Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markers

Abstract

Accurate identification of individual genotypes is important for cacao (Theobroma cacao L.) breeding, germplasm conservation and seed propagation. The development of single nucleotide polymorphism (SNP) markers in cacao offers an effective way to use a high-throughput genotyping system for cacao genotype verification. In the present study, high-throughput genotyping with SNP markers was used to fingerprint 160 cacao trees in the germplasm collection at the Cocoa Research Institute of Ghana (CRIG). These accessions had been originally introduced from international germplasm collections. The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops.

The development of a novel SNP genotyping assay to differentiate cacao clones

“…This is undoubtedly less than the suspected level of mislabeling (15 to 44%) in germplasm collections worldwide 41 . Mislabeling and the verification of genetic identity from introduced as well conserved germplasm is an acute problem, as can be seen in this study and different publications over the last ten years 6,9,16,20,24,42 . Despite the fact that different applications and strategies on tackling cacao mislabeling have been provided by Turnbull et al .…”

“…Various SNP-based genotyping methods are currently available, enabling high-throughput genotype calling without the need for electrophoresis 34 . Many SNP based genotyping methods – specific for cacao research – have been described in literature, ranging from TaqMan-based assays 12,16,23 , over Maldi TOF MS 6,24,35 , and Fluidigm SNP genotyping 15,36,37 to the competitive allele specific PCR KASPar chemistry 9 . Yet, no standard SNP genotyping method has been proposed for cacao genetic diversity studies.…”

“…A total of 115 SNPs were initially selected from literature 6,12,24,27,33,36 , of which 12 have been associated with disease resistance 27 (Supplementary 4). Selection criteria included the distribution across the ten chromosomes, the degree of polymorphism, their inclusion in previous cacao research and the availability of cacao genotyping data in ICGD.…”

“…Alternative molecular markers such as SNPs have been proposed. They constitute the largest class of polymorphisms observed in plant genomes and can be found in coding regions, making associations with traits feasible 23,24 . Approximately 1,560 SNP candidates from a wide range of cacao organs have already been published in Tropgenedb 25 .…”

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

“…resumen, dichos resultados se traducen en la obtención de balances de rendimiento de producción, simplicidad, ahorro de costos y también; transferencia de datos moleculares de vital uso tanto para los productores como genetistas poblacionales.De alguna manera, los análisis de parentesco molecular y filogenia evolutiva de plantas han permitido a los productores de germoplasma y fitomejoradores, la generación de cultivos perennes con patrones de polinización y flujo de genes altamente rentables(Ashley 2010, Lacombe et al 2013, Takrama et al 2014, lo que lleva a comprender mejor los procesos de domesticación y selección de individuos resilientes(Motamayor et al 2002). En el caso del cacao, ciertas variedades de Trinidad y Tobago (que forman parte del germoplasma mejorado de cacao en África occidental) han mostrado altos registros de reproducción lo que se correlaciona en gran parte con la presencia de SNPs(Takrama et al 2014).Mediante análisis genéticos poblacionales que utilizan marcadores microsatelitales se ha descubierto un gran número de grupos genéticos, con una clara diferenciación entre variedades que se encuentran en la cuenca del Amazonas y variedades criollas situadas en América Central. Aunque no sabemos con total certeza cuál es el origen genético de las variedades genotipificadas en este estudio, mediante análisis de unión de secuencias nucleotídicas vecinas (NJ) (Figura Figura 7.A.…”