Background

The extraction of high-quality DNA is a fundamental step in molecular biology and genomics. DNA isolation often requires high levels of concentration, purity, and integrity, particularly for library preparation and sequencing applications [1]. Despite significant advancements in DNA extraction protocols, obtaining high‐molecular‐weight DNA from challenging plant materials, such as older leaves, remains problematic [2], especially when dealing with underexplored species like T. grandiflorum and T. bicolor. Therefore, there is a need to evaluate protocols capable of extracting high-quality DNA from Theobroma species, which have promising applications. Furthermore, obtaining young tissue for DNA extraction is often impractical, particularly in native Theobroma species, which are frequently decades old and grow to considerable heights [3]. Traditional methodologies, such as the cetyltrimethylammonium bromide (CTAB)-based DNA extraction protocol, are widely used due to their adaptability and efficiency in isolating plant DNA. However, conventional CTAB protocols often yield suboptimal results when applied to samples with high levels of contaminants or structural complexity [4]. These limitations underscore the need for protocol modifications to improve DNA yield and purity, especially in underexplored species such as T. bicolor and T. grandiflorum, as well as in economically significant species like T. cacao. The protocol described here addresses these challenges by increasing the CTAB concentration in the lysis buffer and extending the incubation period. These adjustments enhance cell wall disruption and facilitate the removal of secondary metabolites, resulting in higher DNA yield and quality [5]. Additionally, the optimized protocol significantly enhances throughput, enabling the processing of a greater number of samples compared to conventional techniques. This makes it particularly suitable for large-scale genomic studies, such as comparative genomics and population genetics in cacao-related species. Beyond its use in Theobroma species, this protocol may also be adapted for other plant species with similar extraction challenges. By providing a more efficient and reliable method for isolating high-quality DNA, this protocol facilitates the exploration of genetic diversity and supports a range of molecular and genomic investigations.