A Multiplex Microsatellite Marker Kit for Diversity Assessment of Large Cassava (Manihot esculenta Crantz) Germplasm Collections

Abstract

Current methods for molecular fingerprinting of cassava (Manihot esculenta Crantz) have limited throughput or are costly, thus preventing the characterization of large germplasm collections such as those held by the International Agricultural Research Centers or National Research Institutions, which comprise hundreds to thousands of accessions. Here, we report the development of a fluorescence-based multiplex simple sequence repeat (SSR) marker kit that enables accurate and cost-effective cassava fingerprinting. The kit comprises 16 SSR markers assembled into five multiplex panels and was tested on 21 cassava cultivars alongside one accession of Manihot epruinosa, a wild relative. A total of 68 alleles were detected with, on average, 4.25 alleles per locus and a polymorphism information content of 0.53. The marker kit reported here is comparable to previously published amplified fragment length polymorphism and SSR marker systems in terms of discriminating power and informativeness while offering significant advantages in speed and cost of marker analysis. Previous molecular genetic diversity studies have suggested that cassava germplasm collections contain duplicate entries based on the occurrence of identical genetic profiles. Using the newly developed microsatellite kit, three out of six putative duplicate accessions could be readily differentiated, showing that these are distinct genotypes. The relevance of these findings with respect to the characterization and management of large cassava germplasm collections is discussed.

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References

  1. Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631. doi:10.1007/s00299-008-0507-z

    PubMed  Article  CAS  Google Scholar 

  2. Beeching JR, Marmey P, Gavalda MC, Noirot M, Haysom HR, Hughes MA, Charrier A (1993) An assessment of genetic diversity within a collection of cassava (Manihot esculenta Crantz) germplasm using molecular markers. Ann Bot 72:515–520

    Article  CAS  Google Scholar 

  3. Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic-linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331

    PubMed  CAS  Google Scholar 

  4. Campoy JA, Martinez-Gomez P, Ruiz D, Rees J, Celton JM (2010) Developing microsatellite multiplex and megaplex PCR systems for high-throughput characterization of breeding progenies and linkage maps spanning the apricot (Prunus armeniaca L.) genome. Plant Mol Biol Rept 28:560–568. doi:10.1007/s11105-010-0186-0

    Article  CAS  Google Scholar 

  5. Chavarriaga-Aguirre P, Maya MM, Tohme J, Duque MC, Iglesias C, Bonierbale MW, Kresovich S, Kochert G (1999) Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections. Mol Breed 5:263–273. doi:10.1023/A:1009627231450

    Article  CAS  Google Scholar 

  6. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rept 1:19–21. doi:10.1007/BF02712670

    Article  CAS  Google Scholar 

  7. Dixon AGO, Bandyopadhyay R, Coyne D, Ferguson M, Shaun R, Ferris B, Hanna R, Hughes J, Ingelbrecht I, Legg J, Mahungu N, Manyong VMD, Neuenschwander P, Whyte J, Hartmann P, Ortiz R (2003) Cassava: from poor farmers’ crop to pacesetter of African rural development. Chron Hortic 43:8–15

    Google Scholar 

  8. Fregene M, Bernal A, Duque M, Dixon A, Tohme J (2000) AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD). Theor Appl Genet 100:678–685. doi:10.1007/s001220051339

    Article  CAS  Google Scholar 

  9. Hall A, Ballantyne J (2003) The development of an 18-locus Y-STR system for forensic casework. Anal Bioanal Chem 376:1234–1246. doi:10.1007/s00216-003-2039-2

    PubMed  Article  CAS  Google Scholar 

  10. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotech 23:504–511

    CAS  Google Scholar 

  11. Hurtado P, Ospina C, Marin J, Buitrago C, Castelblanco W, Correa A et al (2008) Assessment of the diversity in global cassava genetic resources based on simple sequence repeat (SSR) markers. In: Fauquet CM (ed) Cassava: meeting the challenges of the new millennium. Proceedings of the first scientific meeting of the global cassava partnership, Ghent, Belgium. Institute of Plant Biotechnology for Developing Countries, Ghent University, Ghent, 1p

    Google Scholar 

  12. Kawuki RS, Ferguson M, Labuschagne M, Herselman L, Kim DJ (2009) Identification, characterisation and application of single nucleotide polymorphisms for diversity assessment in cassava (Manihot esculenta Crantz). Mol Breed 23:669–684. doi:10.1007/s11032-009-9264-0

    Article  CAS  Google Scholar 

  13. Koo B, Pardey PG, Wright BD (2003) The economic costs of conserving genetic resources at the CGIAR centres. Agric Econ 29:287–297. doi:10.1016/S0169-5150(03)00056-2

    Google Scholar 

  14. Lerceteau-Kohler E, Weiss S (2006) Development of a multiplex PCR microsatellite assay in brown trout Salmo trutta, and its potential application for the genus. Aquac 258:641–645. doi:10.1016/j.aquaculture.2006.04.028

    Article  Google Scholar 

  15. Lokko Y, Dixon A, Offei S, Danquah E, Fregene M (2006) Assessment of genetic diversity among African cassava Manihot esculenta Crantz accessions resistant to the cassava mosaic virus disease using SSR markers. Genet Resour Crop Evol 53:1441–1453. doi:10.1007/s10722-005-6841-x

    Article  Google Scholar 

  16. Lokko Y, Anderson JV, Rudd S, Raji A, Horvath D, Mikel MA, Kim R, Liu L, Hernandez A, Dixon AGO, Ingelbrecht IL (2007) Characterization of an 18, 166 EST dataset for cassava (Manihot esculenta Crantz) enriched for drought-responsive genes. Plant Cell Rep 26:1605–1618. doi:10.1007/s00299-007-0378-8

    PubMed  Article  CAS  Google Scholar 

  17. Lopez C, Jorge V, Piegu B, Mba C, Cortes D, Restrepo S, Soto M, Laudie M, Berger C, Cooke R, Delseny M, Tohme J, Verdier V (2004) A unigene catalogue of 5700 expressed genes in cassava. Plant Mol Biol 56:541–554. doi:10.1007/s11103-004-0123-4

    PubMed  Article  Google Scholar 

  18. Merdinoglu D, Butterlin G, Bevilacqua L, Chiquet V, Dam-Blondon AF, Decroocq S (2005) Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol Breed 15:349–366. doi:10.1007/s11032-004-7651-0

    Article  CAS  Google Scholar 

  19. Moon CD, Tapper BA, Scott B (1999) Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis. Appl Environ Microbiol 65:1268–1279

    PubMed  CAS  Google Scholar 

  20. Motilal LA, Zhang D, Umaharan P, Mischke S, Boccara M, Pinney S (2009) Increasing accuracy and throughput in large-scale microsatellite fingerprinting of cacao field germplasm collections. Trop Plant Biol 2:23–37. doi:10.1007/s12042-008-9016-z

    Article  CAS  Google Scholar 

  21. Moyib OK, Odunola OA, Dixon AGO (2007) SSR markers reveal genetic variation between improved cassava cultivars and landraces within a collection of Nigerian cassava germplasm. Afr J Biotechnol 6:2666–2674

    CAS  Google Scholar 

  22. Nei M (1972) Genetic distance between populations. Am Nat 106:283–292. doi:10.1086/282771

    Article  Google Scholar 

  23. Nweke FI, Spencer DSC, Lynam JK (2002) The cassava transformation. Africa’s best kept secret. Michigan State University, East Lansing

    Google Scholar 

  24. Olsen KM (2004) SNPs, SSRs and inferences on cassava’s origin. Plant Mol Biol 56:517–526. doi:10.1007/s11103-004-5043-9

    PubMed  Article  CAS  Google Scholar 

  25. Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295. doi:10.1111/j.1471-8286.2005.01155.x

    Article  Google Scholar 

  26. Raji AAJ, Anderson JV, Kolade OA, Ugwu CD, Dixon AGO, Ingelbrecht IL (2009a) Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility. BMC Plant Biol 9:118. doi:10.1186/1471-2229-9-118

    PubMed  Article  Google Scholar 

  27. Raji AAJ, Fawole I, Gedil M, Dixon AGO (2009b) Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers. Ann Appl Biol 155:187–199. doi:10.1111/j.1744-7348.2009.00329.x

    Article  Google Scholar 

  28. Xia L, Peng KM, Yang SY, Wenzl P, de Vicente MC, Fregene M, Kilian A (2005) DArT for high-throughput genotyping of cassava (Manihot esculenta) and its wild relatives. Theor Appl Genet 110:1092–1098. doi:10.1007/s00122-005-1937-4

    PubMed  Article  CAS  Google Scholar 

  29. Zacarias AM, Botha AM, Labuschagne MT, Benesi IRM (2004) Characterization and genetic distance analysis of cassava (Manihot esculenta Crantz) germplasm from Mozambique using RAPD fingerprinting. Euphytica 138:49–53. doi:10.1023/B:EUPH.0000047057.05642.74

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are grateful to Mr. B Odeseye and Ms. T Adenigba for excellent technical assistance and to Mrs. J Plancarte for help with figures and tables. Mr. TC de Bang acknowledges Dansk Agronomforening for a travel grant and Mr. G Backes and Mr. J Orabi for assistance with the GeneMapper software in Copenhagen, Denmark.

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Correspondence to Ivan L. Ingelbrecht.

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de Bang, T.C., Raji, A.A. & Ingelbrecht, I.L. A Multiplex Microsatellite Marker Kit for Diversity Assessment of Large Cassava (Manihot esculenta Crantz) Germplasm Collections. Plant Mol Biol Rep 29, 655–662 (2011). https://doi.org/10.1007/s11105-010-0273-2

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Keywords

  • Cassava
  • Diversity analysis
  • Manihot esculenta Crantz
  • Microsatellite primers
  • Multiplex PCR