Desenvolvimento e validação de painéis multiplex de microssatélites para análise de pureza varietal e diversidade genética em Solanum tuberosum L.

Abstract

Potato (Solanum tuberosum L.) has high economic and nutritional importance, playing a significant role in global food security. In breeding programs, varietal mixtures may occur during mechanized harvesting and seed tuber transportation. In addition, the simultaneous handling of numerous clones increases the risk of mixtures and identification errors, especially in advanced stages, when clones exhibit high phenotypic similarity. Under these conditions, visual distinction becomes limited, compromising the reliability of evaluations. Molecular markers, especially microsatellites (SSRs), are efficient tools for identifying varietal mixtures and characterizing genetic diversity; however, their individual application may be operationally costly. Therefore, the objective of this study was to develop and validate a multiplex panel of microsatellite markers for applications in varietal purity and genetic diversity analyses of potato clones. Genomic DNA was extracted from young leaf tissue. Initially, 24 SSR markers were evaluated for polymorphism and amplification quality. After optimization of PCR conditions, 13 markers were organized into six multiplex reactions, and the amplified products were separated by polyacrylamide gel electrophoresis. The multiplex panel demonstrated high discriminatory power, detecting mixtures in three evaluated clones (CCF04-16, CCF21-03, and CCF01-20). Varietal purity analysis allowed the selection of one molecularly representative plant from each clone for subsequent genetic diversity analyses. A total of 48 polymorphic alleles were identified, with an average of four alleles per locus. The polymorphic information content (PIC) showed a mean value of 0.32, while Nei’s genetic diversity (H*) and Shannon’s index (I*) averaged 0.31 and 0.47, respectively. Genetic dissimilarity estimated using the complement of the Jaccard coefficient ranged from 0.25 to 0.69, and cluster analysis using the UPGMA method resulted in the formation of three distinct groups, with a cophenetic correlation coefficient of 71%. The clones showed sufficient genetic diversity to support parent selection and strategic crosses. The organization of markers into multiplex reactions optimized the genotyping process, reducing the number of reactions by 53.8% and increasing the feasibility of large-scale application. The results demonstrated the potential of the multiplex panel as a practical tool for breeding programs and germplasm banks, contributing to varietal identification and studies of genetic diversity and identity in potato.