Espírito Santo, the largest producer of Coffea canephora in Brazil, faces challenges in the correct identification of clones, resulting from weaknesses in the control and inspection of the seedling trade and the increasing introduction of new genetic materials, which increases labeling errors and compromises the genetic fidelity of clonal materials. Microsatellites (SSRs), frequently used as forensic tools, can support the reliable and early identification of seedlings. This study aimed to determine the multilocus genotypes of commercial and non-commercial C. canephora clones, aiming at the development and characterization of a reference profile database for application in the certification and regulation of clones and seedlings. To this end, a panel of 12 SSRs previously selected for high performance, reproducibility, and discriminatory power was applied in the genotyping of 145 reference genotypes, including 110 commercial and 35 non-commercial clones. PCR products were combined into multiplexes for genotyping by capillary electrophoresis. The obtained multilocus genotypes were used to estimate allele frequencies, number of alleles, observed and expected heterozygosity, genotypic probability, probability of identity (PI), and polymorphic information content (PIC), in addition to performing multilocus correspondence analysis among genotypes. A total of 133 alleles were detected, with an average of 11.08 alleles per locus. Most alleles showed low frequency, concentrated in the rare (0.01-0.05) and moderate (0.05-0.50) classes. The average PIC was 0.66. The genotypic probability showed a progressive reduction with the increase in the number of SSRs, reaching values lower than 10⁻⁹ for most genotypes starting from eight SSRs, while the combined PI for the 12 SSRs was 2.3 x 10⁻¹², indicating high discriminatory power. The evaluation allowed for the identification of 135 unique multilocus genotypes and six clonal groups, enabling the detection of duplicates and genetic discrepancies between theoretically identical clones from different origins. Two multiplex panels were proposed, reducing capillary electrophoresis injections by approximately 82% and optimizing the genotyping process. The evaluated SSRs were efficient, reproducible, and informative, allowing for the construction of a reference DNA profile database and represent a robust tool for genetic identification, seedling certification, and support for the regulation of the C. canephora seedling trade in Brazil.

Authors

• Viviane Alexia Correia Silva (Federal University of Espírito Santo (UFES), Brazil)
• Aline dos Santos Bergamin (Federal University of Espírito Santo (UFES), Brazil)
• Jônatas Gomes Santos (Federal University of Espírito Santo (UFES), Brazil)
• Aléxia Gonçalves Pereira (Federal University of Espírito Santo (UFES), Brazil)
• Adésio Ferreira (Federal University of Espírito Santo (UFES), Brazil)
• Marcia Flores da Silva Ferreira (Federal University of Espírito Santo (UFES), Brazil)