Genetic identification of Coffea canephora by DNA fingerprinting depends on robust, reproducible, polymorphic, and standardized molecular markers. Although microsatellites (SSRs) for the species are available in the literature, applying these markers to obtain multilocus genotypes requires the selection and validation of specific loci to compose a marker panel. This study aimed to select and validate SSR loci with higher polymorphism, efficiency, and reproducibility to compose a panel for the genetic identification of C. canephora. From 76 SSRs selected from the literature and initially tested on 12 divergent genotypes regarding amplification quality and polymorphism, 36 SSRs were selected and fluorescently labeled for polymorphism analysis in another set of 52 genotypes, comprising three genetic groups (20 Robusta, 12 Hybrids, and 20 Conilon). The labeled SSRs were organized into seven multiplex sets for genotyping in post-PCR multiplex via capillary electrophoresis. Based on the quality analysis of electropherograms at this stage, the SSRs were classified into three groups: optimum, intermediate, and unsatisfactory. Additionally, they were analyzed for the number of alleles, observed and expected heterozygosity, and polymorphic information content (PIC). Individuals were grouped using UPGMA. Ten SSRs showed optimum performance with consistent amplification and genotyping, defined peaks, high fluorescent signal, clear distinction between alleles, and low stutter. Seven SSRs showed intermediate performance, and the remainder were discarded due to unsatisfactory performance. Most SSRs with optimum performance featured dinucleotide motifs. Considering the set of 17 SSRs with optimum and intermediate performance, high polymorphism was observed, with an average of nine alleles per locus, 14 of these presented PIC values higher than 0.5, highlighting the high informativeness of the initial panel. These 17 SSRs were validated in the population of 52 genotypes, confirming their robustness and allowing the discrimination of all Robusta genotypes from the others and among all individual genotypes. An initial panel of characterized, validated, and standardized SSRs was obtained, with high potential for application in genetic identification and support for seedling certification of C. canephora. As an important crop in Brazil, C. canephora still lacks specific morphological descriptors recognized by MAPA (Ministry of Agriculture, Livestock, and Food Supply), and cultivar registration often relies on generic names, hindering clone identification.
KEYWORDS: SSR selection. Capillary electrophoresis. Multiplex.

Authors

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