Cr3a, a candidate gene conferring fruit cracking resistance, was fine‐mapped in an introgression line of Solanum lycopersicum L.

SUMMARY In the cultivation and production of tomato ( Solanum lycopersicum L.), fruit cracking is a prevalent and detrimental issue that significantly impacts the esthetic quality and commercial value of the fruit. The complexity of the trait has resulted in a slow advancement in research aimed at identifying genes that influence tomato fruit cracking and the underlying regulatory mechanisms. In this study, a sub‐introgression population for tomato crack‐resistant fruit has been constructed from the cross between S. lycopersicum 1052 and Solanum pennellii LA0716 , followed by 11 generations of selfing. Utilizing specifically designed InDel markers, the tomato crack‐resistant gene, Cr3a , was fine‐mapped, cloned, and its functionality was confirmed through transgenic and gene‐knockout approaches. The precise localization of Cr3a was delineated to a 30 kb genomic region on chromosome 3, corresponding to the gene Sopen03g034650 in S. pennellii and Solyc03g115660.3 in the Heinz1706 variety. An integrated transcriptomic and metabolomic analysis of fruits with and without the Cr3a gene was finally conducted to elucidate the intricate regulatory mechanisms associated with Cr3a . The findings revealed a molecular regulatory network for tomato fruit crack resistance, characterized by 7 key metabolites, 13 pivotal genes, and 4 critical pathways: the phenylpropanoid biosynthesis pathway, the phenylalanine, tyrosine, and tryptophan biosynthesis pathway, the linolenic acid metabolism pathway, and the cysteine and methionine metabolism pathway. In summary, this research provides novel insights into the molecular underpinnings of tomato fruit crack resistance and holds substantial promise for accelerating the molecular breeding of tomatoes with enhanced fruit crack resistance.