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1 Figure S1. Haploid plant produced by centromere-mediated genome elimination Chromosomes containing altered CENH3 in their centromeres (green dots) are eliminated after fertilization in a cross to wild type. Haploid plants produced from such a cross contain only chromosomes from their wild type parent (black dots = centromeres containing wild type CENH3). The cytoplasm of the haploid plant (shaded light blue or light yellow) is derived from its maternal parent. 1
2 I G C J E B H D A F Figure S2. Chromosomal location of genotyping primers. The physical locations of polymorphic genetic markers used to genotype haploid plants are shown. The markers are: A. F5I14 B. MSAT2.9 C. MSAT3.19 D. MSAT4.28 E. nga76 F. nga111 G. MSAT2.36 H. MSAT3.11 I. nga8 J. nga139 All haploids from the GFP-tailswap x Ler cross and diploids from the GFP-tailswap x Wa-1 cross were genotyped with the markers listed above. Haploids from the GFPtailswap x Ws-0 cross were genotyped with markers A-E. Information about markers and primer sequences can be found at the following websites: 2
3 Chromosomes containing altered CENH3 in their centromeres (green dots) are eliminated in a cross to wild type. Haploid plants produced SUPPLEMENTARY from such a cross INFORMATION contain only chromosomes from their wild type parent (black dots = centromeres containing wild type CENH3). The cytoplasm of the haploid plant (shaded light blue or light yellow) is derived from its maternal parent. after fertilization doi: /nature08842 Figure S2. Chromosomal location of genotyping primers. The physical locations of polymorphic genetic markers used to genotype haploid plants are shown. The markers are: A. F5I14 B. MSAT2.9 C. MSAT3.19 D. MSAT4.28 E. nga76 F. nga111 G. MSAT2.36 H. MSAT3.11 I. nga8 J. nga139 All haploids from the GFP-tailswap x Ler cross and diploids from the GFP-tailswap x Wa-1 cross were genotyped with the markers listed above. Haploids from the GFPtailswap x Ws-0 cross were genotyped with markers A-E. Information about markers and primer sequences can be found at the following websites: Figure S3. F1 progeny from a GFP-tailswap x wild type cross Haploid plants (H), diploid plants (D) and aneuploid plants (A) derived from a GFPtailswap x wild type Ler cross. Haploids have leaf shapes typical of Ler. Aneuploids resemble plants with >10 chromosomes described previously1. Figure S4. Haploid Arabidopsis thaliana contain the cytoplasm of their female parent The plastid genome of parental accessions and F1 progeny from various crosses was genotyped with a cleaved amplified polymorphic sequence (CAPs) marker, Atpt Plants genotyped from genetic crosses were haploids, except in the case of GFP-tailswap x Wa-1, in which we genotyped a diploid F1 plant. The female parent is listed first in each of the crosses. Figure S5. Phenotypes of haploid Arabidopsis thaliana Haploid (H) and diploid (D) A. thaliana. A. Col-0 haploids at 18 days after germination, showing the smaller rosette leaves typical of early development. B. Ler haploids also 3
4 Figure S4. Haploid Arabidopsis thaliana contain the cytoplasm of their female parent The plastid genome of parental accessions and F1 progeny from various crosses was genotyped with a cleaved amplified polymorphic sequence (CAPs) marker, Atpt Plants genotyped from genetic crosses were haploids, except in the case of GFP-tailswap x Wa-1, in which we genotyped a diploid F1 plant. The female parent is listed first in each of the crosses. 4
5 doi: /nature08842 Supplementary Figure Legends Figure S1. Haploid plant produced by centromere-mediated genome elimination Chromosomes containing altered CENH3 in their centromeres (green dots) are eliminated after fertilization in a cross to wild type. Haploid plants produced from such a cross contain only chromosomes from their wild type parent (black dots = centromeres containing wild type CENH3). The cytoplasm of the haploid plant (shaded light blue or light yellow) is derived from its maternal parent. Figure S2. Chromosomal location of genotyping primers. The physical locations of polymorphic genetic markers used to genotype haploid plants are shown. The markers are: A. F5I14 B. MSAT2.9 C. MSAT3.19 D. MSAT4.28 E. nga76 F. nga111 G. MSAT2.36 H. MSAT3.11 I. nga8 J. nga139 All haploids from the GFP-tailswap x Ler cross and diploids from the GFP-tailswap x Wa-1 cross were genotyped with the markers listed above. Haploids from the GFPtailswap x Ws-0 cross were genotyped with markers A-E. Information about markers and primer sequences can be found at the following websites: Figure S3. F1 progeny from a GFP-tailswap x wild type cross Haploid plants (H), diploid plants (D) and aneuploid plants (A) derived from a GFPtailswap x wild type Ler cross. Haploids have leaf shapes typical of Ler. Aneuploids resemble plants with >10 chromosomes described previously1. Figure S4. Haploid Arabidopsis thaliana contain the cytoplasm of their female parent The plastid genome of parental accessions and F1 progeny from various crosses was genotyped with a cleaved amplified polymorphic sequence (CAPs) marker, Atpt Plants genotyped from genetic crosses were haploids, except in the case of GFP-tailswap x Wa-1, in which we genotyped a diploid F1 plant. The female parent is listed first in each of the crosses. Figure S5. Phenotypes of haploid Arabidopsis thaliana Haploid (H) and diploid (D) A. thaliana. A. Col-0 haploids at 18 days after germination, showing the smaller rosette leaves typical of early development. B. Ler haploids also have smaller rosette leaves. C. Col-0 haploids and diploids after bolting. D. Col-0 haploids have a bushy appearance, because sterility encourages growth of secondary inflorescences. E. Ler haploids have a bushy appearance. Figure S6. Mitosis and meiosis in Arabidopsis thaliana haploids Mitotic anaphase in A. haploid, B. diploid, and C. tetraploid A. thaliana. D. Metaphase I in haploid A. thaliana. Chromosomes align properly on the metaphase I plate. E. Anaphase I in haploid A. thaliana, showing 4-1 segregation that will ultimately give an aneuploid tetrad. F. Anaphase I in haploid A. thaliana, showing segregation, which is also expected to produce inviable gametophytes. 5
6 Figure S6. Mitosis and meiosis in Arabidopsis thaliana haploids Mitotic anaphase in A. haploid, B. diploid, and C. tetraploid A. thaliana. D. Metaphase I in haploid A. thaliana. Chromosomes align properly on the metaphase I plate. E. Anaphase I in haploid A. thaliana, showing 4-1 segregation that will ultimately give an aneuploid tetrad. F. Anaphase I in haploid A. thaliana, showing segregation, which is also expected to produce inviable gametophytes. 6
7 doi: /nature08842 have smaller rosette leaves. C. Col-0 haploids and diploids after bolting. D. Col-0 haploids have a bushy appearance, because sterility encourages growth of secondary inflorescences. E. Ler haploids have a bushy appearance. Figure S6. Mitosis and meiosis in Arabidopsis thaliana haploids Mitotic anaphase in A. haploid, B. diploid, and C. tetraploid A. thaliana. D. Metaphase I in haploid A. thaliana. Chromosomes align properly on the metaphase I plate. E. Anaphase I in haploid A. thaliana, showing 4-1 segregation that will ultimately give an aneuploid tetrad. F. Anaphase I in haploid A. thaliana, showing segregation, which is also expected to produce inviable gametophytes. Figure S7. Spontaneous diploid progeny from A. thaliana haploids A. Meiotic non reduction produces spontaneous diploid seeds in A. thaliana haploids. A Ws-0 haploid is sterile, but some siliques (inset) contain one or two seeds and thus show limited elongation. The arrowed silique contained two seeds. B. and C. Spontaneous somatic chromosome doubling in haploids is indicated by completely fertile siliques (arrows) on otherwise sterile haploid plants. These siliques give rise to diploid offspring. B. Col-0 haploid. C. Ler haploid. Figure S8. Colchicine induces somatic chromosome doubling in A. thaliana haploids A. Haploid plants before colchicine treatment. B. Plant 1 is untreated, and is a sterile haploid. Plant 2 was treated before bolting. Colchicine applied at this time greatly inhibits plant growth, but after recovery all branches are fertile and produce diploid seeds. All plants (n = 12) treated with colchicine prior to bolting were successfully converted into diploids. The regeneration time was variable. Plant 3 was treated after bolting. In these cases, the primary inflorescence is killed, and secondary inflorescences begin growing out. Treatment after bolting produces a lower frequency of successful somatic chromosome doubling. Two out of ten plants showed fertile diploid secondary branches after treatment. Some regenerated branches were fertile (arrow), while others were still sterile and haploid (arrowhead). C. Plant 2 one month after colchicine treatment, showing 7
8 doi: /nature08842 have smaller rosette leaves. C. Col-0 haploids and diploids after bolting. D. Col-0 haploids have a bushy appearance, because sterility encourages growth of secondary inflorescences. E. Ler haploids have a bushy appearance. Figure S6. Mitosis and meiosis in Arabidopsis thaliana haploids Mitotic anaphase in A. haploid, B. diploid, and C. tetraploid A. thaliana. D. Metaphase I in haploid A. thaliana. Chromosomes align properly on the metaphase I plate. E. Anaphase I in haploid A. thaliana, showing 4-1 segregation that will ultimately give an aneuploid tetrad. F. Anaphase I in haploid A. thaliana, showing segregation, which is also expected to produce inviable gametophytes. Figure S7. Spontaneous diploid progeny from A. thaliana haploids A. Meiotic non reduction produces spontaneous diploid seeds in A. thaliana haploids. A Ws-0 haploid is sterile, but some siliques (inset) contain one or two seeds and thus show limited elongation. The arrowed silique contained two seeds. B. and C. Spontaneous somatic chromosome doubling in haploids is indicated by completely fertile siliques (arrows) on otherwise sterile haploid plants. These siliques give rise to diploid offspring. B. Col-0 haploid. C. Ler haploid. Figure S8. Colchicine induces somatic chromosome doubling in A. thaliana haploids A. Haploid plants before colchicine treatment. B. Plant 1 is untreated, and is a sterile haploid. Plant 2 was treated before bolting. Colchicine applied at this time greatly inhibits plant growth, but after recovery all branches are fertile and produce diploid seeds. All plants (n = 12) treated with colchicine prior to bolting were successfully converted into diploids. The regeneration time was variable. Plant 3 was treated after bolting. In these cases, the primary inflorescence is killed, and secondary inflorescences begin growing out. Treatment after bolting produces a lower frequency of successful somatic chromosome doubling. Two out of ten plants showed fertile diploid secondary branches after treatment. Some regenerated branches were fertile (arrow), while others were still sterile and haploid (arrowhead). C. Plant 2 one month after colchicine treatment, showing fertile diploid branches. D. A plant treated after bolting, showing that one secondary inflorescence is fertile and diploid (arrow), while others are sterile and haploid (arrowhead). Table S1. Number of spontaneous diploid seeds produced by A. thaliana haploids References Henry, I. M. et al. Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response. Genetics 170, (2005). Azhagiri, A. K. & Maliga, P. DNA markers define plastid haplotypes in Arabidopsis thaliana. Curr Genet 51, (2007). 8
9 Table S1. Number of spontaneous diploid seeds produced by A. thaliana haploids Plant Col-0 Ler Ws Mean Standard deviation References 1. Henry, I. M. et al. Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response. Genetics 170, (2005). 2. Azhagiri, A. K. & Maliga, P. DNA markers define plastid haplotypes in Arabidopsis thaliana. Curr Genet 51, (2007). 9
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