Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (Eleusine coracana)
- 467 Downloads
Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05504, NBS-09711, NBS-07688, NBS-03509 and EST-SSR-04241) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.
KeywordsMolecular markers NBS-LRR domains Blast resistance UPGMA Finger millet Magnaporthe grisea
The authors wish to acknowledge the Department of Biotechnology, Govt. of India for providing financial support in the form of Programme Mode Support for research and development in Agricultural Biotechnology at G.B. Pant University of Agriculture and Technology, Pantnagar (Grant No. BT/PR7849/AGR/02/374/2006). The first author was supported by fellowship from DBT, India during the project programme. The authors thank the All India Co-ordinated Small Millets Improvement Project, ICAR, UAS, GKVK, Bangalore and Hill Campus, Ranichauri G. B. Pant University of Agriculture and Technology for providing the seed samples and passport data of the germplasm analyzed in present study. We are highly thankful to Dr. T. Mohapatra for his kind academic inputs in data analysis and interpretation of data.
- 1.Chethan S, Malleshi NG (2007) Finger millet polyphenols, characterization and their nutraceutical potential. Am J Food Technol 27:282–292Google Scholar
- 2.Carine AT, Wang BB, Majesta SO, Shweta D, Zhu HY, Bruce R, Nevin DY, Steven BC (2008) Identification and characterization of nucleotide-binding site-leucine-rich repeat genes in the model plant Medicago truncatula. Plant Physiol 146:5–21Google Scholar
- 3.Li CL, Zhang HY (2004) Research advance of resistance gene analogs in plant. Mol Plant Breed 2(6):853–860Google Scholar
- 18.Lacock L, Van niekerk C, Loots S, Du preez F, Botha AM (2003) Functional and comparative analysis of expressed sequences from Diuraphis noxia infested wheat obtained utilizing the conserved Nucleotide Binding Site. Afr J Biotechnol 2(4):75–81Google Scholar
- 21.Lee SY, Seo JS, Rodriguez-Lanetty M, Lee DH (2003) Comparative analysis of superfamilies of NBSencoding disease resistance gene analogs in cultivated and wild apple species. Mol Gen Genomics 269:101–108Google Scholar
- 31.Maniatis T, Sambrook J, Fritsch EF (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
- 35.Jaccard P (1908) Nouvelles recherches sur la distribuition florale. Bull Soc Vaud Sci Nat 44:223–270Google Scholar
- 36.Rohlf FJ (2001) NTSYS-pc numerical taxonomy and multivariate analysis system. Version 5.1. Exeter Publishing Ltd, SetauketGoogle Scholar
- 37.Yap IV, Nelson R (1995) WinBoot, a program for performing bootstrap analysis of binary data to determine the confidence limits of UPGMA-based dendrograms. IRRI discussion paper series no. 14. IRRI, Los BanosGoogle Scholar
- 41.Breseghello F, Sorrells ME (2005) Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars. Genetics. doi: 10.1534/genetics.105.044586
- 44.Robert NGM, David JB, Franc CB, Candice MRS, Paulo CA, Natalia FM, Roberto CT, Manoel TSJ, Georgios JPJ (2008) Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminate Colla, isolation, RFLP marker development, and physical mapping. BMC Plant Biol 8:1471–2229Google Scholar