Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Effective identification of soybean candidate genes involved in resistance to soybean cyst nematode via direct whole genome re-sequencing of two segregating mutants


Key message

Three soybean candidate genes involved in resistance to soybean cyst nematode race 4 were identified via direct whole genome re-sequencing of two segregating mutants.


The genes conferring resistance to soybean cyst nematode (SCN) race 4 (Hg type in soybean (Glycine max L. Merr.) remains unknown. Next generation sequencing-based methods identify a wide range of targets, it is difficult to identify genes underlying traits. Use of the MutMap and QTL-seq methods to identify trait candidate genes needs backcrossing and is very time-consuming. Here we report a simple method to effectively identify candidate genes involved in resistance to SCN race 4. Two ethane methylsulfonate mutagenized mutants of soybean ‘PI 437654’, whose SCN race 4-infection phenotype altered, were selected. Six relevant whole genomes were re-sequenced, and then calling of genomic variants (SNPs and InDels) was conducted and compared to ‘Williams 82’. The comparison eliminated many genomic variants from the mutant lines that overlapped two non-phenotypic but mutant progeny plants, wild-type PI 437654 and ‘Zhonghuang 13’. Finally, only 27 mutations were found among 10 genes. Of these 10 genes, 3 genes, Glyma.09g054000, Glyma.16g065700 and Glyma.18g192200 were overlapped between two phenotypic mutant progeny plants. Therefore, the three genes may be the candidate genes involved in resistance of PI 437654 to soybean cyst nematode race 4. This method simplifies the effective identification of candidate genes.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2


  1. Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Cano L, Kamoun S, Terauchi R (2012) Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 30:174–178

  2. Afzal AJ, Srour A, Saini N, Hemmati N, El Shemy HA, Lightfoot DA (2012) Recombination suppression at the dominant Rhg1/Rfs2 locus underlying soybean resistance to the cyst nematode. Theor Appl Genet 124:1027–1039

  3. Afzal AJ, Srour A, Goil A, Vasudaven S, Liu T, Samudrala R, Dogra N, Kohli P, Malakar A, Lightfoot DA (2013) Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens. BMC Plant Biol 13:43

  4. Anand SC, Gallo KM, Baker IA, Hartwig EE (1988) Soybean plant introductions with resistance to races 4 or 5 of soybean cyst nematode. Crop Sci 28:563–564

  5. Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Rep 9:208–219

  6. Austin RS, Vidaurre D, Stamatiou G, Breit R, Provart NJ, Bonetta D, Zhang J, Fung P, Gong Y, Wang PW, McCourt P, Guttman DS (2011) Next-generation mapping of Arabidopsis genes. Plant J 67:715–725

  7. Bayless AM, Smith JM, Song J, McMinn PH, Teillet A, August BK, Bent AF (2016) Disease resistance through impairment of alpha-SNAP-NSF interaction and vesicular trafficking by soybean Rhg1. Proc Natl Acad Sci USA 113:E7375–E7382

  8. Bayless AM, Zapotocny RW, Grunwald DJ, Amundson KK, Diers BW, Bent AF (2018) An atypical N-ethylmaleimide sensitive factor enables the viability of nematode-resistant Rhg1 soybeans. Proc Natl Acad Sci USA 115:E4512–E4521

  9. Brucker E, Carlson S, Wright E, Niblack T, Diers B (2005) Rhg1 alleles from soybean PI 437654 and PI 88788 respond differentially to isolates of Heterodera glycines in the greenhouse. Theor Appl Genet 111:44–49

  10. Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890

  11. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Xu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6:80–92

  12. Colgrove AL, Niblack TL (2008) Correlation of female indices from virulence assays on inbred lines and field populations of Heterodera glycines. J Nematol 40:39–45

  13. Concibido VC, Diers BW, Arelli PR (2004) A decade of QTL mapping for cyst nematode resistance in soybean. Crop Sci 44:1121–1131

  14. Cook DE, Lee TG, Guo X, Melito S, Wang K, Bayless AM, Wang J, Hughes TJ, Willis DK, Clemente TE, Diers BW, Jiang J, Hudson ME, Bent AF (2012) Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean. Science 338:1206–1209

  15. Cook DE, Bayless AM, Wang K, Guo X, Song Q, Jiang J, Bent AF (2014) Distinct copy number, coding sequence, and locus methylation patterns underlie Rhg1-mediated soybean resistance to soybean cyst nematode. Plant Physiol 165:630–647

  16. Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-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 8:9

  17. Dalmais M, Schmidt J, Signor CL, Moussy F, Burstin J, Savois V, Aubert G, Brunaud V, Oliveira YD, Guichard C, Thompson R, Bendahmane A (2008) UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biol 9:R43

  18. Deschamps S, Llaca V, May GD (2012) Genotyping-by-sequencing in plants. Biology 1:460–483

  19. Dong C, Dalton-Morgan J, Vincent K, Sharp P (2009) A modified TILLING method for wheat breeding. Plant Genome 2:39–47

  20. Espina MJ, Ahmed CMS, Bernardini A, Adeleke E, Yadegari Z, Arelli P, Pantalone V, Taheri A (2018) Development and phenotypic screening of an ethyl methane sulfonate mutant population in soybean. Front Plant Sci 9:394

  21. Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, Sharma S, Sharma S, Kanzaki H, Matsumura H, Saitoh H, Mitsuoka C, Utsushi H, Uemura A, Kanzaki E, Kosugi S, Yoshida K, Cano L, Kamoun S, Terauchi R (2013) MutMap+: genetic mapping and mutant identification without crossing in rice. PLoS ONE 8:e68529

  22. Ge FY, Zheng N, Zhang LP, Huang WK, Peng DL, Liu SM (2018) Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode. J Integr Agric 17:2734–2744

  23. Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740

  24. Kandoth PK, Liu S, Prenger E, Ludwig A, Lakhssassi N, Heinz R, Zhou Z, Howland A, Gunther J, Warren S, Dhroso A, Lafayette P, Tucker D, Johnson S, Anderson J, Alaswad A, Cianzio SR, Parrott W, Korkin D, Meksem K, Mitchum MG (2017) Systematic mutagenesis of serine hydroxymethyltransferase reveals an essential role in nematode resistance. Plant Physiol 175:1370–1380

  25. Koenning SR, Wrather JA (2010) Suppression of soybean yield potential in the continental United States from plant diseases estimated from 2006 to 2009. Plant Health Progress. https://doi.org/10.1094/PHP-2010-1122-01-RS

  26. Lakhssassi N, Liu S, Bekal S, Zhou Z, Colantonio V, Lambert K, Barakat A, Meksem K (2017) Characterization of the soluble NSF attachment protein gene family identifies two members involved in additive resistance to a plant pathogen. Sci Rep 7:45226

  27. Lee TG, Kumar I, Diers BW, Hudson ME (2015) Evolution and selection of Rhg1, a copy-number variant nematode-resistance locus. Mol Ecol 24:1774–1791

  28. Li H, Durbin R (2010) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760

  29. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079

  30. Li Z, Jiang L, Ma Y, Wei Z, Hong H, Liu Z, Lei J, Liu Y, Guan R, Guo Y, Jin L, Zhang L, Li Y, Ren Y, He W, Liu M, Htwe NM, Liu L, Guo B, Song J, Tan B, Liu G, Li M, Zhang X, Liu B, Shi X, Han S, Hua S, Zhou F, Yu L, Li Y, Wang S, Wang J, Chang R, Qiu L (2017) Development and utilization of a new chemically-induced soybean library with a high mutation density. J Integr Plant Biol 59:60–74

  31. Liu X, Liu A, Jami A, Bendahmane A, Lightfoot DA, Mitchum MG, Meksem K (2011) Soybean cyst nematode resistance in soybean is independent of the Rhg4 locus LRR-RLK gene. Funct Integr Genom 11:539–549

  32. Liu S, Kandoth PK, Warren SD, Yechel G, Heinz R, Alden J, Yang C, Jamai A, Ei-Mellouki T, Juvale PS, Hill J, Baum TJ, Cianzio S, Whitham SA, Korkin D, Mitchum MG, Meksem K (2012a) A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens. Nature 492:256–260

  33. Liu S, Yeh CT, Tang HM, Nettleton D, Schnable PS (2012b) Gene mapping via bulked segregant RNA-Seq (BSR-Seq). PLoS ONE 7:e36406

  34. Liu S, Kandoth PK, Lakhssassi N, Kang J, Colantonio V, Heinz R, Yechel G, Zhou Z, Bekal S, Dapprich J, Rotter B, Cianzio S, Mitchum MG, Meksem K (2017) The soybean GmSNAP18 gene underlies two types of resistance to soybean cyst nematode. Nat Commun 8:14822

  35. Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 18:1509–1517

  36. McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations. Nat Biotechnol 18:455–457

  37. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2010) The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303

  38. Meksem K, Pantazopoulos P, Njiti VN, Hyten LD, Arelli PR, Lightfoot DA (2001) ‘Forrest’ resistance to the soybean cyst nematode is bigenic: saturation mapping of the Rhg1 and Rhg4 loci. Theor Appl Genet 103:710–717

  39. Mitchum MG (2016) Soybean resistance to the soybean cyst nematode Heterodera glycines: an update. Phytopathology 106:1444–1450

  40. Mitchum MG, Wrather JA, Heinz RD, Shannon JG, Danekas G (2007) Variability in distribution and virulence phenotypes of Heterodera glycines in Missouri during 2005. Plant Dis 91:1473–1476

  41. Mou S, Liu Z, Guan D, Qiu A, Lai Y, He S (2013) Functional analysis and expression characterization of rice ankyrin repeat-containing protein, OsPIANK1, in basal defense against Magnaporthe oryzae attack. PLoS ONE 8:e59699

  42. Ng P, Kirkness E (2010) Whole genome sequencing. Methods Mol Biol 628:215–226

  43. Ngaki MN, Wang B, Sahu BB, Srivastava SK, Farooqi MS, Kambakam S, Swaminathan S, Bhattacharyya MK (2016) Tanscriptomic study of the soybean-Fusarium virguliforme interaction revealed a novel ankyrin-repeat containing defense gene, expression of whose during infection led to enhanced resistance to the fungal pathogen in transgenic soybean plants. PLoS ONE 11:e0163106

  44. Patil GB, Lakhssassi N, Wan J, Song L, Zhou Z, Klepadlo M, Vuong TD, Stec AO, Kahil SS, Colantonio V, Valliyodan B, Rice HJ, Piya S, Hewezi T, Stupar RM, Meksem K, Nguyen HT (2019) Whole genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode. Plant Biotechnol J. https://doi.org/10.1111/pbi.13086

  45. Peng DL, Pen H, Wu DQ, Huang WK, Ye WX, Cui JK (2016) First report of soybean cyst nematode (Heterodera glycines) on soybean from Gansu and Ningxia China. Plant Dis 100:229

  46. Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol 131:866–871

  47. Porceddu A, Panara F, Calderini O, Molinari L, Taviani P, Lanfaloni L, Scotti C, Carelli M, Scaramelli L, Bruschi G, Cosson V, Ratet P, Larembergue HD, Duc G, Piano E, Arcioni S (2008) An Italian functional genomic resource for Medicago truncatula. BMC Res Note 1:129

  48. Pottorff M, Wanamaker S, Ma YQ, Ehlers JD, Roberts PA, Close TJ (2012) Genetic and physical mapping of candidate genes for resistance to Fusarium oxysporum f.sp. tracheiphilum race 3 in cowpea [Vigna unguiculata (L.) Walp]. PLoS ONE 7: e41600

  49. Schneeberger K, Ossowski S, Lanz C, Juul T, Petersen AH, Nielsen KL, Jørgensen JE, Weigel D, Andersen SU (2009) SHOREmap: simultaneous mapping and mutation identification by deep sequencing. Nat Methods 6:550–551

  50. Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145

  51. Shi Z, Liu S, Noe J, Arelli P, Meksem K, Li Z (2015) SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance. BMC Genom 16:314

  52. Srour A, Afzal AJ, Blahut-Beatty L, Hemmati N, Simmonds DH, Li W, Liu M, Town CD, Sharma H, Arelli P, Lightfoot DA (2012) The receptor like kinase at Rhg1-a/Rfs2 caused pleiotropic resistance to sudden death syndrome and soybean cyst nematode as a transgene by altering signaling responses. BMC Genom 13:368

  53. Stranger B, Stahl E, Raj T (2011) Progress and promise of genome-wide association studies for human complex trait genetics. Genetics 187:367–383

  54. Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genom 279:213–223

  55. Swaminathan S, Das A, Assefa T, Knight JM, Da Silva AF, Carvalho JPS, Hartman GL, Huang X, Leandro LF, Cianzio SR, Bhattacharyya MK (2019) Genome wide association study identifies novel single nucleotide polymorphic loci and candidate genes involved in soybean sudden death syndrome resistance. PLoS ONE 14:e0212071

  56. Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano LM, Kamoun S, Terauchi R (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J 74:174–183

  57. Takagi H, Tamiru M, Abe A, Yoshida K, Uemura A, Yaegashi H, Obara T, Oikawa K, Utsushi H, Kanzaki E, Mitsuoka C, Natsume S, Kosugi S, Kanzaki H, Matsumura H, Urasaki N, Kamoun S, Terauchi R (2015) MutMap accelerates breeding of a salt-tolerant rice cultivar. Nat Biotechnol 33:445–449

  58. Talamè V, Bovina R, Sanguineti MC, Tuberosa R, Lundqvist U, Salvi S (2008) TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotechnol J 6:477–485

  59. Teer JK, Mullikin JC (2010) Exome sequencing: the sweet spot before whole genomes. Hum Mol Genet 19:R145–151

  60. Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4:12

  61. Wang N, Wang Y, Tian F, King GJ, Zhang C, Long Y, Shi L, Meng J (2008) A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytol 180:751–765

  62. Wu X, Blake S, Sleper DA, Shannon JG, Cregan P, Nguyen HT (2009) QTL, additive and epistatic effects for SCN resistance in PI 437654. Theor Appl Genet 118:1093–1105

  63. Xin Z, Wang ML, Barkley NA, Burow G, Franks C, Pederson G, Burke J (2008) Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biol 8:103

  64. Yan J, Wang J, Zhang H (2002) An ankyrin repeat-containing protein plays a role in both disease resistance and antioxidation metabolism. Plant J 29:193–202

  65. Yang Y, Zhang Y, Ding P, Johnson K, Li X, Zhang Y (2012) The ankyrin-repeat transmembrane protein BDA1 functions downstream of the receptor-like protein SNC2 to regulate plant immunity. Plant Physiol 159:1857–1865

  66. Yu N, Lee TG, Rosa DP, Hudson M, Diers BW (2016) Impact of Rhg1 copy number, type, and interaction with Rhg4 on resistance to Heterodera glycines in soybean. Theor Appl Genet 129:2403–2412

Download references


This work was financially supported by the Innovation Program and Youth Elite Program of Chinese Academy of Agricultural Sciences and the Special Fund for Agro-scientific Research in the Public Interest of China (201503114). We thank the staff at OE Biotech, China, for sequencing and analyzing the original sequencing data.

Author information

Correspondence to Shiming Liu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Istvan Rajcan.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, S., Ge, F., Huang, W. et al. Effective identification of soybean candidate genes involved in resistance to soybean cyst nematode via direct whole genome re-sequencing of two segregating mutants. Theor Appl Genet 132, 2677–2687 (2019). https://doi.org/10.1007/s00122-019-03381-6

Download citation


  • Soybean
  • Soybean cyst nematode
  • Chemically mutagenized mutants
  • Whole genome re-sequencing
  • Resistant candidate genes