A phylogenetic approach to prioritizing crop wild relatives in Brassiceae (Brassicaceae) for breeding applications

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BACKGROUND AND AIMS: Crop wild relatives (CWRs) are key resources for enhancing agricultural resilience, providing genetic traits that can improve pest resistance, abiotic stress tolerance and nutritional composition in domesticated crops. Within the mustard family (Brassicaceae) this is especially significant in the Brassiceae tribe, which includes economically important genera for agriculture such as Brassica and Sinapis. However, while breeding programmes have historically focused on major crops within this tribe, the potential of their wild relatives, particularly for underutilized and minor crops, remains insufficiently explored. METHODS: We sequenced 175 accessions from six genera, Brassica, Crambe, Diplotaxis, Eruca, Raphanus and Sinapis, using herbarium, seed and living collections. We combined those sequences with 30 nuclear internal transcribed spacer (ITS) sequences from GenBank. Libraries were prepared with Angiosperms353 and Brassicaceae bait kits to build a phylogenetic tree to calculate the phylogenetic distances between tips. KEY RESULTS: We studied the ITS region to build a robust phylogeny for 189 accessions spanning 90 taxa, incorporating newly generated sequences, which included a total of 30 taxa not sequenced before, alongside publicly available sequence data. Phylogenetic distances derived from an ultrametric tree were used to infer cross-compatibility and identify 23 candidate CWRs across the six genera. Our results confirm known relationships based on gene pool classifications of CWRs but also highlight previously overlooked or misclassified taxa that may hold value for crop improvement. CONCLUSIONS: This work demonstrates the efficacy of ITS markers for CWR identification and supports their use as a scalable tool for integrating biological collections into modern breeding and conservation strategies. It provides a comprehensive framework for targeting wild genetic diversity in Brassiceae crops and prioritizing species for future pre-breeding research.

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