Whole-Genome Resequencing Identifies the Molecular Genetic Cause for the Absence of a Gy5 Glycinin Protein in Soybean PI 603408

Abstract

During ongoing proteomic analysis of the soybean (Glycine max (L.) Merr) germplasm collection, PI 603408 was identified as a landrace whose seeds lack accumulation of one of the major seed storage glycinin protein subunits. Whole genomic resequencing was used to identify a two-base deletion affecting glycinin 5 The newly discovered deletion was confirmed to be causative through immunological, genetic, and proteomic analysis, and no significant differences in total seed protein content were found to be due to the glycinin 5 loss-of-function mutation per se In addition to focused studies on this one specific glycinin subunit-encoding gene, a total of 1,858,185 nucleotide variants were identified, of which 39,344 were predicted to affect protein coding regions. In order to semiautomate analysis of a large number of soybean gene variants, a new SIFT 4G (Sorting Intolerant From Tolerated 4 Genomes) database was designed to predict the impact of nonsynonymous single nucleotide soybean gene variants, potentially enabling more rapid analysis of soybean resequencing data in the future.

Keywords: Glycine max; amino acid changes (AAC); genomic resequencing; glycinin; seed proteomics; single nucleotide variants (SNV); soybean.

Figure 1

SDS-PAGE analysis of mature seed from lines Patriot and PI 603408. (A) SDS-PAGE analysis of total seed proteins (samples 1 and 2) or calcium carbonate enrichment of seed storage proteins (samples 3 and 4), and (B) immunoblot analysis of total seed proteins (samples 1 and 2) or calcium carbonate enrichment of seed storage proteins (samples 3 and 4) with anti-glycinin 4/5 antibodies. Samples 1 and 3 are from seed of cultivar Patriot and samples 2 and 4 are from seed of PI 603408. Arrow indicates protein band polymorphism between genotypes. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 2

SDS-PAGE analysis of progeny of a F_3:4 Patriot × PI 603408 cross. (A) SDS-PAGE analysis of total seed proteins. Samples 1 and 2 are seed from PI 603408, samples 3 and 4 are seed from line Patriot. Samples 5–13 are selected progeny from a Patriot × PI 603408 cross. The genotype of the plant that produced seed is indicated below the gel image: “+” indicates homozygosity for wild-type alleles and “−” indicates homozygosity for mutant alleles. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3

SDS-PAGE and immunoblot analysis of F_4:5 progeny of a 10/81b × PI 603408 cross. (A) SDS-PAGE analysis of total seed proteins and (B) immunoblot analysis of total seed proteins with anti-glycinin 5 antibodies. Samples 1, 13, and 14 are from line Patriot and sample 2 is seed from PI 603408 plants. Samples 3–12 are selected progeny from a 10/81b × PI 603408 cross. The genotype of plant that produced seed is indicated below figure: “+” indicates homozygosity for wild-type alleles, “H” indicates heterozygosity, and “−” indicates homozygosity for mutant alleles. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 4

Overlay of two separate 2D gels of soybean seed proteins using Delta2D software. Isoelectric focusing (pI 3–10) followed by second dimension SDS-PAGE resulted in the separation of seed proteins and visualization of those proteins using Coomassie Blue. Gels were scanned and the resulting images were assigned two different colors (green = Patriot and red = gy1/gy4/gy5 mutant) in order to visualize the differences between the two. Delta2D software provides an overlay of both, with spot matching, where yellow demonstrates similar protein quantities in each. Green color demonstrates absence of that particular protein species in the gy1/gy4/gy5 mutant. Spots 1, 2, and 4 represent the 7S β-conglycinin subunits and spots 5–10, 11–17, and 19 represents the different glycinin subunits. Spots 3, 11, and 18 are the sucrose-binding proteins, KTi and BBi, respectively. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.