Functional & Integrative Genomics

, Volume 11, Issue 4, pp 539–549 | Cite as

Soybean cyst nematode resistance in soybean is independent of the Rhg4 locus LRR-RLK gene

  • Xiaohong Liu
  • Shiming Liu
  • Aziz Jamai
  • Abdelhafid Bendahmane
  • David A. Lightfoot
  • Melissa G. Mitchum
  • Khalid Meksem
Original Paper


To test the function of candidate genes in soybean for resistance to the soybean cyst nematode (SCN), a large collection of EMS-mutants from the SCN-resistant soybean cultivar “Forrest” was developed for Targeting Induced Local Lesions IN Genomes (TILLING). Additionally, due to the complexity of the soybean genome, an integrated set of genomic and genetic analysis tools was employed to complement the TILLING approach. The efficiency of this integrated set of tools was tested using a candidate soybean gene for resistance to SCN, encoding a leucine-rich repeat receptor-like kinase (LRR-RLK) that was identified by map-based cloning at the Rhg4 locus. The Rhg4 locus is one of the major quantitative trait loci controlling soybean resistance against SCN race 3 (HG type 0) in cv. Forrest, but the gene(s) sequence for resistance remains to be determined. Using TILLING, a Forrest mutant containing a nonsense mutation in the LRR domain of the candidate resistance protein was identified and confirmed; however, the SCN-resistant phenotype of the mutant was not altered. Haplotyping and EcoTILLING of recombinant inbred lines along with complementation analysis corroborated the TILLING result and ruled out the possibility of functional redundancy by a second copy of the LRR-RLK gene identified in the soybean genome. This study validates the use of TILLING, in combination with an integrated set of genomic tools, as an efficient means of testing candidate genes for SCN resistance in soybean.


Cyst nematode Genetics Genomics Heterodera glycines Resistance Soybean TILLING 

Supplementary material

10142_2011_225_MOESM1_ESM.doc (16 kb)
Table S1EcoTILLING of the EXF RILs population using the LRR-kinase sequences at each, the Rhg1 locus (Rhg1 allele) and the Rhg4 locus (Rhg4 allele) and the corresponding SCN phenotype of each line. RIL recombinant inbred line of EXF population, E allele from Essex, F allele from Forrest, R resistant, S susceptible (DOC 16 kb)
10142_2011_225_MOESM2_ESM.ppt (109 kb)
Fig. S1(PPT 109 kb)


  1. Cho H-J, Farrand SK, Noel GR, Widholm JM (2000) High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta 210:195–204PubMedCrossRefGoogle Scholar
  2. Concibido VC, Diers BW, Arelli PR (2004) A decade of QTL mapping for cyst nematode resistance in soybean. Crop Sci 44:1121–1131CrossRefGoogle Scholar
  3. Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, EI-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD, Meksem K, Comai L, Henikoff S (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8:123–132CrossRefGoogle Scholar
  4. Dierking EC, Bilyeu KD (2009) New sources of soybean seed meal and oil composition traits identified through TILLING. BMC Plant Biol 9:89PubMedCrossRefGoogle Scholar
  5. Faghihi J, Ferris JM (2000) An efficient new device to release eggs from Heterodera glycines. J Nematol 32:411–413PubMedGoogle Scholar
  6. Hartwig EF, Epps JM (1973) Registration of ‘Forrest’ soybeans. Crop Sci 13:287CrossRefGoogle Scholar
  7. Hauge BM, Wang ML, Parsons JD et al. (2003) Nucleic acid molecules and other molecules associated with soybean cyst nematode resistance. US Patent 20030005491, 2 Jan 2003Google Scholar
  8. Hauge BM, Wang ML, Parsons JD et al. (2006) Nucleic acid molecules and other molecules associated with soybean cyst nematode resistance. US Patent 7154021, 26 Dec 2006Google Scholar
  9. Hauge BM, Wang ML, Parsons JD et al. (2009) Methods of introgressing nucleic acid molecules associated with soybean cyst nematode resistance into soybean. US Patent 7485770, 3 Feb 2009Google Scholar
  10. Henikoff S, Comai L (2003) Single-nucleotide mutations for plant functional genomics. Ann Rev Plant Biol 54:375–401CrossRefGoogle Scholar
  11. Koenning SR, Wrather JA (2010) Suppression of soybean yield potential in the continental 12 United States from plant diseases estimated from 2006 to 2009. Plant Health Progress. doi:10.1094/PHP-2010-1122-01-RS Google Scholar
  12. Lightfoot DA, Meksem K (2002) Isolation of polynucleotides and polypeptides relating to loci underlying resistance to soybean cyst nematode and soybean sudden death syndrome and methods employing same. US Patent 2002144310, 3 Oct 2002Google Scholar
  13. Lightfoot DA, Njiti VN, Gibson PT, Kassem MA, Iqbal JM, Meksem K (2005) Registration of the Essex × Forrest recombinant inbred line mapping population. Crop Sci 45:1678–1681CrossRefGoogle Scholar
  14. Meksem K, Doubler TW, Chancharoenchai VN, Njiti VN, Chang SJC, Rao-Arelli AP, Cregan PE, Gray LE, Gibson PT, Lightfoot DA (1999) Clustering among loci underlying soybean resistance to Fusarium solani, SDS and SCN in near-isogenic lines. Theor Appl Genet 99:1161–1171CrossRefGoogle Scholar
  15. Meksem K, Zobrist K, Ruben E, Hyten D, Quanzhou T, Zhang HB, Lightfoot DA (2000) Two large-insert soybean genomic libraries constructed in a binary vector: applications in chromosome walking and genome wide physical mapping. Theor Appl Genet 101:747–755CrossRefGoogle Scholar
  16. Meksem K, Pantazopoulos P, Njiti VN, Hyten LD, Arelli PR, Lightfoot DA (2001a) ‘Forrest’ resistance to the soybean cyst nematode is bigenic: saturation mapping of the Rhg1 and Rhg4 loci. Theor Appl Genet 103:710–717CrossRefGoogle Scholar
  17. Meksem K, Hyten D, Ruben E, Lightfoot DA (2001b) High-through genotyping for a polymorphism linked to soybean cyst nematode resistance Rhg4 by using Taqman ™ probes. Mol Breed 77:63–71CrossRefGoogle Scholar
  18. Meksem K, Njiti VN, Banz WJ, Iqbal MJ, Kassem MA, Hyten DL, Yuang J, Winters TA, Lightfoot DA (2001c) Genomic regions that underlie soybean seed isoflavone content. J Biomed Biotechnol 1:1–8CrossRefGoogle Scholar
  19. Meksem K, Liu S, Liu X, Jamai A, Mitchum M, Bendahmane A, El-Mellouki T (2008) TILLING: a reverse genetics and a functional genomics tool in soybean. In: Kahl G, Meksem K (eds) The handbook of plant functional genomics: concepts and protocols. Wiley, Weinheim, pp 251–265CrossRefGoogle Scholar
  20. Melito S, Heuberger AL, Cook D, Diers BW, MacGuidwin AE, Bent AF (2010) A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance. BMC Plant Biol 10:104PubMedCrossRefGoogle Scholar
  21. Myers GO, Anand SC (1991) Inheritance of resistance and genetic relationships among soybean plant introductions to races of soybean cyst nematodes. Euphytica 55:197–201CrossRefGoogle Scholar
  22. Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14:387–392PubMedCrossRefGoogle Scholar
  23. Niblack TL, Heinz RD, Smith GS, Donald PA (1993) Distribution, density, and diversity of Heterodera glycines in Missouri. J Nematol 25:880–886PubMedGoogle Scholar
  24. Ruben E, Aziz J, Afzal J, Njiti VN, Triwitayakorn K, IqbalMJ YS, Arelli P, Town C, Meksem K et al (2006) Genomic analysis of the ‘Peking’ rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode. Mol Genet Genomics 276:320–330CrossRefGoogle Scholar
  25. Schlueter JA, Lin JY, Schlueter SD, Vasylenko-Sanders IF, Deshpande S, Yi J, O’Bleness M, Roe BA, Nelson RT, Scheffler BE, Jackson SA, Shoemaker RC (2007) Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing. BMC Genomics 8:330PubMedCrossRefGoogle Scholar
  26. Schmutz J, Cannon S, Schlueter J, Ma J, Mitros T, Nelson W, Hyten D, Song Q, Thelen J, Cheng J, Xu D, Hellsten U, May G, Yu Y, Sakurai T, Umezawa T, Bhattacharyya M, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs MDJ, Tian Z, Zhu L, Gill N, Trupti J, Libault M, Sethuraman A, Zhang X, Shinozaki S, Nguyen H, Wing R, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker R, Jackson S (2010) Genome sequence of the paleopolyploid soybean (Glycine max (L.) Merr.). Nature 463:178–183PubMedCrossRefGoogle Scholar
  27. Smith TJ, Camper HM (1973) Registration of Essex soybeans. Crop Sci 13:495CrossRefGoogle Scholar
  28. Song W-Y, Wang G-L, Chen L-L, Kim H-S, Pi L-Y, Holsten T, Gardner J, Wang B, Zhai W-X, Zhu L-H, Fauquet C, Ronald P (1995) A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270:1804–1806PubMedCrossRefGoogle Scholar
  29. Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530PubMedCrossRefGoogle Scholar
  30. Till BJ, Zerr T, Bowers E, Greene EA, Comai L, Henikoff S (2006) High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling. Nucleic Acids Res 34(13):e99PubMedCrossRefGoogle Scholar
  31. Webb DM (1996) Soybean cyst nematode resistant soybeans and methods of breeding and identifying resistant plants. US Patent 5491081, 13 Feb 1996Google Scholar
  32. Webb DM (2000) Positional cloning of soybean cyst nematode resistance genes. US Patent 6162967, 19 Dec 2000Google Scholar
  33. Webb DM (2003) Quantitative trait loci associated with soybean cyst nematode resistance and uses thereof. US Patent 6538175, 25 Mar 2003Google Scholar
  34. Weisman J, Matthews B, Devine T (1992) Molecular markers located proximal to the soybean cyst nematode resistance gene, Rhg4. Theor Appl Genet 85:136–138Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Xiaohong Liu
    • 1
  • Shiming Liu
    • 2
  • Aziz Jamai
    • 2
  • Abdelhafid Bendahmane
    • 3
    • 4
  • David A. Lightfoot
    • 5
  • Melissa G. Mitchum
    • 1
  • Khalid Meksem
    • 2
  1. 1.Division of Plant Sciences and Bond Life Sciences CenterUniversity of MissouriColumbiaUSA
  2. 2.Plants and Microbes Genomics and Genetics Lab, Department of Plant Soil and Agricultural SystemsSouthern Illinois University at CarbondaleCarbondaleUSA
  3. 3.Unité de Recherche en Génomique Végétale, UMR INRA-CNRSEvry CedexFrance
  4. 4.Department of Plant Production, College of Food and Agricultural SciencesKing Saud UniversityRiyadhSaudi Arabia
  5. 5.Center for Excellence, The Illinois Soybean CenterCarbondaleUSA

Personalised recommendations