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AbstractFreezing stress is a major limiting factor in crop production. To increase frost-hardiness of crops via breeding, deciphering the genes conferring freezing-tolerance is vital. Potato cultivars (Solanum tuberosum) are generally freezing-sensitive, but some potato wild species are freezing-tolerant, including S. commersonii, S. malmeanum, and S. acaule. However, the underlying molecular mechanisms conferring the freezing-tolerance to the wild species remain to be deciphered. In the present study, five representative genotypes of the above-mentioned species with distinct freezing-tolerance were investigated. Comparative transcriptomics analysis showed that SaCBL1?like (calcineurin B-like protein) was up-regulated substantially in all of the freezing-tolerant genotypes. Transgenic over-expression and known-down lines of SaCBL1?like were examined. SaCBL1?like was shown to confer freezing-tolerance without significantly impacting main agricultural traits. A functional mechanism analysis showed that SaCBL1?like increases the expression of the CBF-regulon (C-repeat binding factor) as well as causes a prolonged higher expression of CBF1 after exposure to cold conditions. Furthermore, SaCBL1?like was found to only interact with SaCIPK3?1 (CBL-interacting protein kinase) among all apparent cold-responsive SaCIPKs. Our study identifies SaCBL1?like to play a vital role in conferring freezing tolerance in potato, which may provide a basis for a targeted potato breeding for frost-hardiness.This article is protected by copyright. All rights reserved.