Editing multiple genes in Physalis pubescens provides valuable lessons and implications for creating new germplasm and varieties of Physalis crops.

Physalis pubescens, as a staple horticultural crop of Physalis in China, has shortcomings of single variety, low yield, and unique fruit shape and color. In attempt to overcome these disadvantages, we employed gene editing technology in the main cultivar 'Jinhuang 1' ('JH1') of P. pubescens to reprogram the related traits. Six orthologous genes related to tomato domestication and improvement, which include fruit shape gene OVATE, inflorescence branching gene COMPOUND INFLORESCENCE (S), fruit color gene LYCOPENE BETA CYCLASE (LCY1), and fruit size regulating genes CLAVATA3 (CLV3), FRUIT WEIGHT2.2 (FW2.2), and EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), were identified and edited in 'JH1'. Phenotypic variations observed in ovate, s, and clv3 of P. pubescens were mainly consistent with those found in tomato mutants, whereas phenotypic variations exhibited by lcy1, eno, and fw2.2 mutants in 'JH1' were significantly different from these observed in the corresponding tomato mutants. Moreover, most of these gene edited mutants showed inferiority to 'JH1'. Our results mainly revealed that functions of the orthologous genes among close relatives may significantly diverge and that pure gene-editing for loss-of-function mutation is insufficient to yield elite varieties for P. pubescens, thus offering valuable lessons and insights for genetic and germplasm improvement of Physalis crops.