The present study has successfully obtained genetic information that allow the identification from direct descendants of Christopher Columbus, dating from 1526 to 1734. This represents the first achievement of this unique objective. The results obtained from the analysed samples, characterized by their significant antiquity, yield exceptional insights into forensic genetics and its newest protocols and methodologies applied to the study of ancient skeletal remains. This work presents the preliminary results of the whole family study using massive parallel sequencing, focusing on the autosomal Single Nucleotide Polymorphism (SNP) and Short Tandem Repeats (STR) markers, including their flanking region sequences, as well as ancestry informative markers (AIMS) of the great-great-grandson of the discoverer. The results of this project pave the way towards the application of new sequencing technologies for the study of genetic evolution, biogeographical origins and forensic identification of historical characters.

The generation of DNA profiles from skeletal remains is a core challenge in forensic genetics, particularly for the identification of missing persons and the resolution of cold cases. Factors such as diagenesis, DNA fragmentation, and the presence of PCR inhibitors severely compromise traditional methodologies, limiting the potential for successful identification. Similarly, in many archaeological studies, the presence of multiple individuals within a single burial site presents a significant challenge for accurate identification. This study shows how the most innovative forensic genetic techniques can be integrated into archaeological research, enabling the identification of individuals through the establishment of familial relationships between remains recovered from an archaeological site. A large-scale SNP assay, Forenseq® Kintelligence HT, has been applied to a familial group dating back to the 16th-17th century, allowing robust kinship calculations and lineage studies, that, in a first stage, lead to the identification of four individuals spanning four generations within the same family.

The geographical and familial origins of Christopher Columbus have remained a subject of intense historiographical debate for over five centuries. Despite numerous hypotheses, empirical genetic evidence capable of resolving his ancestral history or place of birth has been absent from the literature until now. This study presents the third stage of the first forensic genetic analysis performed on skeletal remains belonging to several direct descendants of Columbus, spanning the 16 ^th to 18 ^th centuries. By applying Massively Parallel Sequencing (MPS) to analyse autosomal, X- and Y- chromosome DNA markers, and integrating the results with multidisciplinary evidence from historical, genealogical, archaeological, and anthropological research implicated in this project, the identification of several individuals founded in the Crypt of Santa María de Gracia located in Gelves (Sevilla, Spain) has been achieved. The analysis of their biological relatedness enabled the reconstruction of kinship networks among the individuals interred in the crypt, which, when interpreted in the context of documented genealogical lineages, provides indirect but consistent evidence pointing toward the debated origin of the discoverer.