Molecular Breeding

, Volume 33, Issue 4, pp 895–907 | Cite as

Characterization of the fan1 locus in soybean line A5 and development of molecular assays for high-throughput genotyping of FAD3 genes

  • Anh-Tung Pham
  • Kristin Bilyeu
  • Pengyin Chen
  • H. Roger Boerma
  • Zenglu LiEmail author


Soybean is one of the most important oil crops worldwide, and reducing the linolenic acid content of soybean oil will provide increased stability of the oil to consumers and limit the amount of trans fat in processed foods. The linolenic content in soybean seed is controlled by three fatty acid desaturase (FAD) three enzymes, FAD3A, B, and C. The soybean lines with 1 % linolenic acid content which are widely used in breeding for reduced linolenic acid in the USA have mutations in each of the three FAD genes derived from lines A5 (deletion of FAD3A), A26, and A23 (missense mutations in FAD3B and C, respectively). Although soybean line A5 has been released for 30 years, the extent and definition of the deletion of the FAD3A gene has not been characterized, which has prevented researchers from designing robust molecular markers for effective marker-assisted selection (MAS). Using a PCR-based genomic strategy, we have identified a 6.4-kbp deletion of the FAD3A gene in A5 and developed a TaqMan detection assay by targeting the deletion junction in A5, which could be used to distinguish the homozygotes and heterozygotes of the gene. In addition, based on mutant single nucleotide polymorphisms in FAD3B and FAD3C identified in A26 and A23, respectively, we have also developed TaqMan assays for high-throughput MAS. The TaqMan assays have proven to be a very effective platform for detecting the mutant FAD3 alleles and thus will greatly facilitate high-throughput MAS for development of soybean lines with reduced linolenic acid content.


FAD3 Low linolenic TaqMan assay genotyping Marker-assisted selection 



The authors wish to acknowledge the excellent technical assistance provided by Christine Cole, and two undergraduate helpers Jonathan Serrano and Bryan Grommersch. Funding for this research was provided by the United Soybean Board.

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  1. Abney SA, Crochet WD (2006) The uniform soybean tests, northern states 2006. USDA-ARS, Department of Agronomy, Purdue University, West Lafayette, INGoogle Scholar
  2. Anai T, Yamada T, Kinoshita T, Rahman SM, Takagi Y (2005) Identification of corresponding genes for three low-α-linolenic acid mutants and elucidation of their contribution to fatty acid biosynthesis in soybean seed. Plant Sci 168:1615–1623CrossRefGoogle Scholar
  3. Ascherio A, Willett WC (1997) Health effects of trans fatty acids. Am J Clin Nutr 66(Suppl):1006S–1010SPubMedGoogle Scholar
  4. Beuselinck PR, Sleper DA, Bilyeu KD (2006) An assessment of phenotype selection for linolenic acid using genetic markers. Crop Sci 46:747–750CrossRefGoogle Scholar
  5. Bilyeu KD, Palavalli L, Sleper DA, Beuselinck PR (2003) Three microsomal omega-3 fatty-acid desaturase genes contribute to soybean linolenic acid levels . Crop Sci 43:1833–1838CrossRefGoogle Scholar
  6. Bilyeu K, Palavalli L, Sleper DA, Beuselinck P (2006) Molecular genetic resources for development of 1% linolenic acid soybeans. Crop Sci 46:1913–1918CrossRefGoogle Scholar
  7. Bilyeu K, Gillman JD, LeRoy AR (2011) Novel FAD3 mutant allele combinations produce soybeans containing 1% linolenic acid in the seed oil. Crop Sci 51:259–264CrossRefGoogle Scholar
  8. Boerma HR, Hussey RS, Phillips DV, Wood ED, Rowan GB, Finnerty SL (1997) Registration of ‘Benning’ soybean. Crop Sci 37:1982CrossRefGoogle Scholar
  9. Byrum JR, Kinney AJ, Stecca KL, Grace DJ, Diers BW (1997) Alteration of the omega-3 fatty acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype. Theor Appl Genet 94:356–359CrossRefGoogle Scholar
  10. Chen P, Sneller CH, Rupe JC, Riggs RD, Robbins RT (2006) Registration of ‘UA 4805’ soybean. Crop Sci 46:974CrossRefGoogle Scholar
  11. Chen P, Ishibashi T, Dombek DG, Rupe JC (2011) Registration of R05-1415 and R05-1772 high-protein soybean germplasm lines. J Plant Reg 5:410–413CrossRefGoogle Scholar
  12. Chung G, Chiu R, Cheung J, Jin Y, Chim S, Chan P, Lo Y (2005) A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study. BMC Infect Dis 5:1–7CrossRefGoogle Scholar
  13. Dibb NJ, Brown DM, Karn J, Moerman DG, Bolten SL, Waterston RH (1985) Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans. J Mol Biol 183:543–551CrossRefPubMedGoogle Scholar
  14. Drummond A, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011) Geneious v5.4. Available from
  15. Fedick A, Su J, Treff NR (2012) Development of TaqMan allelic discrimination based genotyping of large DNA deletions. Genomics 99:127–131CrossRefPubMedGoogle Scholar
  16. Fehr WR (2007) Breeding for modified fatty acid composition in soybean. Crop Sci 47:S-72–S-87Google Scholar
  17. Fehr WR, Hammond EG (1998) A16 soybeans having low linolenic acid and descendants. U.S. Patent Number 5 710 369. Date issued: 20 JanuaryGoogle Scholar
  18. Fehr WR, Hammond EG (2000) Reduced linolenic acid production in soybeans. U.S. Patent No. 6 133 509. Issued October 17, 2000Google Scholar
  19. Fehr WR, Welke GA, Hammond EG, Duvick DN, Cianzio SR (1992) Inheritance of reduced linolenic acid content in soybean genotype-A16 and genotype-A17. Crop Sci 32:903–906CrossRefGoogle Scholar
  20. Flores T, Karpova O, Su X, Zeng P, Bilyeu K, Sleper D, Nguyen H, Zhang Z (2008) Silencing of GmFAD3 gene by siRNA leads to low α-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (L.) Merr.]. Transgenic Res 17:839–850CrossRefPubMedGoogle Scholar
  21. Gómez-Fernández N, Castellví-Bel S, Fernández-Rozadilla C, Balaguer F, Muñoz J, Madrigal I, Milà M, Graña B, Vega A, Castells A, Carracedo Á, Ruiz-Ponte C (2009) Molecular analysis of the APC and MUTYH genes in Galician and Catalonian FAP families: a different spectrum of mutations? BMC Med Genet 10:1–12CrossRefGoogle Scholar
  22. Ha B-K, Boerma HR (2008) High-throughput SNP genotyping by melting curve analysis for resistance to southern root-knot nematode and frogeye leaf spot in soybean. J Crop Sci Biotechnol 11:91–100Google Scholar
  23. Hammond EG, Fehr WR (1983) Registration of A5 germplasm line of soybean1 (Reg. No. GP44). Crop Sci 23:192Google Scholar
  24. Ishii Y, Kondo S (1975) Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvra-, pola-, reca-) by various mutagens. Mutat Res/Fundam Mol Mech Mutagen 27:27–44CrossRefGoogle Scholar
  25. Keim P, Olson TC, Shoemaker RC (1988) A rapid protocol for isolating soybean DNA. Soybean Genetic Newsl 15:150–152Google Scholar
  26. Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protocols 4:1073–1081CrossRefGoogle Scholar
  27. Lieber MR (2008) The mechanism of human nonhomologous DNA end joining. J Biol Chem 283:1–5CrossRefPubMedGoogle Scholar
  28. Liu LX, Spoerke JM, Mulligan EL, Chen J, Reardon B, Westlund B, Sun L, Abel K, Armstrong B, Hardiman G, King J, McCague L, Basson M, Clover R, Johnson CD (1999) High-throughput isolation of Caenorhabditis elegans deletion mutants. Genome Res 9:859–867PubMedCentralCrossRefPubMedGoogle Scholar
  29. Mao Z, Bozzella M, Seluanov A, Gorbunova V (2008) Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair 7:1765–1771PubMedCentralCrossRefPubMedGoogle Scholar
  30. Maughan PJ, Smith SM, Raney JA (2012) Utilization of super BAC pools and Fluidigm access array platform for high-throughput BAC clone identification: proof of concept. J Biomed Biotechnol 2012:7Google Scholar
  31. Mozaffarian D, Aro A, Willett WC (2009) Health effects of trans-fatty acids: experimental and observational evidence. Eur J Clin Nutr 63:S5–S21CrossRefPubMedGoogle Scholar
  32. Oliva M, Shannon JG, Sleper DA, Ellersieck MR, Cardinal AJ, Paris RL, Lee JD (2006) Stability of fatty acid profile in soybean genotypes with modified seed oil composition. Crop Sci 46:2069–2075CrossRefGoogle Scholar
  33. Paris RL (2004) The uniform soybean tests, southern states 2004. USDA-ARS, Crop Genetics and Production Research Unit, Stoneville, MSGoogle Scholar
  34. Rennie BD, Tanner JW (1991) New allele at the fan locus in the soybean line A5. Crop Sci 31:297–301CrossRefGoogle Scholar
  35. Robledo R, Beggs W, Bender P (2003) A simple and cost-effective method for rapid genotyping of insertion/deletion polymorphisms. Genomics 82:580–582CrossRefPubMedGoogle Scholar
  36. Ross AJ, Fehr WR, Welke GA, Cianzio SR (2000) Agronomic and seed traits of 1%-linolenate soybean genotypes. Crop Sci 40:383–386CrossRefGoogle Scholar
  37. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X-C, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183CrossRefPubMedGoogle Scholar
  38. Sega GA (1984) A review of the genetic effects of ethyl methanesulfonate. Mutat Res/Rev Genetic Toxicol 134:113–142Google Scholar
  39. Shannon JG, Lee J-D, Wrather JA, Sleper DA, Mian MAR, Bond JP, Robbins RT (2009) Registration of S99-2281 soybean germplasm line with resistance to frogeye leaf spot and three nematode species. J Plant Reg 3:94–98CrossRefGoogle Scholar
  40. Thompson LH, Schild D (2001) Homologous recombinational repair of DNA ensures mammalian chromosome stability. Mutat Res/Fundam Mol Mech Mutagen 477:131–153CrossRefGoogle Scholar
  41. Walker JB, Fehr WR, Welke GA, Hammond EG, Duvick DN, Cianzio SR (1998) Reduced-linolenate content associations with agronomic and seed traits of soybean. Crop Sci 38:352–355CrossRefGoogle Scholar
  42. Warner K, Fehr W (2008) Mid-oleic/ultra low linolenic acid soybean oil: a healthful new alternative to hydrogenated oil for frying. J Am Oil Chem Soc 85:945–951CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Anh-Tung Pham
    • 1
  • Kristin Bilyeu
    • 2
  • Pengyin Chen
    • 3
  • H. Roger Boerma
    • 1
    • 4
  • Zenglu Li
    • 1
    Email author
  1. 1.Department of Crop and Soil Sciences, Center for Applied Genetic TechnologiesUniversity of GeorgiaAthensUSA
  2. 2.Plant Genetics Research UnitUSDA-ARSColumbiaUSA
  3. 3.Department of Crop, Soil, and Environmental SciencesUniversity of ArkansasFayettevilleUSA
  4. 4.Georgia Seed Development CommissionAthensUSA

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