Figure 1.

orf137 triggers cytoplasmic male sterility in tomato. A, mTAL137 construct and binding sites of each TALEN in the orf137 sequence. LB, left border; RB, right border; 35S pro, cauliflower mosaic virus (CaMV) 35S promoter; 35S ter, CaMV terminator; HSP ter, Arabidopsis heat shock protein terminator; MLS, mitochondrial localization signal. B, PCR analysis of orf137 in mTAL137 T₀ lines. “Micro-Tom”, which does not have the orf137 gene, and water served as negative controls; cytochrome oxidase subunit 2 (cox2) served as the mitochondrial genome control; and neomycin phosphotransferase II (NPTII) served as the marker gene for the mTAL137 vector. C, Long-read sequencing coverage of orf137 and its vicinity (CMS-PMt002: 205,000–230,000 bp; accession number LC613119 in the DDBJ database) in mTAL137 T₀ lines. The location of orf137 is indicated by the red dashed lines. The read coverage of “CMS[P]”, in which the mTAL137 construct was not introduced, was obtained from our previous report (Kuwabara et al., 2021). Cov., read coverage. D, Fruit and seed formation in mTAL137 T₀ lines. Red arrows indicate the fruits on the plants. E, Phenotypes of pollen after 4 h of incubation in germination media for “Micro-Tom”, Dwarf “CMS[P]”, and mTAL137 T₁ lines. Black arrows indicate the positions of apertures. PG, pollen grain; PT, pollen tube; BA, burst aperture. F, Phenotypes of pollen stained with aniline blue 24 h after pollination on stigma for “Micro-Tom”, Dwarf “CMS[P]”, and mTAL137 T₁ lines. White arrows indicate the positions of apertures. PG, pollen grain; PT, pollen tube; BA, burst aperture. G, Germinated pollen ratio 4 h after incubation in germination media for “Micro-Tom”, Dwarf “CMS[P]”, and mTAL137 T₁ lines. The boxplots represent interquartile ranges, the black center lines indicate the medians, and the whiskers represent 1.5 × interquartile ranges. Each point indicates the value for each experiment. Values were calculated from three independent experiments (n = 3). H, Abnormal pollen ratio 4 h after incubation in germination media for “Micro-Tom”, Dwarf “CMS[P]”, and mTAL137 T₁ lines. The boxplots represent interquartile ranges, the black center lines indicate the medians, and the whiskers represent 1.5 × interquartile ranges. Each point indicates the value for each experiment. Values were calculated from three independent experiments (n = 3).