProbe analysis. Underline indicates region to which SimpleProbe binds. Gray background with black text indicates differences between Lpa2 and Lpa1. (B) Typical Lpa2 genotyping result with SimpleProbe on DNA Engine Opticon 2 (MJ Research/Bio-Rad). Lpa2 (W82/5601T) yields a characteristic peak at 62°C, lpa2-a (CX1834) gives a characteristic peak at 57°C, and lpa2-b (M766) yields a characteristic peak at 58.5°C. Heterozygotes show both peaks (Lpa2/lpa2-a peaks shown in orange).

 


Figure 4.
Figure 4.

SequenceLOGO of region of multidrug resistance proteins from National Center for Biotechnology Information BLAST matches (limited to Viridiplantae). Lpa2 protein sequence surrounding the region affected by the lpa2-a mutation (1 = residue 1019 of Lpa2) was used as a tBLASTn query against NCBI Viridiplantae mRNA entries. The top 100 best matches were downloaded and duplicate entries removed to yield a total of 84 entries. Sequences were then aligned and used as input for sequence LOGO (http://weblogo.berkeley.edu/ [verified 4 May 2009]). Height is proportionate to occurrence of that residue in the 84 entries used. Arrow indicates residue changed due to the lpa2-a mutation (R1039K).

 


Figure 5.
Figure 5.

Seed inorganic phosphate (Pi) phenotype and Lpa1 and Lpa2 genotype association analyses. (A) Average inorganic phosphate content of soybean seeds from a segregating F5:8 recombinant inbred line population. Labels indicate Lpa1 and Lpa2 genotype, respectively: T = 5601T allele of Lpa1 or Lpa2, cx = lpa1-a or lpa2-a. Error bars indicate one standard deviation. n = the number of lines corresponding to a genotypic class found within this population. (B) Average inorganic phosphate content of soybean seeds from a segregating population derived from one F9:10 near-isogenic line population heterozygous for both Satt237 and Satt561 (see Materials and Methods). Labels indicate Lpa1 and Lpa2 genotype, respectively: T = 5601T allele Lpa1 or Lpa2, cx = lpa1-a or lpa2-a. Error bars indicate one standard deviation. n = the number of seeds corresponding to a genetypic class found within this population.

 


Figure 6.
Figure 6.

Recombination events between simple sequence repeat (SSR) marker Satt237 and Lpa1. Representation of the genotypes and inorganic phosphate (Pi) phenotype of two recombinant lines in independent F9:10 near-isogenic lines (NILs) displaying recombination resulting in the lpa1-a alleles donated from CX1834 and the SSR marker Satt237 donated from 5601T. Two representative independent F9:10 NILs displaying each of the parental genotypes are presented for comparison. Labels indicate the homozygous genotypes. Satt237 and Satt561: 5601T (5601T) or CX1834 (CX) donated alleles. Lpa1 and Lpa2: 5601T (5601T) or CX1834 lpa1-a or lpa2-a (cx) donated mutant alleles. Inorganic phosphate phenotype is the average of four seeds individually analyzed for each NIL. Shading is intended to illustrate the genotypic and phenotypic categories. LG = linkage group; Chr = chromosome.