Molecular Breeding

, Volume 32, Issue 2, pp 291–298 | Cite as

Identification of a plant introduction soybean line with genetic lesions affecting two distinct glycinin subunits and evaluation of impacts on protein content and composition

  • Won-Seok Kim
  • Jason D. Gillman
  • Hari B. Krishnan


Unlike other oilseeds, soybean (Glycine max [L.] Merr) is also valuable due to its direct conversion into human food. One notable example is the cheese-like product tofu. The quality of tofu is improved when protein subunits derived from two glycinin genes, Gy1 and Gy4, are reduced or absent. Here we report the discovery that one exotic soybean plant introduction line, PI 605781 B, has not only a previously described loss-of-expression mutation affecting one glycinin gene (gy4), but also bears an extremely rare, potentially unique, frameshift mutation in the Glycinin1 gene (gy1-a). We analyzed glycinin gene expression via qRT-PCR with mRNA from developing seeds, which revealed that the novel allele dramatically reduced Gy1 mRNA accumulation. Similarly, both A4A5B3 and A1aB1a protein subunits were absent or at undetectable levels, as determined by two-dimensional protein fractionation. Despite the reduction in glycinin content, overall seed protein levels were unaffected. The novel gy1-a allele was found to be unique to PI 605871B in a sampling of 247 diverse germplasm lines drawn from a variety of geographic origins.


Glycinin Soybean Tofu Seed proteins 



The authors would like to acknowledge the superb technical assistance of Sungchan Jang, Jeremy Mullis and Nathan Oehrle. This research was supported by the USDA-Agricultural Research Service.

Supplementary material

11032_2013_9870_MOESM1_ESM.pptx (154 kb)
Supplementary Figure 1 Typical genotyping “melting peak” analyses for Gy1 and Gy4 SimpleProbes. Colors: Light blue line indicates negative control, dark blue indicates homozygote mutant, red indicates homozygote wild type, green indicates heterozygote. Analysis was run on a Lightcycler 480 II (Roche). (PPTX 153 kb)
11032_2013_9870_MOESM2_ESM.pptx (351 kb)
Supplementary Figure 2 One dimensional protein gel electrophoresis of lines selected via genotyping assays. 40 micrograms of seed protein were separated on 12.5 % SDS-PAGE gels. C indicates protein from ‘Patriot’ seeds and PI indicates protein from PI 605871 B seeds, numbered samples were F2:3 seeds from F2 selections for gy4 or Gy4 homozygotes identified by SimpleProbe assay (genotype is indicated at bottom of chart). * indicates either A4 or B5 glycinin subunit, encoded by the Gy4 gene. (PPTX 351 kb)
11032_2013_9870_MOESM3_ESM.xlsx (12 kb)
Supplemental Table 1 Glycinin primers used in this work (XLSX 12 kb)
11032_2013_9870_MOESM4_ESM.xlsx (33 kb)
Supplemental Table 2 Distribution of Gy1 alleles in a diverse sampling of 247 genotypes (XLSX 32 kb)


  1. Beilinson V, Chen Z, Shoemaker RC, Fischer RL, Goldberg RB, Nielsen NC (2002) Genomic organization of glycinin genes in soybean. Theor Appl Genet 104(6–7):1132–1140PubMedGoogle Scholar
  2. Bilyeu KD, Zeng P, Coello P, Zhang ZJ, Krishnan HB, Bailey A, Beuselinck PR, Polacco JC (2008) Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase. Plant Physiol 146(2):468–477PubMedCrossRefGoogle Scholar
  3. Bilyeu K, Gillman JD, LeRoy AR (2011) Novel FAD3 mutant allele combinations produce soybeans containing 1% linolenic acid in the seed oil. Crop Sci 51(1):1–5CrossRefGoogle Scholar
  4. Boland MJ, Rae AN, Vereijken JM, Meuwissen MPM, Fischer ARH, van Boekel MAJS, Rutherfurd SM, Gruppen H, Moughan PJ, Hendriks WH (2013) The future supply of animal-derived protein for human consumption. Trends Food Sci Technol 29(1):62–73CrossRefGoogle Scholar
  5. Chang Y-F, Imam JS, Wilkinson MF (2007) The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem 76:51–74PubMedCrossRefGoogle Scholar
  6. Cho TJ, Davies CS, Fischer RL, Turner NE, Goldberg RB, Nielsen NC (1989a) Molecular characterization of an aberrant allele for the Gy3 glycinin gene: a chromosomal rearrangement. Plant Cell 1(3):339–350PubMedGoogle Scholar
  7. Cho TJ, Davies CS, Nielsen NC (1989b) Inheritance and organization of glycinin genes in soybean. Plant Cell 1(3):329–337PubMedGoogle Scholar
  8. Derbyshire E, Wright D, Boulter D (1976) Legumin and vicilin, storage proteins of legume seed. Phytochemistry 15(1):3–24CrossRefGoogle Scholar
  9. Gillman JD, Baxter I, Bilyeu K (2013) Phosphorus partitioning of soybean lines containing different mutant alleles of two soybean seed-specific adenosine triphosphate-binding cassette phytic acid transporter paralogs. Plant Genome 6(1):1–10CrossRefGoogle Scholar
  10. Gizlice Z, Carter TE Jr, Burton JW (1994) Genetic base for North American public soybean cultivars released between 1947 and 1988. Crop Sci 34(5):1143–1151CrossRefGoogle Scholar
  11. Gizlice Z, Carter TE Jr, Gerig TM, Burton JW (1996) Genetic diversity patterns in North American public Soybean cultivars based on coefficient of parentage. Crop Sci 36(3):753–765CrossRefGoogle Scholar
  12. Jegadeesan S, Yu K, Woodrow L, Wang Y, Shi C, Poysa V (2012) Molecular analysis of glycinin genes in soybean mutants for development of gene-specific markers. Theor Appl Genet 124(2):365–372PubMedCrossRefGoogle Scholar
  13. Jenkinson JE, Fehr WR (2010) Agronomic and seed characteristics of soybean lines with alleles for modified glycinin concentration. Crop Sci 50(5):1896–1903CrossRefGoogle Scholar
  14. Kim W-S, Ho HJ, Nelson RL, Krishnan HB (2008) Identification of several gy4 nulls from the USDA soybean germplasm collection provides new genetic resources for the development of high-quality tofu cultivars. J Agric Food Chem 56(23):11320–11326PubMedCrossRefGoogle Scholar
  15. Kinney AJ, Jung R, Herman EM (2001) Cosuppression of the α subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies. Plant Cell 13(5):1165–1178PubMedGoogle Scholar
  16. Kitamura K, Takagi T, Shibasaki K (1976) Subunit structure of soybean 11S globulin. Agric Biol Chem Tokyo 40(9):1837–1844CrossRefGoogle Scholar
  17. Krishnan HB, Natarajan SS, Mahmoud AA, Nelson RL (2007) Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines. J Agric Food Chem 55(5):1839–1845PubMedCrossRefGoogle Scholar
  18. Lam H-M, Xu X, Liu X, Chen W, Yang G, Wong F-L, Li M-W, He W, Qin N, Wang B, Li J, Jian M, Wang J, Shao G, Wang J, Sun SS-M, Zhang G (2010) Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nat Genet 42(12):1053–1059PubMedCrossRefGoogle Scholar
  19. Li C, Zhang YM (2011) Molecular evolution of glycinin and beta-conglycinin gene families in soybean (Glycine max L. Merr.). Heredity 106(4):633–641PubMedCrossRefGoogle Scholar
  20. Libault M, Thibivilliers S, Bilgin DD, Radwan O, Benitez M, Clough SJ, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Gen 1(1):44–54CrossRefGoogle Scholar
  21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4):402–408PubMedCrossRefGoogle Scholar
  22. Mahmoud AA, Natarajan SS, Bennett JO, Mawhinney TP, Wiebold WJ, Krishnan HB (2006) Effect of six decades of selective breeding on soybean protein composition and quality: a biochemical and molecular analysis. J Agric Food Chem 54(11):3916–3922PubMedCrossRefGoogle Scholar
  23. Peltz SW, Brown AH, Jacobson A (1993) mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor. Genes Dev 7(9):1737–1754PubMedCrossRefGoogle Scholar
  24. Pieretti MM, Chung D, Pacenza R, Slotkin T, Sicherer SH (2009) Audit of manufactured products: use of allergen advisory labels and identification of labeling ambiguities. J Allergy Clin Immun 124(2):337–341PubMedCrossRefGoogle Scholar
  25. Poysa V, Woodrow L, Yu K (2006) Effect of soy protein subunit composition on tofu quality. Food Res Int 39(3):309–317CrossRefGoogle Scholar
  26. Samuels ML, Witmer JA (1999) Statistics for the life sciences, 2nd edn. Prentice-Hall, LondonGoogle Scholar
  27. Scallon BJ, Dickinson CD, Nielsen NC (1987) Characterization of a null-allele for the Gy4 glycinin gene from soybean. Mol Gen Genet 208(1):107–113CrossRefGoogle Scholar
  28. Schmidt MA, Barbazuk WB, Sandford M, May G, Song Z, Zhou W, Nikolau BJ, Herman EM (2011) Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome. Plant Physiol 156(1):330–345PubMedCrossRefGoogle Scholar
  29. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183PubMedCrossRefGoogle Scholar
  30. Severin A, Woody J, Bolon Y-T, Joseph B, Diers B, Farmer A, Muehlbauer G, Nelson R, Grant D, Specht J, Graham M, Cannon S, May G, Vance C, Shoemaker R (2010) RNA-Seq Atlas of Glycine max: a guide to the soybean transcriptome. BMC Plant Biol 10(1):160PubMedCrossRefGoogle Scholar
  31. Takahashi K, Banba H, Kikuchi A, Ito M, Nakamura S (1994) An induced mutant line lacking the α-subunit of β-conglycinin in soybean (Glycine max (L.) Merrill). Jpn J Breed 44(1):65–66Google Scholar
  32. Takahashi M, Uematsu Y, Kashiwaba K, Yagasaki K, Hajika M, Matsunaga R, Komatsu K, Ishimoto M (2003) Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency. Planta 217(4):577–586PubMedCrossRefGoogle Scholar
  33. Thanh V, Shibasaki K (1978) Major proteins of soybean seeds: subunit structure of β-conglycinin. J Agric Food Chem 26(3):692–695CrossRefGoogle Scholar
  34. Yagasaki K, Kalzuma N, Kitamura K (1996) Inheritance of glycinin subunits and characterization of glycinin molecules lacking the subunits in soybean (Glycine max (L.) Merr.). Jpn J Breed 46(1):11–15Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht (outside the USA) 2013

Authors and Affiliations

  • Won-Seok Kim
    • 1
  • Jason D. Gillman
    • 1
  • Hari B. Krishnan
    • 1
  1. 1.United States Department of Agriculture-Agricultural Research ServiceUniversity of Missouri-ColumbiaColumbiaUSA

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