Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation1,2,3,4. In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events5,6. It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication6,7. Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes8. Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting—a mechanism of plant–plant interaction that is widespread in nature—entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.
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We thank the MPI-MP Green Team for help with plant transformation, K. Köhl for providing the hpt vector and S. Ruf (MPI-MP) for discussions. We are grateful to J. Fuchs (IPK Gatersleben) for advice on flow cytometry measurements. This research was financed by the Max Planck Society.
The authors declare no competing financial interests.
Extended data figures and tables
a, Chromosome preparations of mitotic cells. Along with preparations from each of the two graft partners (Nt-kan:yfp and Nt-hyg), four examples of mitotic cells from four individual progeny plants of the self-pollinated line NGT-2 are shown. Chromosomes are visualized by hot tissue hydrolysis in HCl and staining with toluidine blue. Chromosome numbers are given in parentheses. Chr, chromosomes. b, Chromosome counts for individual seedlings from the two graft partners and the selfed line NGT-2. Mitotic cells from the root tips were analysed. The total number of metaphases investigated was 32 for Nt-kan:yfp (blue), 35 for Nt-hyg (purple) and 86 for NGT-2 (pink). c, Absolute genome size determination by flow cytometry. Leaf samples were mixed with tomato leaves (Solanum lycopersicum, Sl) that served as internal standard, nuclei were isolated and the relative fluorescence intensity of propidium iodide (PI) was measured. Each peak corresponds to a population of nuclei. For each sample, the ratio between the peak of the analysed tobacco line and the peak of the internal standard (4C tomato nuclei) was calculated to determine the absolute genome size. Sample 1: Nt-hyg; sample 2: Nt-kan:yfp; sample 3: seedling from a cross between NGT-1 and wild type; sample 4: seedling from a self-pollinated NGT-2 plant.
a–c,Young flower buds were fixed and stained, either with aceto-orceine (a, b) or with DAPI (c). The anthers were collected and the pollen mother cells (PMC) were examined under the microscope. Two plants from the F1 generation of self-pollinated NGT plants were used. a, Four representative PMCs of a wild-type tobacco plant. Scale bar, 50 µm. b, Six PMC from NGT plants. The upper three cells are from line NGT-2, the lower three from line NGT-3. Scale bar, 20 µm. c, Two PMCs from line NGT-3 stained with DAPI. Scale bar, 5 µm. Mis-segregating chromosomes are indicated by arrowheads in b and c.
a, b, Wild-type tobacco (Nt-wt), the two transgenic graft partners (Nt-hyg and Nt-kan:yfp) and the second generation of three NGT lines (NGT-1, NGT-2 and NGT-3) were grown under greenhouse conditions. A total of 21 NGT progeny plants were investigated, 14 of them resulted from self-pollinated lines (NGT-2 and NGT-3) and the remaining 7 from the cross of the NGT-1 line with a wild-type plant (which was male sterile and could not be selfed). Pictures were taken 30 (a) and 45 (b) days after sowing.
a, b, Wild-type tobacco (Nt-wt), the two transgenic graft partners (Nt-hyg and Ng-kan) and the F1 generation of an N. tabauca line were raised from seeds and grown under greenhouse conditions. Pictures were taken 28 (a) and 47 (b) days after sowing.
Extended Data Figure 5 Detection of allopolyploid and aberrant karyotypes in Nicotiana tabauca F1 progeny plants.
a, Two examples of DAPI-stained metaphases with fewer than 72 chromosomes. b, As an alternative to the DAPI staining shown in Fig. 4c and in panel a, a method based on hot tissue hydrolysis in HCl and staining with toluidine blue was used (see Methods). Shown is a Nicotiana tabauca F1 plant with the full allopolyploid chromosome set of 72 chromosomes.
Extended Data Figure 6 Molecular analysis of four polymorphic regions in the plastid genome of Nicotiana tabauca lines.
a, Physical map of the Nicotiana tabacum plastid genome showing the four plastid polymorphic regions analysed (pt1, pt2, pt3 and pt4). pt1: polymorphism amplified with oligonucleotides Ppt1F and Ppt1R (Extended Data Table 2) resulting in a 211 bp fragment in N. tabacum cv. SNN and a 203 bp fragment in N. glauca; pt2: polymorphism amplified with oligonucleotides Ppt2F and Ppt2R resulting in a 221 bp fragment in N. tabacum cv. SNN and a 229 bp fragment in N. glauca; pt3: polymorphic region of ∼3 kb (containing altogether 25 polymorphisms) amplified using the primer pairs Ppt3-1F + Ppt3-1R, Ppt3-2F + Ppt3-2R and Ppt3-3F + Ppt3-3R; pt4: polymorphic region of ∼3 kb (containing altogether 32 polymorphisms) amplified with the primer pairs Ppt4-1F + Ppt4-1R, Ppt4-2F + Ppt4-2R and Ppt4-3F + Ppt4-3R. The polymorphisms were selected based on published sequence information of the plastid genomes of N. tabacum and N. glauca16,36. b, Overview of the plant lines and the polymorphic regions analysed. Nicotiana tabacum sequence is represented with Nt on yellow background, Nicotiana glauca sequence is represented with Ng on pink background. c, A 4% agarose gel showing the size difference of the PCR fragments for the pt1 and pt2 polymorphisms between N. tabacum, N. glauca and five N. tabauca lines.
Extended Data Figure 7 Molecular analysis of five mitochondrial genome polymorphisms in Nicotiana tabauca lines by sequencing of amplified PCR products.
a, Physical map of the Nicotiana tabacum mitochondrial genome showing the five mitochondrial polymorphic regions analysed (mt1, mt2, mt3, mt4 and mt5). mt1: polymorphic region of 693 bp (containing 5 polymorphisms) amplified with oligonucleotides Pmt1F and Pmt1R (Extended Data Table 2); mt2: polymorphism (SNP) amplified with oligonucleotides Pmt2F and Pmt2R; mt3: polymorphism (SNP) amplified with oligonucleotides Pmt3F and Pmt3R; mt4: polymorphism (SNP) amplified with oligonucleotides Pmt4F and Pmt4R; mt5: polymorphic region amplified with oligonucleotides Pmt5F and Pmt5R and resulting in a 298 bp fragment in N. tabacum and a 288 bp fragment in N. glauca. The polymorphisms were selected based on published sequence information of the mitochondrial genomes of N. tabacum and N. glauca16,37. b, Overview of the plant lines and the polymorphic regions analysed. Nicotiana tabacum sequence is represented with Nt on yellow background, Nicotiana glauca sequence is represented with Ng on dark pink background. Heteroplasmy (that is, detectability of both the N. tabacum and the N. glauca sequence in an N. tabauca plant) is indicated by Nt/Ng on light pink background. A and B denote two different plants from the same N. tabauca line.
a, Example of sequences amplified from the mt1 polymorphic region in Nt-hyg, Ng-kan and in an N. tabauca (Ntca) line. The mt1 polymorphic region was amplified with the oligonucleotide combination Pmt1F and Pmt1R (Extended Data Table 2) resulting in a 693 bp fragment (corresponding to nucleotide positions 36,746 to 37,438 in the Nicotiana tabacum mitochondrial genome, accession number: NC_006581.1). The fragment contains five single nucleotide polymorphisms (SNPs) that are denoted with an arrow in the sequence chromatograms and the nucleotide position in the N. tabacum mitochondrial genome. A mixed nucleotide position indicating heteroplasmy is denoted by two letters above each other. b, A 4% agarose gel analysing the mt4 polymorphism in N. tabacum, N. glauca and nine N. tabauca plants (representing five different lines). A 310 bp fragment was amplified by PCR and digested with the two restriction enzymes NcoI and BsaBI. Digestion of the PCR product amplified from the N. tabacum mitochondrial genome yields three restriction fragments (25 bp, 185 bp and 100 bp), whereas digestion of the PCR product amplified from the N. glauca mitochondrial genome results in two fragments (25 bp and 285 bp). This difference between the two species is due to a T to G substitution in N. glauca (relative to N. tabacum; position in the N. tabacum mitochondrial genome: 305,402), resulting in the loss of a BsaBI site. The 25 bp restriction fragment is not detectable in the gel because of its small size. Note that the low level of heteroplasmy detected by restriction-fragment length polymorphism (RFLP) analysis was not reliably detectable by DNA sequencing (compare with Extended Data Fig. 7b). A and B denote two different plants from the same N. tabauca line.
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Fuentes, I., Stegemann, S., Golczyk, H. et al. Horizontal genome transfer as an asexual path to the formation of new species. Nature 511, 232–235 (2014). https://doi.org/10.1038/nature13291
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