Skip to main content
SpringerLink
Log in

Optimization of a SNP assay for genotyping Theobroma cacao under field conditions

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

The tropical tree crop Theobroma cacao L. is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. Although the upper Amazon region is the center of origin for cacao, 70% of the world’s supply of cacao beans currently comes from small farms in West Africa. While cacao breeding programs in producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. While DNA extraction can be routinely performed, the equipment needed to analyze simple sequence repeats (SSRs) is seldom available, forcing the outsourcing of genotyping to foreign laboratories and delaying the breeding process. We describe a 5′ nuclease (TaqMan)-based single nucleotide polymorphism (SNP) assay for genotyping cacao plants under conditions similar to those found in most cacao-producing areas. The assay was tested under field conditions by planting open pollinated seeds of seven pods from four different maternal plants. The resulting 171 seedlings were successfully genotyped with 18 SNP markers representing 12 loci. The ability to use temperature-stable reagents and rapid DNA extraction methods is also explored. Additionally, by examining the seedling genotypes for the SNP markers and 14 additional SSR markers, we investigated whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method of genotyping cacao seedlings in the field should allow for more efficient resource management of seed gardens and is currently being implemented in Ghana.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Adu-Ampomah Y, Novak FJ, Klu GYP, Lamptey TVO (1990) Use of irradiated pollen as mentor pollen to induce self-fertilization of two self-incompatible Upper Amazon cacao clones. Euphytica 51:219–225

    Article  Google Scholar 

  • Bartley BGD (1969) Selfing of self-incompatible trees. Ann Rep Cacao Res (1968), Trinidad, pp 22–23

  • Bartley BGD (2005) The Genetic diversity of cacao and its utilization. CABI Publishing, Wallingford, p 341

    Book  Google Scholar 

  • Borrone J, Kuhn D, Schnell R (2004) Isolation, characterization, and development of WRKY genes as useful genetic markers in Theobroma cacao. Theor Appl Genet 109:495–507

    Article  PubMed  CAS  Google Scholar 

  • Borrone JW, Brown JS, Kuhn DN, Motamayor JC, Schnell RJ (2007) Microsatellite markers developed from Theobroma cacao L. expressed sequence tags. Mol Ecol Notes 7:236–239

    Article  CAS  Google Scholar 

  • Brown JS, Schnell RJ, Motamayor JC, Lopes U, Kuhn DN, Borrone JW (2005) Resistance gene mapping for witches’ broom disease in Theobroma cacao L. in an F2 population using SSR markers and candidate genes. J Am Soc Hort Sci 130:366–373

    CAS  Google Scholar 

  • Brown J, Sautter R, Olano C, Borrone J, Kuhn D, Motamayor J, Schnell R (2008) A composite linkage map from three crosses between commercial clones of cacao, Theobroma cacao L. Trop Plant Biol 1:120–130

    Article  Google Scholar 

  • Chan KL, Linley JR (1989) A new Florida species of Forcipomyia (Euprojoannisia) (Diptera: Ceratopogonidae) from leaves of the water lettuce, Pistia stratiotes. Fla Entomol 72:252–262

    Article  Google Scholar 

  • Decazy B, Coulibaly N (1981) Behaviour of cacao cultivars with respect to biting-sucking insects: possibility of early selection of tolerant cacao trees. In: Proceedings of 8th international Cocoa Res conference, Cartagena. COPAL, Nigeria

  • Duguma B, Gockowski J, Bakala J (2001) Smallholder cacao (Theobroma cacao Linn.) cultivation in agroforestry systems of West and Central Africa: challenges and opportunities. Agrofor Syst 51:177–188

    Article  Google Scholar 

  • Efombagn I, Motamayor J, Sounigo O, Eskes A, Nyassé S, Cilas C, Schnell R, Manzanares-Dauleux M, Kolesnikova-Allen M (2008) Genetic diversity and structure of farm and GenBank accessions of cacao (Theobroma cacao L.) in Cameroon revealed by microsatellite markers. Tree Genet Genom 4:821–831

    Article  Google Scholar 

  • Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194

    PubMed  CAS  Google Scholar 

  • Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185

    PubMed  CAS  Google Scholar 

  • Faleiro FG, Queiroz VT, Lopes UV, Guimaraes CT, Pires JL, Yamada MM, Araujo IS, Pereira MG, Schnell R, de Souza GA, Ferreira CF, Barros EG, Moreira MA (2006) Mapping QTLs for witches’ broom (Crinipellis perniciosa) resistance in cacao (Theobroma cacao L.). Euphytica 149:227–235

    Article  CAS  Google Scholar 

  • Falque M, Vincent A, Vaissiere B, Eskes A (1995) Effect of pollination intensity on fruit and seed set in cacao (Theobroma cacao L.). Sex Plant Reprod 8:354–360

    Article  Google Scholar 

  • Frimpong EA, Gordon I, Kwapong PK, Gemmill-Herren B (2009) Dynamics of cocoa pollination: tools and applications for surveying and monitoring cocoa pollinators. Int J Trop Insect Sci 29:62–69

    Article  Google Scholar 

  • Glendinning DR (1960) Selfing of self-incompatible cocoa. Nature 187:170

    Article  Google Scholar 

  • Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202

    PubMed  CAS  Google Scholar 

  • Hirotsu N, Murakami N, Kashiwagi T, Ujiie K, Ishimaru K (2010) Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species. Plant Methods 6:12

    Article  PubMed  Google Scholar 

  • ICCO (2010) Latest quarterly bulletin of cocoa statistics. http://www.icco.org/about/press2.aspx?Id=onl12753. Accessed 8 Feb 2011

  • Irish BM, Goenaga R, Zhang D, Schnell R, Brown JS, Motamayor JC (2010) Microsatellite fingerprinting of the USDA-ARS Tropical Agriculture Research Station cacao (Theobroma cacao L.) germplasm collection. Crop Sci 50:656–667

    Article  CAS  Google Scholar 

  • Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program cervus accommodates genotyping error increases success in paternity assignment. Mol Ecol 16:1099–1106

    Article  PubMed  Google Scholar 

  • Kuhn DN, Heath M, Wisser RJ, Meerow A, Brown JS, Lopes U, Schnell RJ (2003) Resistance gene homologues in Theobroma cacao as useful genetic markers. Theor Appl Genet 107:191–202

    Article  PubMed  CAS  Google Scholar 

  • Kuhn DN, Motamayor JC, Meerow AW, Borrone JW, Schnell RJ (2008) SSCP markers provide a useful alternative to microsatellites in genotyping and estimating genetic diversity in populations and germplasm collections of plant specialty crops. Electrophoresis 29:1–14

    Article  Google Scholar 

  • Kuhn D, Figueira A, Lopes U, Motamayor J, Meerow A, Cariaga K, Freeman B, Livingstone D, Schnell R (2010) Evaluating Theobroma grandiflorum for comparative genomic studies with Theobroma cacao. Tree Genet Genom 6:783–792. doi:10.1007/s11295-010-0291-0

    Article  Google Scholar 

  • Lanaud C, Sounigo O, Amefia YK, Paulin D, Lachenaud P, Clément D (1987) New data on the mechanisms of incompatibility in cocoa and its consequences on breeding. Café Cacao Thé 31:278–282

    Google Scholar 

  • Lanaud C, Risterucci AM, Pieretti I, Falque M, Bouet A, Lagoda PJL (1999) Isolation and characterization of microsatellites in Theobroma cacao L. Mol Ecol 8:2141–2143

    Article  PubMed  CAS  Google Scholar 

  • Lanaud C, Fouet O, Clément D, Boccara M, Risterucci A, Surujdeo-Maharaj S, Legavre T, Argout X (2009) A meta–QTL analysis of disease resistance traits of Theobroma cacao L. Mol Breed 24:361–374

    Article  Google Scholar 

  • Leggate J, Allain R, Isaac L, Blais B (2006) Microplate fluorescence assay for the quantification of double-stranded DNA using SYBR Green I dye. Biotechnol Lett 28:1587–1594

    Article  PubMed  CAS  Google Scholar 

  • Lijavetzky D, Cabezas J, Ibanez A, Rodriguez V, Martinez-Zapater J (2007) High-throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology. BMC Genomics 8:424

    Article  PubMed  Google Scholar 

  • Lima L, Gramacho K, Carels N, Novais R, Gaiotto F, Lopes U, Gesteira A, Zaidan H, Cascardo J, Pires J, Micheli F (2009) Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches’ broom disease in cacao. Genet Mol Res 8:799–808

    Article  PubMed  CAS  Google Scholar 

  • Livingstone D III, Freeman B, Tondo CL, Cariaga KA, Oleas NH, Meerow AW, Schnell RJ, Kuhn DN (2009) Improvement of high-throughput genotype analysis after implementation of a dual-curve Sybr Green I-based quantification and normalization procedure. Hort Sci 44:1228–1232

    Google Scholar 

  • Livingstone D, Motamayor J, Schnell R, Cariaga K, Freeman B, Meerow A, Brown J, Kuhn D (2011) Development of single nucleotide polymorphism markers in Theobroma cacao and comparison to simple sequence repeat markers for genotyping of Cameroon clones. Mol Breed 27:93–106

    Article  Google Scholar 

  • Motamayor JC, Risterucci AM, Lopez PA, Ortiz CF, Moreno A, Lanaud C (2002) Cacao domestication I: the origin of the cacao cultivated by the Mayas. Heredity 89:380–386

    Article  PubMed  CAS  Google Scholar 

  • Motamayor JC, Lachenaud P, e Mota JW, Loor R, Kuhn DN, Brown JS, Schnell RJ (2008) Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L.). PLoS ONE 3:e3311

    Article  PubMed  Google Scholar 

  • Opeke LK, Jacob VJ (1967) Studies on methods of overcoming self-incompatibility in Theobroma cacao Linn. In: 2e Conférence Internationale sur les Recherches Cacaoyères, pp 356–359

  • Posnette AF (1940) Self-incompatibility in cocoa (Theobroma spp.). Trop Agric 17:67–71

    Google Scholar 

  • Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100

    Article  PubMed  CAS  Google Scholar 

  • Rice RA, Greenberg R (2000) Cacao cultivation and the conservation of biological diversity. Ambio 29:167–173

    Google Scholar 

  • Schnell RJ, Kuhn DN, Brown JS, Olano CT, Phillips-Mora W, Amores FM, Motamayor JC (2007) Development of a marker assisted selection program for cacao. Phytopathology 97:1664–1669

    Article  PubMed  CAS  Google Scholar 

  • Schnell RJ, Motamayor JC, Brown JS, Kuhn DN, Tondo CL, Livingstone D, III, Royaert S, Nagai C, Phillips W, Amores FM, Suarez-Capello C, Lopes U, Takrama J, Padi F, Opoku S, Efombagn IB, Aikpokpodion P, Pokou D, Epaina P, Marfu J (in press) The international marker-assisted selection program for cacao. In: Proceedings of 16th international cocoa research conference, Bali, 2009. COPAL, Nigeria

  • Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088

    Article  PubMed  CAS  Google Scholar 

  • The Bovine HapMap Consortium (2009) Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds. Science 324:528–532

    Article  Google Scholar 

  • Van Tassell CP, Smith TPL, Matukumalli LK, Taylor JF, Schnabel RD, Lawley CT, Haudenschild CD, Moore SS, Warren WC, Sonstegard TS (2008) SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat Meth 5:247–252

    Article  Google Scholar 

  • Van K, Hwang EY, Kim MY, Park HJ, Lee SH, Cregan PB (2005) Discovery of SNPs in soybean genotypes frequently used as the parents of mapping populations in the United States and Korea. J Hered 96:529–535

    Article  PubMed  CAS  Google Scholar 

  • Vitzthum F, Geiger G, Bisswanger H, Brunner H, Bernhagen J (1999) A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system. Anal Biochem 276:59–64

    Article  PubMed  CAS  Google Scholar 

  • Yoon M, Song Q, Choi I, Specht J, Hyten D, Cregan P (2007) BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification. Theor Appl Genet 114:885–899

    Article  PubMed  CAS  Google Scholar 

  • Young A, Severson D (1994) Comparative analysis of steam-distilled floral oils of cacao cultivars (Theobroma cacao L., Sterculiaceae) and attraction of flying insects: implications for a Theobroma pollination syndrome. J Chem Ecol 20:2687–2703

    Article  CAS  Google Scholar 

  • Zhang D, Mischke S, Johnson E, Phillips-Mora W, Meinhardt L (2009) Molecular characterization of an international cacao collection using microsatellite markers. Tree Genet Genom 5:1–10

    Article  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. Nancy Epsky and Dr. Paul Kendra for their discussions about midges and cacao pollination. The authors are also grateful to Mike Winterstein, Carol Lee, and Paul Kuhn for their assistance maintaining and planting the cacao seedlings used in this study. The authors would also like to thank MARS, Inc. for their continued financial support.

Author information

Authors and Affiliations

  1. Subtropical Horticulture Research Station, USDA-ARS, 13601 Old Cutler Rd., Miami, FL, 33158, USA

    Donald S. Livingstone III, Barbie Freeman, Raymond J. Schnell, Stefan Royaert, Alan W. Meerow & David N. Kuhn

  2. Mars, Inc., Hackettstown, NJ, 07840, USA

    Juan Carlos Motamayor

  3. Cocoa Research Institute of Ghana (CRIG), Tafo, Ghana

    Jemmy Takrama

Authors
  1. Donald S. Livingstone III
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbie Freeman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juan Carlos Motamayor
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raymond J. Schnell
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefan Royaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jemmy Takrama
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alan W. Meerow
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David N. Kuhn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David N. Kuhn.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

Table of collected SNP assay genotypes (PDF 53 kb)

Online Resource 2

Table of collected SSR assay genotypes (PDF 48 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Livingstone, D.S., Freeman, B., Motamayor, J.C. et al. Optimization of a SNP assay for genotyping Theobroma cacao under field conditions. Mol Breeding 30, 33–52 (2012). https://doi.org/10.1007/s11032-011-9596-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-011-9596-4

Keywords

Search

Navigation