The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type.
References
[1]
Steiner FA, Henikoff S. Diversity in the organization of centromeric chromatin. Current Opinion in Genetics & Development. 2015;31(0):28–35. doi: http://dx.doi.org/10.1016/j.gde.2015.03.？010.
[2]
Fukagawa T, Earnshaw William C. The Centromere: Chromatin Foundation for the Kinetochore Machinery. Developmental Cell. 30(5):496–508. doi: 10.1016/j.devcel.2014.08.016. pmid:25203206
[3]
Cheeseman IM. The Kinetochore. Cold Spring Harbor Perspectives in Biology. 2014;6(7). doi: 10.1101/cshperspect.a015826.
[4]
Duro E, Marston AL. From equator to pole: splitting chromosomes in mitosis and meiosis. Genes & Development. 2015;29(2):109–22. doi: 10.1101/gad.255554.114.
[5]
Allshire RC, Karpen GH. Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nat Rev Genet. 2008;9(12):923–37. doi: 10.1038/nrg2466. pmid:19002142
[6]
Sekulic N, Black BE. Molecular underpinnings of centromere identity and maintenance. Trends in Biochemical Sciences. 37(6):220–9. doi: 10.1016/j.tibs.2012.01.003. pmid:22410197
[7]
Palmer DK, O'Day K, Trong HL, Charbonneau H, Margolis RL. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proceedings of the National Academy of Sciences. 1991;88(9):3734–8. doi: 10.1073/pnas.88.9.3734.
[8]
Earnshaw W, Migeon B. Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome. Chromosoma. 1985;92(4):290–6. doi: 10.1007/BF00329812. pmid:2994966
[9]
Talbert PB, Masuelli R, Tyagi AP, Comai L, Henikoff S. Centromeric Localization and Adaptive Evolution of an Arabidopsis Histone H3 Variant. The Plant Cell Online. 2002;14(5):1053–66. doi: 10.1105/tpc.010425.
[10]
Dalal Y, Furuyama T, Vermaak D, Henikoff S. Structure, dynamics, and evolution of centromeric nucleosomes. Proceedings of the National Academy of Sciences. 2007;104(41):15974–81. doi: 10.1073/pnas.0707648104.
[11]
Maheshwari S, Tan EH, West A, Franklin FCH, Comai L, Chan SWL. Naturally Occurring Differences in CENH3 Affect Chromosome Segregation in Zygotic Mitosis of Hybrids. Plos Genet. 2015;11(1):e1004970. doi: 10.1371/journal.pgen.1004970. pmid:25622028
[12]
Malik HS, Henikoff S. Phylogenomics of the nucleosome. Nat Struct Mol Biol. 2003;10(11):882–91. doi: 10.1038/nsb996
[13]
Stoler S, Keith KC, Curnick KE, Fitzgerald-Hayes M. A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes & Development. 1995;9(5):573–86. doi: 10.1101/gad.9.5.573.
[14]
Buchwitz BJ, Ahmad K, Moore LL, Roth MB, Henikoff S. Cell division: A histone-H3-like protein in C. elegans. Nature. 1999;401(6753):547–8. pmid:10524621 doi: 10.1038/44062
[15]
Howman EV, Fowler KJ, Newson AJ, Redward S, MacDonald AC, Kalitsis P, et al. Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Proceedings of the National Academy of Sciences. 2000;97(3):1148–53. doi: 10.1073/pnas.97.3.1148.
[16]
Ravi M, Kwong PN, Menorca RMG, Valencia JT, Ramahi JS, Stewart JL, et al. The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana. Genetics. 2010;186(2):461–71. doi: 10.1534/genetics.110.120337. pmid:20628040
[17]
Dunleavy EM, Roche D, Tagami H, Lacoste N, Ray-Gallet D, Nakamura Y, et al. HJURP Is a Cell-Cycle-Dependent Maintenance and Deposition Factor of CENP-A at Centromeres. Cell. 2009;137(3):485–97. doi: http://dx.doi.org/10.1016/j.cell.2009.02？.040. doi: 10.1016/j.cell.2009.02.040. pmid:19410545
[18]
Mishra PK, Au W-C, Choy JS, Kuich PH, Baker RE, Foltz DR, et al. Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells. Plos Genet. 2011;7(9):e1002303. doi: 10.1371/journal.pgen.1002303. PMC3183075. pmid:21980305
[19]
Lermontova I, Kuhlmann M, Friedel S, Rutten T, Heckmann S, Sandmann M, et al. Arabidopsis KINETOCHORE NULL2 is an upstream component for centromeric histone H3 variant cenH3 deposition at centromeres. The Plant Cell. 2013;25(9):3389–404. doi: 10.1105/tpc.113.114736. pmid:24014547
[20]
Tan EH, Henry IM, Ravi M, Bradnam KR, Mandakova T, Marimuthu MP, et al. Catastrophic chromosomal restructuring during genome elimination in plants. eLife. 2015:e06516. doi: 10.7554/elife.06516
[21]
Ravi M, Chan SWL. Haploid plants produced by centromere-mediated genome elimination. Nature. 2010;464(7288):615–8. doi: http://www.nature.com/nature/journal/v46？4/n7288/suppinfo/nature08842_S1.html. doi: 10.1038/nature08842. pmid:20336146
[22]
Lermontova I, Koroleva O, Rutten T, Fuchs J, Schubert V, Moraes I, et al. Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation. The Plant Journal. 2011;68(1):40–50. doi: 10.1111/j.1365-313X.2011.04664.x. pmid:21635586
[23]
Forster BP, Thomas WTB. Doubled Haploids in Genetics and Plant Breeding. Plant Breeding Reviews: John Wiley & Sons, Inc.; 2010. p. 57–88.
[24]
W？dzony M, Forster BP, ？ur I, Golemiec E, Szechyńska-Hebda M, Dubas E, et al. Progress in Doubled Haploid Technology in Higher Plants. In: Touraev A, Forster B, Jain SM, editors. Advances in Haploid Production in Higher Plants: Springer Netherlands; 2009. p. 1–33.
[25]
Tester M, Langridge P. Breeding Technologies to Increase Crop Production in a Changing World. Science. 2010;327(5967):818–22. doi: 10.1126/science.1183700. pmid:20150489
[26]
Ng PC, Henikoff S. Predicting the Effects of Amino Acid Substitutions on Protein Function. Annual Review of Genomics and Human Genetics. 2006;7(1):61–80. doi: 10.1146/annurev.genom.7.080505.115630 pmid:16824020.
[27]
Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protocols. 2009;4(8):1073–81. doi: 10.1038/nprot.2009.86
[28]
Henry IM, Dilkes BP, Miller ES, Burkart-Waco D, Comai L. Phenotypic Consequences of Aneuploidy in Arabidopsis thaliana. Genetics. 2010;186(4):1231–45. doi: 10.1534/genetics.110.121079. pmid:20876566
[29]
Cifuentes M, Rivard M, Pereira L, Chelysheva L, Mercier R. Haploid Meiosis in Arabidopsis: Double-Strand Breaks Are Formed and Repaired but Without Synapsis and Crossovers. PLoS ONE. 2013;8(8):e72431. doi: 10.1371/journal.pone.0072431. pmid:23951324
[30]
Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, et al. Large-scale discovery of induced point mutations with high-throughput TILLING. Genome research. 2003;13(3):524–30. pmid:12618384
[31]
Ravi M, Marimuthu MPA, Tan EH, Maheshwari S, Henry IM, Marin-Rodriguez B, et al. A haploid genetics toolbox for Arabidopsis thaliana. Nat Commun. 2014;5. doi: 10.1038/ncomms6334.
[32]
Cao J, Schneeberger K, Ossowski S, Gunther T, Bender S, Fitz J, et al. Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat Genet. 2011;43(10):956–63. doi: http://www.nature.com/ng/journal/v43/n10？/abs/ng.911.html—supplementary-informati？on. doi: 10.1038/ng.911. pmid:21874002
[33]
Hirsch CD, Wu Y, Yan H, Jiang J. Lineage-Specific Adaptive Evolution of the Centromeric Protein CENH3 in Diploid and Allotetraploid Oryza Species. Molecular Biology and Evolution. 2009;26(12):2877–85. doi: 10.1093/molbev/msp208. pmid:19741004
[34]
Tachiwana H, Kagawa W, Shiga T, Osakabe A, Miya Y, Saito K, et al. Crystal structure of the human centromeric nucleosome containing CENP-A. Nature. 2011;476(7359):232–5. doi: http://www.nature.com/nature/journal/v47？6/n7359/abs/nature10258.html—supplementa？ry-information. doi: 10.1038/nature10258. pmid:21743476
[35]
Sekulic N, Bassett EA, Rogers DJ, Black BE. The structure of (CENP-A-H4)2 reveals physical features that mark centromeres. Nature. 2010;467(7313):347–51. doi: http://www.nature.com/nature/journal/v46？7/n7313/abs/nature09323.html—supplementa？ry-information. doi: 10.1038/nature09323. pmid:20739937
[36]
Zhang W, Colmenares Serafin U, Karpen Gary H. Assembly of Drosophila Centromeric Nucleosomes Requires CID Dimerization. Molecular Cell. 2012;45(2):263–9. doi: http://dx.doi.org/10.1016/j.molcel.2011.？12.010. doi: 10.1016/j.molcel.2011.12.010. pmid:22209075
[37]
Bassett Emily A, DeNizio J, Barnhart-Dailey Meghan C, Panchenko T, Sekulic N, Rogers Danielle J, et al. HJURP Uses Distinct CENP-A Surfaces to Recognize and to Stabilize CENP-A/Histone H4 for Centromere Assembly. Developmental Cell. 2012;22(4):749–62. doi: http://dx.doi.org/10.1016/j.devcel.2012.？02.001. doi: 10.1016/j.devcel.2012.02.001. pmid:22406139
[38]
Ingouff M, Rademacher S, Holec S, ？olji？ L, Xin N, Readshaw A, et al. Zygotic Resetting of the HISTONE 3 Variant Repertoire Participates in Epigenetic Reprogramming in Arabidopsis. Current Biology. 2010;20(23):2137–43. doi: http://dx.doi.org/10.1016/j.cub.2010.11.？012. doi: 10.1016/j.cub.2010.11.012. pmid:21093266
[39]
Sanei M, Pickering R, Kumke K, Nasuda S, Houben A. Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proceedings of the National Academy of Sciences. 2011;108(33):E498–E505. doi: 10.1073/pnas.1103190108.
[40]
Forster BP, Heberle-Bors E, Kasha KJ, Touraev A. The resurgence of haploids in higher plants. Trends in Plant Science. 2007;12(8):368–75. doi: http://dx.doi.org/10.1016/j.tplants.2007？.06.007. pmid:17629539
[41]
Chan SWL. Chromosome engineering: power tools for plant genetics. Trends in Biotechnology. 28(12):605–10. doi: 10.1016/j.tibtech.2010.09.002. pmid:20933291
[42]
Dunwell JM. Haploids in flowering plants: Origins and exploitation. Plant Biotechnol Journal. 2010; 8:377–424. doi: 10.1111/j.1467-7652.2009.00498.x
[43]
Murovec J, Bohanec B. Haploids and Doubled Haploids in Plant Breeding. In: Abdurakhmonov I, editor. Plant Breeding2012.
[44]
Feuillet C, Leach JE, Rogers J, Schnable PS, Eversole K. Crop genome sequencing: lessons and rationales. Trends in Plant Science. 2011;16(2):77–88. doi: http://dx.doi.org/10.1016/j.tplants.2010？.10.005. doi: 10.1016/j.tplants.2010.10.005. pmid:21081278
[45]
Seymour DK, Filiault DL, Henry IM, Monson-Miller J, Ravi M, Pang A, et al. Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping. Proceedings of the National Academy of Sciences. 2012:4227–32. doi: 10.1073/pnas.1117277109.
[46]
Wijnker E, van Dun K, de Snoo CB, Lelivelt CLC, Keurentjes JJB, Naharudin NS, et al. Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nat Genet. 2012;44(4):467–70. doi: http://www.nature.com/ng/journal/v44/n4/？abs/ng.2203.html—supplementary-informati？on. doi: 10.1038/ng.2203. pmid:22406643
[47]
Marimuthu MPA, Jolivet S, Ravi M, Pereira L, Davda JN, Cromer L, et al. Synthetic Clonal Reproduction Through Seeds. Science. 2011;331(6019):876. doi: 10.1126/science.1199682. pmid:21330535
[48]
Ravi M, Marimuthu MPA, Tan EH, Maheshwari S. A haploid genetics toolbox for Arabidopsis thaliana. Nature Communications. In Press.
[49]
Comai L, Henikoff S. TILLING: practical single-nucleotide mutation discovery. The Plant Journal. 2006;45(4):684–94. doi: 10.1111/j.1365-313X.2006.02670.x. pmid:16441355
[50]
Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen CB, Murata M, et al. Meiosis-Specific Loading of the Centromere-Specific Histone CENH3 in Arabidopsis thaliana. Plos Genet. 2011;7(6). doi: Artn E1002121 doi: 10.1371/Journal.Pgen.1002121 pmid:WOS:000292386300037.
[51]
Clough SJ, Bent AF. Floral dip: a simplified method forAgrobacterium‐mediated transformation ofArabidopsis thaliana. The plant journal. 1998;16(6):735–43. pmid:10069079 doi: 10.1046/j.1365-313x.1998.00343.x
[52]
Henry IM, Dilkes BP, Young K, Watson B, Wu H, Comai L. Aneuploidy and Genetic Variation in the Arabidopsis thaliana Triploid Response. Genetics. 2005;170(4):1979–88. doi: 10.1534/genetics.104.037788. pmid:15944363
[53]
Armstrong SJ, Franklin FCH, Jones GH. Nucleolus-associated telomere clustering and pairing precede meiotic chromosome synapsis in Arabidopsis thaliana. Journal of Cell Science. 2001;114(23):4207–17.
[54]
Peterson R, Slovin JP, Chen C. A simplified method for differential staining of aborted and non-aborted pollen grains. International Journal of Plant Biology. 2010;1(2):13. doi: 10.4081/pb.2010.e13
[55]
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9. doi: 10.1093/bioinformatics/bts199. pmid:22543367
