A novel variant of Gh_D02G0276 is required for root-knot nematode resistance on chromosome 14 (D02) in Upland cotton

  • Martin J. WubbenEmail author
  • Gregory N. Thyssen
  • Franklin E. Callahan
  • David D. Fang
  • Dewayne D. Deng
  • Jack C. McCarty
  • Ping Li
  • Md Sariful Islam
  • Johnie N. Jenkins
Original Article


Key message

MAGIC population sequencing and virus-induced gene silencing identify Gh_D02G0276 as a novel root-knot nematode resistance gene on chromosome 14 in Upland cotton.


The southern root-knot nematode [RKN; Meloidogyne incognita (Kofoid & White)] remains the primary yield-limiting biotic stress to Upland cotton (Gossypium hirsutum L.) throughout the southeastern USA. While useful genetic markers have been developed for two major RKN resistance loci on chromosomes 11 (A11) and 14 (D02), these markers are not completely effective because the causative genes have not been identified. Here, we sequenced 550 recombinant inbred lines (RILs) from a multi-parent advanced generation intercross (MAGIC) population to identify five RILs that had informative recombinations near the D02-RKN resistance locus. The RKN resistance phenotypes of these five RILs narrowed the D02-RKN locus to a 30-kb region with four candidate genes. We conducted virus-induced gene silencing (VIGS) on each of these genes and found that Gh_D02G0276 was required for suppression of RKN egg production conferred by the Chr. D02 resistance gene. The resistant lines all possessed an allele of Gh_D02G0276 that showed non-synonymous mutations and was prematurely truncated. Furthermore, a Gh_D02G0276-specific marker for the resistance allele variant was able to identify RKN-resistant germplasm from a collection of 367 cotton accessions. The Gh_D02G0276 peptide shares similarity with domesticated hAT-like transposases with additional novel N- and C-terminal domains that resemble the target of known RKN effector molecules and a prokaryotic motif, respectively. The truncation in the resistance allele results in a loss of a plant nuclear gene-specific C-terminal motif, potentially rendering this domain antigenic due to its high homology with bacterial proteins. The conclusive identification of this RKN resistance gene opens new avenues for understanding plant resistance mechanisms to RKN as well as opportunities to develop more efficient marker-assisted selection in cotton breeding programs.



The authors would like to acknowledge Drs. Marilyn Warburton (USDA-ARS) and Osman Gutierrez (USDA-ARS) for their critical review of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

122_2019_3289_MOESM1_ESM.docx (24 kb)
Supplementary material 1 (DOCX 23 kb)
122_2019_3289_MOESM2_ESM.docx (270 kb)
Supplementary material 2 (DOCX 270 kb)
122_2019_3289_MOESM3_ESM.docx (58 kb)
Supplementary material 3 (DOCX 57 kb)


  1. Bundock P, Hooykaas P (2005) An Arabidopsis hAT-like transposase is essential for plant development. Nature 436:282–284CrossRefGoogle Scholar
  2. Fang DD, Xiao J, Canci PC, Cantrell RG (2010) A new SNP haplotype associated with blue disease resistance gene in cotton (Gossypium hirsutum L.). Theor Appl Genet 120:943–953CrossRefGoogle Scholar
  3. Fernandez-Pozo N, Rosli HG, Martin GB, Mueller LA (2015) The SGN VIGS tool: user friendly software to design virus-induced gene silencing (VIGS) constructs for functional genomics. Mol Plant 8:486–488CrossRefGoogle Scholar
  4. Gao X, Britt RC Jr, Shan L, He P (2011) Agrobacterium-mediated virus-induced gene silencing assay in cotton. J Vis Exp 54:e2938–e2938Google Scholar
  5. Gutierrez OA, Jenkins JN, McCarty JC, Wubben MJ, Hayes RW, Callahan FE (2010) SSR markers closely associated with genes for resistance to root-knot nematode on chromosomes 11 and 14 of Upland cotton. Theor Appl Genet 121:1323–1337CrossRefGoogle Scholar
  6. He Y, Kumar P, Shen X, Davis RF, Becelaere GV, May OL, Nichols RL, Chee PW (2014) Re-evaluation of the inheritance for root-knot nematode resistance in the Upland cotton germplasm line M-120 RNR revealed two epistatic QTLs conferring resistance. Theor Appl Genet 127:1343–1351CrossRefGoogle Scholar
  7. Huang G, Dong R, Allen R, Davis EL, Baum TJ, Hussey RS (2006) A root-knot nematode secretory peptide functions as a ligand for a plant transcription factor. Mol Plant-Microbe Interact 19:463–470CrossRefGoogle Scholar
  8. Hussey RS, Barker KR (1973) A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Dis Rep 57:1025–1028Google Scholar
  9. Islam MS, Thyssen GN, Jenkins JN, Zeng L, Delhom CD, McCarty JC, Deng DD, Hinchliffe DJ, Jones DC, Fang DD (2016) A MAGIC population-based genome-wide association study reveals functional association of GhRBB1_A07 gene with superior fiber quality in cotton. BMC Genom 17:903CrossRefGoogle Scholar
  10. Jenkins JN, Creech RG, Tang B, Lawrence GW, McCarty JC (1995) Cotton resistance to root-knot nematode: II. Post-penetration development. Crop Sci 35:369–373CrossRefGoogle Scholar
  11. Jenkins JN, McCarty JC Jr, Gutiérrez OA, Hayes RW, Bowman DT, Watson CE, Jones DC (2008) Registration of RMUP-C5, a random mated population of Upland cotton germplasm. J Plant Regist 2:239–242CrossRefGoogle Scholar
  12. Jenkins JN, McCarty JC, Wubben MJ, Hayes R, Gutierrez OA, Callahan F, Deng D (2012) SSR markers for marker assisted selection of root-knot nematode (Meloidogyne incognita) resistant plants in cotton (Gossypium hirsutum L.). Euphytica 183:49–54CrossRefGoogle Scholar
  13. Kirkpatrick TL, van Iersel MW, Oosterhuis DM (1995) Influence of Meloidogyne incognita on the water relations of cotton growth in microplots. J Nematol 27:465–471Google Scholar
  14. Knip M, de Pater S, Hooykaas PJJ (2012) The SLEEPER genes: a transposase-derived angiosperm-specific gene family. BMC Plant Biol 12:192CrossRefGoogle Scholar
  15. Knip M, Hiemstra S, Sietsma A, Castelein M, de Pater S, Hooykaas P (2013) DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues. BMC Plant Biol 13:211CrossRefGoogle Scholar
  16. Kumar P, He Y, Singh R, Davis RF, Guo H, Paterson AH, Peterson DG, Shen X, Nichols R, Chee PW (2016) Fine mapping and identification of candidate gene for a QTL affecting Meloidogyne incognita reproduction in Upland cotton. BMC Genom 17:567CrossRefGoogle Scholar
  17. Lu P, Davis RF, Kemerait RC, van Iersel MW, Scherm H (2014) Physiological effects of Meloidogyne incognita infection on cotton genotypes with differing levels of resistance in the greenhouse. J Nematol 46:352–359Google Scholar
  18. McClure MA, Ellis KC, Nigh EL (1974) Post-infection development and histopathology of Meloidogyne incognita in resistant cotton. J Nematol 6:21–26Google Scholar
  19. McPherson GR, Jenkins JN, McCarty JC, Watson CE (1995) Combining ability analysis of root-knot nematode resistance in cotton. Crop Sci 35:373–375CrossRefGoogle Scholar
  20. McPherson MG, Jenkins JN, Watson CE, McCarty JC Jr (2004) Inheritance of root-knot nematode resistance in M-315 RNR and M78-RNR cotton. J Cotton Sci 8:154–161Google Scholar
  21. Nakajima K, Benfey PN (2002) Signaling in and out: control of cell division and differentiation in the shoot and root. Plant Cell 14:S265–S276CrossRefGoogle Scholar
  22. Shen X, Becelaere GV, Kumar P, Davis RF, May OL, Chee P (2006) QTL mapping for resistance to root-knot nematodes in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. Theor Appl Genet 113:1539–1549CrossRefGoogle Scholar
  23. Shen X, He Y, Lubbers EL, Davis RF, Nichols RL, Chee PW (2010) Fine mapping QMi-C11 a major QTL controlling root-knot nematodes resistance in Upland cotton. Theor Appl Genet 121:1623–1631CrossRefGoogle Scholar
  24. Shepherd RL (1974) Transgressive segregation for root-knot nematode resistance in cotton. Crop Sci 14:872–875CrossRefGoogle Scholar
  25. Shepherd RL (1982) Registration of three germplasm lines of cotton (Reg. Nos. GP 164–166). Crop Sci 22:692Google Scholar
  26. Shepherd RL, McCarty JC Jr, Jenkins JN, Parrott WL (1996) Registration of nine cotton germplasm lines resistant to root-knot nematode. Crop Sci 36:820CrossRefGoogle Scholar
  27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  28. Thyssen GN, Fang DD, Turley RB, Florane C, Li P, Naoumkina M (2014) Next generation genetic mapping of the Ligon-lintless-2 (Li2) locus in upland cotton (Gossypium hirsutum L.). Theor Appl Genet 127:183–192CrossRefGoogle Scholar
  29. Walker NR, Kirkpatrick TL, Rothrock CS (1998) Interaction between Meloidogyne incognita and Thielaviopsis basicola on cotton (Gossypium hirsutum). J Nematol 30:415–422Google Scholar
  30. Wan C-Y, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12CrossRefGoogle Scholar
  31. Wubben MJ, Callahan FE, Jenkins JN, Deng DD (2016) Coupling of MIC-3 overexpression with the chromosome 11 and 14 root-knot nematode (RKN) (Meloidogyne incognita) resistance QTLs provides insights into the regulation of the RKN resistance response in Upland cotton (Gossypium hirsutum). Theor Appl Genet 129:1759–1767CrossRefGoogle Scholar
  32. Ynturi P, Jenkins JN, McCarty JC, Gutierrez OA, Saha S (2006) Association of root-knot nematode resistance genes with simple sequence repeat markers on two chromosomes in cotton. Crop Sci 46:2670–2674CrossRefGoogle Scholar
  33. Zhang T, Hu Y, Jiang W, Fang L, Guan X, Chen J, Zhang J, Saski CA, Scheffler BE, Stelly DM, Hulse-Kempe AM, Wan Q, Liu B, Liu C, Wang S, Pan M, Wang Y, Wang D, Ye W, Chang L, Zhang W, Song Q, Kirkbride RC, Chen X, Dennis E, Llewellyn DJ, Peterson DG, Thaxton P, Jones DC, Wang Q, Xu X, Zhang H, Wu H, Zhou L, Mei G, Chen S, Tian Y, Xiang D, Li X, Ding J, Zuo Q, Tao L, Liu Y, Li J, Lin Y, Hui Y, Cao Z, Cai C, Zhu X, Jiang Z, Zhou B, Guo W, Li R, Chen ZJ (2015) Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol 33:531–540CrossRefGoogle Scholar

Copyright information

© This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2019

Authors and Affiliations

  • Martin J. Wubben
    • 1
    Email author
  • Gregory N. Thyssen
    • 2
    • 3
  • Franklin E. Callahan
    • 1
  • David D. Fang
    • 2
  • Dewayne D. Deng
    • 1
  • Jack C. McCarty
    • 1
  • Ping Li
    • 2
  • Md Sariful Islam
    • 4
  • Johnie N. Jenkins
    • 1
  1. 1.Crop Science Research Laboratory, Genetics and Precision Agriculture Research UnitUSDA-ARSMississippi StateUSA
  2. 2.Southern Regional Research Center, Cotton Fiber Bioscience Research UnitUSDA-ARSNew OrleansUSA
  3. 3.Southern Regional Research Center, Cotton Chemistry and Utilization Research UnitUSDA-ARSNew OrleansUSA
  4. 4.Sugarcane Field StationUSDA-ARSCanal PointUSA

Personalised recommendations