Otimização de protocolo para a amplificação via PCR de marcadores SSR em Eugenia uniflora l. (Myrtaceae)

Abstract

The species Eugenia uniflora L., popularly known as pitangueira, belongs to the Myrtaceae family and is widely distributed throughout South America, being native to the Mata Atlântica and Pampa biomes. The pitangueira has significant importance for the great appreciation of its fruits for both the man and the fauna in general. Having been employed by popular medicine for several generations, nowadays the economic sector has noticed its quality for the manufacture of food, pharmacological and cosmetic products. Due to this possibility of exploitation, consequently there will be reduction of the areas where it occurs naturally. Geneticmolecular studies are fundamental to understanding the effects of fragmentation and degradation of the environment, reflected in the population behavior of the species as a whole. For this, it is necessary to develop tools capable of elucidating the variations of individuals at the population level. For these reasons, the present work had as objective to test and to evaluate the efficiency of the amplification of the microsatellite locus specific for the species E. uniflora, previously characterized and validated in silico. For amplification of the 24 loci evaluated, we used individuals belonging to a natural population of a forest fragment of Atlantic Forest. Among the evaluated loci, 12 were efficient and could be directly indicated for future applications. For those that did not present good results, it is necessary to perform new tests and adjustments in relation to the adopted protocols. Finally, only seven were discarded for lack of amplification. This evaluation was based on amplification of loci visualized by 2% agarose gel electrophoresis. In addition, a PCR (Polymerase Chain Reaction) protocol was optimized aiming at increasing the number of amplified loci. The genomic origin of the loci was characterized by a BLAST (Basic alignment search tool) performed in the online database NCBI, demonstrating a high similarity of the sequences aligned with the species Eucalyptus grandis, also belonging to the family Myrtaceae.