Fig. 2: Precise engineering of tetraploid plants with four nonrecombined haplotypes via polyploid genome design.

a, Schematic of generation of tetraploid 4-Hap plants that contain the complete genetic repertoire of their four inbred grandparents. Created with BioRender.com. b,c, Presence of haplotype-specific markers in 13 MbTMV-MT^MiMe ⨯ Maxeza^MiMe offspring, 2 Maxeza F₁ plants, 5 MbTMV-MT^MiMe ⨯ Funtelle^MiMe offspring, 2 Funtelle F₁ plants and 3 MbTMV-MT F₁ plants. Colors represent each haplotype tested and the size of the circle represents the percentage of markers found (a circle completely filling the square denotes that 100% of markers were found). d, Chromosome spreads of male meiocytes from a tetraploid MbTMV-MT^MiMe ⨯ Funtelle^MiMe offspring 4-Hap plant at the diakinesis stage (n = 36). Scale bar, 10 µm. e, The expected gene dosage (histogram) and whole-genome sequencing-based genotyping (box-and-whisker plot; solid black line is the median, boxes show quartiles and whiskers show values within 1.5⨯ the interquartile range above and below the quartiles) of MbTMV-MT-Funtelle^MiMe (n = 5) and MbTMV-MT-Maxeza^MiMe (n = 13) 4-Hap plants. The genes tested encode tobacco mosaic virus resistance (Tm-2², Solyc09g018220), M. incognita resistance (Mi, Solyc06g008720), self-pruning (SP, Solyc06g074350), dwarfism (D, Solyc02g089160) and Fusarium wilt resistance (I, Solyc11g011180). f, Genomic rearrangements on chromosome 6 at the Mi-1 resistance locus. The haplotypes are depicted as horizontal lines and are (from top to bottom) MbTMV, Micro-Tom and Funtelle-2 and Funtelle-1 haplotypes. g, Frequency of genes derived from S. peruvianum (donor of Tm-2² and Mi-1) per genome haplotype. h, Images of a tetraploid MbTMV-MT^MiMe ⨯ Funtelle^MiMe offspring 4-Hap plant. Whole-plant morphology (left), structure of a branch with ripening fruits (middle), fully ripened fruits (top right), harvested fruits (middle right) and cut fruits (bottom right) are shown. Scale bars, 2.5 cm. i, Left, single-fruit weights of MbTMV-MT F₁ (n = 40), Funtelle F₁ (n = 34) and four tetraploid MbTMV-MT-Funtelle^MiMe plants (1, n = 41; 2, n = 53; 3, n = 46; 5, n = 52). Right, single-fruit weights of MbTMV-MT F₁ (n = 40), Maxeza F₁ (n = 17) and 12 tetraploid MbTMV-MT-Maxeza^MiMe plants (1, n = 27; 2, n = 31; 3, n = 36; 4, n = 18; 5, n = 35; 6, n = 28; 8, n = 11; 9, n = 23; 10, n = 15; 11, n = 33; 12, n = 30; 13, n = 25). The same MbTMV-MT F₁ data are used twice in this panel for comparison. We used ordinary one-way ANOVA followed by Šídák’s multiple-comparison test: P < 0.05, ‘NS’ (not significant); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See the ‘Statistics and reproducibility’ section (Methods) for more information.