TECHNICAL MANUAL FOR FOLIAR DNA EXTRACTION OF CARYOCAR BRASILIENSE (CARYOCARACEAE). STANDARDIZED PROTOCOL FOR OBTAINING HIGH-QUALITY GENOMIC DNA IN CERRADO SPECIES

Abstract

The pequi tree (Caryocar brasiliense Cambess.) is a native species of the Brazilian Cerrado with significant ecological, economic, and sociocultural importance. Despite its relevance, molecular studies involving this species often face challenges related to obtaining high-quality DNA, mainly due to the high concentration of phenolic compounds and other secondary metabolites present in plant tissues. This technical manual presents a standardized and optimized protocol for genomic DNA extraction from C. brasiliense leaves, based on the classical method proposed by Doyle and Doyle (1987) and adapted to the specific characteristics of the species. The procedure includes steps for appropriate collection and preservation of plant material, tissue maceration using liquid nitrogen, CTAB-based extraction, purification with chloroform:isoamyl alcohol, and alcoholic DNA precipitation. Guidelines for DNA quantification and quality assessment using spectrophotometry and agarose gel electrophoresis are also provided. Additionally, the use of bovine serum albumin (BSA) in PCR reactions is discussed as a strategy to reduce the inhibitory effects of secondary metabolites and improve amplification efficiency. The proposed protocol demonstrates good reproducibility and yield, producing high-integrity and high-purity DNA suitable for several molecular analyses, including ISSR and SSR markers. Therefore, this manual represents an important methodological resource for studies in conservation genetics, population structure, biotechnology, and sustainable management of C. brasiliense.