double mutants are necessary and sufficient for the compact plant architecture of butterhead lettuce.

Lettuce, an important leafy vegetable crop worldwide, has rich variations in plant architecture. Butterhead lettuce, a popular horticultural type, has a unique plant architecture with loose leafy heads. The genetic and molecular mechanisms for such a compact plant architecture remain unclear. In this study we constructed a segregating population through crossing a butterhead cultivar and a stem lettuce cultivar. Genetic analysis identified the gene, which encodes a kinase, as the candidate gene controlling butterhead plant architecture. The gene in the butterhead parent had a nonsense mutation, leading to a partial predicted protein. CRISPR/Cas9 and complementation tests verified its functions in plant architecture. We showed that the loss of function of is necessary but not sufficient for the butterhead plant architecture. To identify additional genes required for butterhead lettuce, we crossed a butterhead cultivar and a crisphead cultivar, both with the mutated gene. Genetic mapping identified a new gene encoding an ATPase contributing to butterhead plant architecture. Knockout and complementation tests showed that loss of function of is also required for the development of butterhead plant architecture. The double mutation could reduce leaf size and leaf angle, leading to butterhead plant architecture. Expression and cytology analysis indicated that the loss of function of and contributed to butterhead plant architecture by regulating cell wall development, a regulatory mechanism different from that for crisphead. This study provides new gene resources and theory for the breeding of the crop ideotype.