Theoretical and Applied Genetics

, Volume 127, Issue 2, pp 481–488 | Cite as

Mapping and identification of a Cicer arietinum NSP2 gene involved in nodulation pathway

  • L. AliEmail author
  • E. Madrid
  • R. K. Varshney
  • S. Azam
  • T. Millan
  • J. Rubio
  • J. Gil
Original Paper


Key message

For the first time the putative NSP2 gene in chickpea has been identified using pairs of NILs differing for the Rn1 / rn1 nodulation gene that was located in LG5 of chickpea genetic map.


An intraspecific cross between the mutant non-nodulating genotype PM233, carrying the recessive gene rn1, and the wild-type CA2139 was used to develop two pairs of near-isogenic lines (NILs) for nodulation in chickpea. These pairs of NILs were characterized using sequence tagged microsatellite site (STMS) markers distributed across different linkage groups (LGs) of the chickpea genetic map leading to the detection of polymorphic markers located in LG5. Using this information, together with the genome annotation in Medicago truncatula, a candidate gene (NSP2) known to be involved in nodulation pathway was selected for mapping in chickpea. The full length sequence obtained in chickpea wild-type (CaNSP2) was 1,503 bp. Linkage analysis in an F3 population of 118 plants derived from the cross between the pair of NILS NIL7-2A (nod) × NIL7-2B (non-nod) revealed a co-localization between CaNSP2 and Rn1 gene. These data implicate the CaNSP2 gene as a candidate for identity to Rn1, and suggest that it could act in the nodulation signaling transduction pathway similarly to that in other legumes species.


Cicer Arietinum Nodulation Pathway Chickpea Genome Nodulin Gene GRAS Domain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work has been supported by the project INIA contract RTA2010-00059, co-financed by EU funds (FEDER). We are also indebted to Dr. F. Temprano (IFAPA, Spain) for his support with Rhizobium inoculations. Ali L acknowledges PhD fellowship from Syrian Ministry of High Education and ICRISAT for supporting 6 months stay at the Center of Excellence in Genomics, ICRISAT, Patancheru, India (

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Arrighi JF, Barre A, Ben Amor B, Bersoult A, Soriano LC, Mirabella R, de Carvalho-Niebel F, Journet EP, Ghérardi M, Huguet T, Geurts R, Dénarié J, Rougé P, Gough C (2006) The Medicago truncatula lysine motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. J Plant Physiol 142:265–279. doi: 10.1104/pp.106.084657 CrossRefGoogle Scholar
  2. Asad S, Fang Y, Wycoff KL, Hirsch A (1994) Isolation and characterization of cDNA and genomic clones of MsENOD40; transcripts are detected in meristematic cells of alfalfa. Protoplasma 183:10–23. doi: 10.1007/bf01276808 CrossRefGoogle Scholar
  3. Aslam M, Mahmood IA, Peoples MB, Schwenke GD, Herridge DF (2003) Contribution of chickpea nitrogen fixation to increased wheat production and soil organic fertility in rain-fed cropping. Biol Fertil Soils 38:59–64. doi: 10.1007/s00374-003-0630-5 CrossRefGoogle Scholar
  4. Baek KH, Skinner DZ (2003) Alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. Plant Sci 165:1221–1227. doi: 10.1016/S0168-9452(03)00329-7 CrossRefGoogle Scholar
  5. Catoira R, Galera C, de Billy F, Penmetsa RV, Journet EP, Maillet F, Rosenberg C, Cook D, Gough C, Dénarié J (2000) Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell 12:1647–1665. doi: 10.1105/tpc.12.9.1647 PubMedCentralPubMedGoogle Scholar
  6. Cook D, Dreyer D, Bonnet D, Howell M, Nony E, VandenBosch K (1995) Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti. Plant Cell 7:43–55. doi: 10.1105/tpc.7.1.43 PubMedCentralPubMedGoogle Scholar
  7. Crespi MD, Jurkevitch E, Poiret M, D’Aubenton-Carafa Y, Petrovics G, Kondorosi E, Kondorosi A (1994) Enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth. EMBO J 13:5099–5112PubMedGoogle Scholar
  8. Davis TM (1988) Two genes that confer ineffective nodulation in chickpea (Cicer arietinum L.). J Hered 79:476–478Google Scholar
  9. Davis TM, Foster KW, Phillips DA (1985) Nodulation mutants in chickpea. Crop Sci 25:345–348. doi: 10.2135/cropsci1985.0011183X002500020033x CrossRefGoogle Scholar
  10. Davis TM, Foster KW, Phillips DA (1986) Inheritance and expression of three genes controlling root nodule formation in chickpea. Crop Sci 26:719–723. doi: 10.2135/cropsci1986.0011183X002600040017x CrossRefGoogle Scholar
  11. Drummond AJ, Ashton B, Cheung M, Heled J, Kearse M, Moir R, Stones-Havas S, Thierer T, Wilson A (2010) Geneious v5.0. Available from
  12. Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev Phytopathol 42:185–209PubMedCrossRefGoogle Scholar
  13. FAOSTAT (2013) Last update January 2013
  14. Fisher RF, Long SR (1992) Rhizobium–plant signal exchange. Nature 357:655–660PubMedCrossRefGoogle Scholar
  15. Hirsch S, Kim J, Munoz A, Heckmann AB, Downie JA, Oldroyd GED (2009) GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell 21:545–557. doi: 10.1105/tpc.108.064501 PubMedCentralPubMedCrossRefGoogle Scholar
  16. Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes Genom Genomics 3:1–14Google Scholar
  17. Kalo P, Gleason C, Edwards A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J, Kiss GB, Downie JA, Oldroyd GED (2005) Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308:1786–1789. doi: 10.1126/science.1110951 PubMedCrossRefGoogle Scholar
  18. Kosambi DD (1944) The estimation of map distance from recombination values. Ann Eugen 12:172–175. doi: 10.1111/j.1469-1809.1943.tb02321.x CrossRefGoogle Scholar
  19. Lichtenzveig J, Scheuring C, Dodge J, Abbo S, Zhang HB (2005) Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L. Theor Appl Genet 110:492–510. doi: 10.1007/s00122-004-1857-8 PubMedCrossRefGoogle Scholar
  20. Long SR (1996) Rhizobium symbiosis: nod factors in perspective. Plant Cell 8:1885–1898. doi: 10.1105/tpc.8.10.1885 PubMedCentralPubMedGoogle Scholar
  21. Manickavelu A, Kawaura K, Oishi K, Shin IT, Kohara Y, Yahiaoui N, Keller B, Suzuki A, Yano K, Ogihara Y (2010) Comparative gene expression analysis of susceptible and resistant near-isogenic lines in common wheat infected by Puccinia triticina. DNA Res 17:211–222. doi: 10.1093/dnares/dsq009 PubMedCentralPubMedCrossRefGoogle Scholar
  22. Matthews LJ, Davis TM (1990) Anatomical comparison of wild-type and non-nodulating mutant chickpea (Cicer arietinum). Can J Bot 68:1201–1207. doi: 10.1139/b90-152 CrossRefGoogle Scholar
  23. Millan T, Winter P, Jüngling R, Gil J, Rubio J, Cho S, Cobos MJ, Iruela M, Rajesh PN, Tekeoglu M, Kahl G, Muehlbauer FJ (2010) A consensus genetic map of chickpea (Cicer arietinum L.) based on 10 mapping populations. Euphytica 175:175–189. doi: 10.1007/s10681-010-0157-4 CrossRefGoogle Scholar
  24. Murakami Y, Miwa H, Imaizumi-Anraku H, Kouchi H, Downie JA, Kawaguchi M, Kawasaki S (2007) Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation. DNA Res 13:255–265. doi: 10.1093/dnares/dsl017 CrossRefGoogle Scholar
  25. Nayak S, Zhu H, Varghese N, Datta S, Choi H-K, Horres R, Jüngling R, Singh J, Kavi Kishor PB, Sivaramakrishnan S, Hoisington D, Kahl G, Winter P, Cook D, Varshney R (2010) Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome. Theor Appl Genet 120:1415–1441. doi: 10.1007/s00122-010-1265-1 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Oh JM, Balkunde S, Yang P, Yoon DB, Ahn SN (2011) Fine mapping of grain weight QTL, tgw11 using near isogenic lines from a cross between Oryza sativa and O. grandiglumis. Genes Genom 33:259–265. doi: 10.1007/s13258-011-0038-9 CrossRefGoogle Scholar
  27. Oldroyd GED, Long SR (2003) Identification and characterization of nodulation-signaling pathway 2, a gene of Medicago truncatula involved in nod factor signaling. J Plant Physiol 131:1027–1032. doi: 10.1104/pp.102.010710 CrossRefGoogle Scholar
  28. Paruvangada V, Davis T (1999) A dominant, host plant mutation conferring ineffective nodulation in the chickpea–Rhizobium symbiosis. J Hered 90:297–299. doi: 10.1093/jhered/90.2.297 CrossRefGoogle Scholar
  29. Pichon M, Journet EP, Dedieu A, de Billy F, Truchet G, Barker DG (1992) Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa. Plant Cell 4:1199–1211. doi: 10.1105/tpc.4.10.1199 PubMedCentralPubMedGoogle Scholar
  30. Rajesh P, Tullu A, Gil J, Gupta V, Ranjekar P, Muehlbauer F (2002) Identification of an STMS marker for the double-podding gene in chickpea. Theor Appl Genet 105:604–607. doi: 10.1007/s00122-002-0930-4 PubMedCrossRefGoogle Scholar
  31. Rozen S, Skaletsky H (1999) Bioinformatics methods and protocols. Methods in molecular biology™. In: Misener S, Krawetz S (eds) Primer3 on the WWW for general users and for biologist programmers, vol 132. Humana Press, New York, pp 365–386. doi: 10.1385/1-59259-192-2:365 Google Scholar
  32. Sabatini S, Heidstra R, Wildwater M, Scheres B (2003) SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev 17:354–358PubMedCrossRefGoogle Scholar
  33. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  34. Seres A, Deák G, Tóth G, Aubert G, Burstin J, Ellis N, Kiss GB (2007) Comparative mapping. In: Mathesius U, Journet EP, Sumner LW (eds) The Medicago truncatula handbook. ISBN 0-9754303-1-9.
  35. Singh O, Rheenen HAV, Rupela OP (1992) Inheritance of a new nonnodulation gene in chickpea. Crop Sci 32:41–43. doi: 10.2135/cropsci1992.0011183X003200010009x CrossRefGoogle Scholar
  36. Štajner N, Bohanec B, Javornik B (2002) Genetic variability of economically important Asparagus species as revealed by genome size analysis and rDNA ITS polymorphisms. Plant Sci 162:931–937. doi: 10.1016/S0168-9452(02)00039-0 CrossRefGoogle Scholar
  37. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092 PubMedCrossRefGoogle Scholar
  38. Van Ooijen JW (2006) MAPQIL® 5, software for the mapping of quantitative trait loci in experimental populations. Plant Research International, WagenningenGoogle Scholar
  39. Varshney RK, Song C, Saxena RK, Azam S et al (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat Biotechnol.
  40. Vernoud V, Journet EP, Barker DG (1999) MtENOD20, a nod factor-inducible molecular marker for root cortical cell activation. Mol Plant Microbe Interact 12:604–614CrossRefGoogle Scholar
  41. Winter P, Pfaff T, Udupa SM, Hüttel B, Sharma PC, Sahi S, Arreguin-Espinoza R, Weigand F, Muehlbauer FJ, Kahl G (1999) Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol Gen Genet 262:90–101. doi: 10.1007/s004380051063 PubMedCrossRefGoogle Scholar
  42. Zuckercandl E, Pauling L (1965) Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ (eds) Evolving genes and proteins. Academic Press, USA, pp 97–165 (article-id:2345322)Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • L. Ali
    • 1
    Email author
  • E. Madrid
    • 2
  • R. K. Varshney
    • 3
  • S. Azam
    • 3
  • T. Millan
    • 1
  • J. Rubio
    • 4
  • J. Gil
    • 1
  1. 1.Dpto GenéticaUniv Córdoba, Campus de excelencia internacional CeiA3, Campus de RabanalesCórdobaSpain
  2. 2.Institute for Sustainable Agriculture, CSICCórdobaSpain
  3. 3.International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)HyderabadIndia
  4. 4.Área de Mejora y BiotecnologíaIFAPA Centro “Alameda del Obispo”CórdobaSpain

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