New highly polymorphic simple sequence repeat (SSR) markers for cacao genetic diversity research, and the discovery of accessions with Criollo genetic background in the Marañón seasonally dry tropical forests of Northern Peru

Peruvian cacao ( Theobroma cacao L.) is highly diverse and is recognized for its fine-flavor attributes. However, various geographical regions within its native range remain insufficiently characterized at the molecular level. In this study, the genetic diversity of 151 cacao accessions from seven populations in Amazonas and Cusco regions (Northern and Southern Peru, respectively) was evaluated with 15 simple sequence repeat (SSR) markers, six of which were in-house designed using publicly available genomic data. A total of 175 alleles were detected, ranging from 2 to 25 alleles per locus, with the newly developed nuclear SSR marker INDES_mTC98 showing the highest polymorphism. Each accession had a unique multilocus genotype, i.e., there were no clones in this study. Population structure analysis revealed four genetic clusters comprising 2, 13, 25, and 111 individuals, which accounted for 64.90% of the total intraspecific variation and supported that their genetic background derived from five ancestral populations. Notably, the Cusco population (composed mostly of Chuncho cacao samples) showed high genetic differentiation from the Amazonas populations, highlighting its distinct genetic composition. Linkage equilibrium patterns in the Luya population (composed of samples from within or in the proximities of the Marañón Seasonally Dry Tropical Forests protected area) were consistent with past random sexual recombination events, free of clonal propagation as commonly mediated by humans. Further genotyping of Luya accessions using 96 single-nucleotide polymorphism markers placed them closely related with the Mesoamerican Criollo and Piura Porcelana genetic groups. These results revealed Criollo-related genetic affinities among cacao accessions from the Marañón Seasonally Dry Tropical Forests, deriving from one single ancestral population. The observed relationships suggested historical connections between northern Peruvian cacao populations and Criollo-related germplasm, although further research is required to clarify their evolutionary history. The limited accessibility of the dry forest areas along the Marañón River highlights this region as an underexplored reservoir of cacao genetic diversity with importance for conservation and breeding programs.