Map-based Cloning And Functional Analysis Of FIS1,a Fruit Firmness Regulator In Tomato

Fruit firmness is an important quality trait in tomato because it influences fruit compression resistance,transportation and storage.The fruit firmness is a complex trait,challenging to fully uncover its molecular genetic mechanism.Although dozens of fruit firmness QTLs have been discovered,few genes have been cloned.At present,the main method to improve fruit firmness is to delay fruit ripening and softening by using tomato ripening mutants.These mutants increased the fruit firmness but damaged tomato taste flavor and other important qualities.Therefore,the genes controlling fruit firmness need to be discovered for potential utility in breeding.Here we used a RILs population which is constructed by crossing between cultivated tomatoes Moneymaker?MM?and cherry tomatoes LA1310?CC?to identify the loci associated with fruit firmness.We modified a method to evaluate fruit firmness by fruit compression resistance?CR?.According to the CR of RILs,a key QTL,qFIRM SKIN 1?qFIS1?,was found to by GWAS.For comfirming the role of qFIS1 locus in fruit firmness,we constructed the NILs and found that the qFIS1improved fruit firmness and prolonged storage time by affecting the thickness of fruit epicuticle.However,fruit shape,weight,developmental process and fruit flavor were no significant differences between the NILs.The qFIS1 was mapped to a 17.8kb genomic region.And Solyc10g007570 was the candidate gene.Sequence analysis revealed that there was a 1-bp insertion in the exon of Solyc10g007570 in MM.The function of Solyc10g007570 was confirmed by CRISPR/Cas9 and complementary transgenic technology and disruption of Solyc10g007570 function could enhance tomato fruit firmness.FIS1 encodes a GA2-oxidase,a gibberellin deactivating enzyme that catalyzes active GAs into inactive products and its mutation leads to a causal sequence of changes:increased bioactive gibberellin content,enhanced cutin and wax biosynthesis,and increased fruit firmness and shelf life.The same result was got from the studies after GA₃ treatment and complementary transgenic.Importantly,FIS1 has no unfavorable effect on fruit weight or quality,making it an ideal target during tomato domestication.FIS1 gene was edited using CRISPR/Cas9 technology in wild tomato?Solanum pimpinellifolium,PP?and cherry tomato?TS205?.The genetic materials with significantly improved fruit firmness were obtained.In summary,FIS1 mutation leads to a causal sequence of changes:increased bioactive gibberellin content,enhanced cuticle and wax biosynthesis,and increased fruit firmness and shelf life.Our study demonstrates that FIS1-mediated gibberellin catabolism is critical for fruit firmness,providing a new strategy to improve fruit firmness in tomato breeding.