The wild Solanum pimpinellifolium (SP) and the weedy S. lycopersicum var. cerasiforme (SLC) are largely unexploited genetic reservoirs easily accessible to breeders, as they are fully cross-compatible with cultivated tomato (S. lycopersicum var. lycopersicum). We performed a comprehensive morphological and genomic characterization of four wild SP and four weedy SLC accessions, selected to maximize the range of variation of both taxa. These eight accessions are the founders of the first tomato interspecific multi-parent advanced generation inter-cross (MAGIC) population. The morphoagronomic characterization was carried out with 39 descriptors to assess plant, inflorescence, fruit and agronomic traits, revealing the broad range of diversity captured. Part of the morphological variation observed in SP was likely associated to the adaptation of the accessions to different environments, while in the case of SLC to both human activity and adaptation to the environment. Whole-genome resequencing of the eight accessions revealed over 12 million variants, ranging from 1.2 to 1.9 million variants in SLC and from 3.1 to 4.8 million in SP, being 46.3% of them (4,897,803) private variants. The genetic principal component analysis also confirmed the high diversity of SP and the complex evolutionary history of SLC. This was also reflected in the analysis of the potential footprint of common ancestors or old introgressions identified within and between the two taxa. The functional characterization of the variants revealed a significative enrichment of GO terms related to changes in cell walls that would have been negatively selected during domestication and breeding. The comprehensive morphoagronomic and genetic characterization of these accessions will be of great relevance for the genetic analysis of the first interspecific MAGIC population of tomato and provides valuable knowledge and tools to the tomato community for genetic and genomic studies and for breeding purposes.

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