Genotipado por secuenciación de variedades tradicionales de Theobroma cacao (Malvaceae) del Estado de Tabasco, México

  • Jorge Ricaño-Rodríguez Universidad Veracruzana
  • Enrique Hipólito-Romero Universidad Veracruzana
  • José M. Ramos-Prado Universidad Veracruzana
  • Eliezer Cocoletzi-Vásquez Universidad Veracruzana


Background: Single nucleotide polymorphisms (SNPs) have been identified in Theobroma cacao through a genotyping-by-sequencing approach. Through this research it is shared for the first time a set of results related to genetic variability and nature of conserved coding regions of reduced nucleotide sequences of mexican native varieties of cocoa.

Hypothesis: Obtaining reduced genomes of T. cacao specimens by restriction enzymes (REs) allows the characterization of single nucleotide polymorphisms (SNPs) as well as conserved coding regions (CDs).

Species of study and dates: Theobroma cacao L. (Malvaceae)

Study site: Theobroma cacao twigs came from traditional agroforestry plots located in the municipalities of Cardenas, Huimanguillo, Comalcalco, Paraiso, Jalpa de Mendez and Cunduacan, Tabasco, as well as Ixtacomitan and Pichucalco, Chiapas, Mexico; and they were collected and grafted among May and June from 2018.

Methods: A method of genotyping-by-sequencing for the characterization of biobanks was developed. Filtering of crude sequences, genomic assembly, identification of SNPs, taxonomic molecular characterization and characterization of coding regions as well as minimum evolution of protein transcripts were performed.

Results: Theobroma cacao samples showed different SNPs percentages (2 – 11 %) and the molecular evolution analyzes suggested similar maximum compound probabilities respect to their phylogeny. Conserved sequences were observed in the genomes´ coding regions, which suggest heuristic ontological predictions that have been evolutionarily regrouped in five clusters related to transcription processes and secondary metabolism.

Conclusions: The GBS method allows to identify SNPs in cocoa. The characterization of reduced genomes determined the structural and transcriptional correlation between the samples and the reference genome of cacao Criollo.


Download data is not yet available.

Author Biographies

Jorge Ricaño-Rodríguez, Universidad Veracruzana
Centro Ecodiálogo
Enrique Hipólito-Romero, Universidad Veracruzana
Centro Ecodiálogo. Profesor Investigador de Tiempo Completo
José M. Ramos-Prado, Universidad Veracruzana
Centro Ecodiálogo. Profesor Investigador de Tiempo Completo
Eliezer Cocoletzi-Vásquez, Universidad Veracruzana
Centro Ecodiálogo. Investigador postdoctoral
Genotipado por secuenciación de variedades tradicionales de <em>Theobroma cacao</em> (Malvaceae) del Estado de Tabasco, México


Allegre M, Argout X, Boccara M, Fouet O, Roguet Y, Be?rard A, The?venin JM, Chauveau A, Rivallan R, Clement D, Courtois B, Gramacho K, Boland-Auge? A, Tahi M, Umaharan P, Brunel D, Lanaud C. 2012. Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L. DNA Research 19: 23-35. DOI:

Argout X, Martin G, Droc G, Fouet O, Labadie K, Rivals E, Aury JM, Lanaud C. 2017. The cacao Criollo genome v2.0: an improved version of the genome for genetic and functional genomic studies. BMC Genomics 18: 730. DOI:

Ashley MV. 2010. Plant parentage, pollination, and dispersal: how DNA microsatellites have altered the landscape. Critical Reviews in Plant Sciences 29: 148-161. DOI:

Bartley BGD. 2005. The genetic diversity of cacao and its utilization. Oxfordshire, UK: CABI Publishing. ISBN: 184593024X, 9781845930240

Buckler ES, Thornsberry J. 2002. Plant molecular diversity and applications to genomics. Current Opinion in Plant Biology 5: 107-111. DOI:

Carvalho-Santos R, Pires JL, Correa RX. 2012. Morphological characterization of leaf, flower, fruit and seed traits among Brazilian Theobroma L. species. Genetic Resources and Crop Evolution 59: 327-345. DOI:

Collard BC, Mackill DJ. 2008. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philosophical Transactions of the Royal Society 363: 557-572. DOI:

Craig DW, Pearson JV, Szelinger S, Sekar A, Redman M, Corneveaux JJ, Pawlowski TL, Laub T, Nunn G, Stephan DA, Homer N, Huentelman MJ. 2008. Identification of genetic variants using bar-coded multiplexed sequencing. Nature Methods 10: 887-893. DOI:

Cronn R, Liston A, Parks M, Gernandt DS, Shen R, Mockler T. 2008. Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology. Nucleic Acids Research 36: e122. DOI:

Dadzie AM, Livingstone DS III, Opoku SY, Takrama J, Padi FK, Offei SK, Danquah EY, Motamayor JC, Schnell RJ, Kuhn DN. 2013. Conversion of microsatellite markers to single nucleotide polymorphism (SNP) markers for genetic fingerprinting of Theobroma cacao L. Journal of Crop Improvement 27: 215-241. DOI:

Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler E, Mitchell SE. 2011. A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLOS ONE 6: e19397. DOI:

Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791. DOI:

Gesteira AS, Micheli F, Carels N, Da Silva AC, Gramacho KP, Schuster I, Macêdo JN, Pereira GA, Cascardo JC. 2007. Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa. Annals of Botany 100: 129-140. DOI:

Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S, Thomas SM, Toulmin C. 2010. Food Security: The Challenge of Feeding 9 Billion People. Science 327: 812-818. DOI:

Gore M, Bradbury P, Hogers R, Kirst M, Verstege E, Oeveren JV, Peleman J, Buckler E, van Eijk M. 2007. Evaluation of target preparation methods for single-feature polymorphism detection in large complex plant genomes. Crop Science 47: S135-S148. DOI:

Gore MA, Chia JM, Elshire RJ, Sun Q, Ersoz ES, Hurwitz BL, Peiffer JA, McMullen MD, Grills GS, Ross-Ibarra J, Ware DH, Buckler ES. 2009. A first-generation haplotype map of maize. Science 326: 1115-1117. DOI:

Hipólito-Romero E, Carcaño-Montiel MG, Ramos-Prado JM, Vázquez-Cabañas EA, López-Reyes L, Ricaño-Rodríguez J. 2017. Efecto de inoculantes bacterianos edáficos mixtos en el desarrollo temprano de cultivares mejorados de cacao (Theobroma cacao L.) en un sistema agroforestal tradicional del norte de Oaxaca, México. Revista Argentina de Microbiología 94: 356-365. DOI:

Huang X, Feng Q, Qian Q, Zhao Q, Wang L, Wang A, Guan J, Fan D, Weng Q, Huang T, Dong G, Sang T, Han B. 2009. High-throughput genotyping by whole-genome resequencing. Genome Research 19: 1068-1076. DOI:

Ji K, Zhang D, Motilal LA, Boccara M, Lachenaud P, Meinhardt LW. 2013. Genetic diversity and parentage in farmer varieties of cacao (Theobroma cacao L.) from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markers. Genetic Resources and Crop Evolution 60: 441-453. DOI:

Jones JD, Dangl JL. 2006. The plant immune system. Nature 444: 323-329. DOI:

Jones PG, Allaway D, Gilmour DM, Harris C, Rankin D, Retzel ER, Jones CA. 2002. Gene discovery and microarray analysis of cacao (Theobroma cacao L.) varieties. Planta 216: 255-264. DOI:

Kuhn DN, Livingstone DS III, Main D, Zheng P, Saski C, Feltus FA, Mockaitis K, Farmer AD, May GD, Schnell RJ, Motamayor JC. 2012. Identification and mapping of conserved ortholog set (COS) II sequences of cacao and their conversion to SNP markers for marker- assisted selection in Theobroma cacao and comparative genomics studies. Tree Genetics & Genomes 8: 97-111. DOI:

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: 4096-4108. DOI:

Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874. DOI:

Lacombe T, Boursiquot J-M, Laucou V, Di Vecchi-Staraz M, Péros J-P, This P. 2013. Large-scale parentage analysis in an extended set of grapevine cultivars (Vitis vinifera L.). Theoretical and Applied Genetics 126: 401-414. DOI:

Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754-1760. DOI:

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. Genetics and Molecular Research 8: 799-808. DOI:

Lin R-C, Ding Z-S, Li L-B, Kuang T-Y. 2001. A rapid and efficient DNA minipreparation suitable for screening transgenic plants. Plant Molecular Biology Reporter 19: 379a-379e. DOI:

Lindo AA, Robinson DE, Tennant PF, Meinhardt LW, Zhang D. 2018. Molecular Characterization of Cacao (Theobroma cacao) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers. Tropical Plant Biology 11: 93-106. DOI:

Livingstone DS III, Freeman B, Motamayor JC, Schnell RJ, Royaert S, Takrama J, Meerow AW, Kuhn D. 2012. Optimization of a SNP assay for genotyping Theobroma cacao under field conditions. Molecular Breeding 30: 33-52. DOI:

Livingstone DS III, 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. Molecular Breeding 27: 93-106. DOI:

Motamayor JC, Mockaitis K, Schmutz J, Haiminen N, Livingstone III D, Cornejo O, Findley SD, Zheng P, Utro F, Royaert S, Saski C, Jenkins J, Podicheti R, Zhao M, Scheffler BE, Stack JC, Feltus FA, Mustiga GM, Amores F, Phillips W, Marelli JP, May GD, Shapiro H, Ma J, Bustamante CD, Schnell RJ, Main D, Gilbert D, Parida L, Kuhn DN. 2013. The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color. Genome Biology 14:r53. DOI:

Motamayor JC, Lachenaud P, da Silva 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. DOI:

Motamayor JC, Risterucci AM, Heath M, Lanaud C. 2003. Cacao domestication II: progenitor germplasm of the Trinitario cacao cultivar. Heredity 91: 322-330: DOI:

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. DOI:

Nei M, Kumar S. 2000. Molecular Evolution and Phylogenetics. New York: Oxford University Press, ISBN-10: 0195135857; ISBN-13: 978-0195135855

Papadopoulos JS, Agarwala R. 2007. COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics 23: 1073-1079. DOI:

Peakall R, Smouse PE. 2006. Genalex 6: genetic analysis in excel. Population genetic software for teaching and research. Molecular Ecology Resources 6: 288-295. DOI:

Peakall R, Smouse PE. 2012. Genalex 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics 28: 2537-2539. DOI:

Poland JA, Rife TW. 2012. Genotyping-by-Sequencing for plant breedings and genetics. The Plant Genome 5: 92-102. DOI:

Powis TG, Cyphers A, Gaikwad NW, Grivetti L, Cheong K. 2011. Cacao use and the San Lorenzo Olmec. Proceedings of the National Academy of Sciences (USA) 108: 8595-8600. DOI:

Rafalski A. 2002. Applications of single nucleotide polymorphisms in crop genetics. Current Opinion in Plant Biology 5: 94-100. DOI:

Ricaño-Rodríguez J, Ramos-Prado JM, Cocoletzi-Vásquez E, Hipólito-Romero E. 2018. El estudio genómico del cacao; breve recopilación de sus bases conceptuales. Agroproductividad 11: 29-35. DOI:

Richardson JE, Whitlock BA, Meerow AW, Madrin?an S. 2015. The age of chocolate: a diversification history of Theobroma and Malvaceae. Frontiers in Ecology and Evolution 3: 120. DOI:

Rzhetsky A, Nei M. 1992. A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution 9: 945-967. DOI:

Saitou N, Nei M. 1987. The Neighbor-Joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425.

Santana JO, Freire L, de Sousa AO, Fontes Soares VL, Gramacho KP, Pirovani CP. 2016. Characterization of the legumains encoded by the genome of Theobroma cacao L. Plant Physiology and Biochemistry 98: 162-170. DOI:

Sneath PHA, Sokal RR. 1973. Numerical Taxonomy: the principles and practice of numerical classification. San Francisco: WH. Freeman. ISBN 0716706970, 9780716706977

Stone D, Mck Bird R, Ford RI, Leon J, Pickersgill B, Plowman T, Prance GT, Roosevelt A, Evans Schultes R. 1984. Pre-Columbian Plant Migration. Cambridge, UK: Papers of the Peabody Museum of Archaeology and Ethnology. ISBN-10: 0873652029; ISBN-13: 978-0873652025

Takrama J, Kun J, Meinhardt L, Mischke S, Opoku SY, Padi FK, Zhang D. 2014. Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markers. African Journal of Biotechnology 13: 2127-2136. DOI:

Takrama J, Dadzie AM, Opoku FK, Padi FK, Adomako B, Asu-Ampomah Y, Livingstone DS III, Motamayor JC, Schnell RJ, Kuhn RJ. 2012. Applying SNP marker technology in the cacao breeding program in Ghana. African Crop Science Journal 20: 67-75.

Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using the Neighbor-Joining method. Proceedings of the National Academy of Sciences 101: 11030-11035. DOI:

Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: 512-526. DOI:

Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander ES. 1998. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280: 1077-1082. DOI:

Weber JL, May PE. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. American Journal of Human Genetics 44: 388-396. DO

Zhang D, Martínez WJ, Johnson ES, Somarriba EJ, Phillips-Mora W, Astorga CM, Mischke S, Meinhardt LW. 2012. Genetic diversity and spatial structure in a new distinct Theobroma cacao L. population in Bolivia. Genetic Resources and Crop Evolution 59: 239-252. DOI:

How to Cite
Ricaño-Rodríguez, J., Hipólito-Romero, E., Ramos-Prado, J. M., & Cocoletzi-Vásquez, E. (2019). Genotipado por secuenciación de variedades tradicionales de Theobroma cacao (Malvaceae) del Estado de Tabasco, México. Botanical Sciences, 97(3), 381-397.