Intrinsic karyotype stability and gene copy number variations may have laid the foundation for tetraploid wheat formation Huakun Zhanga,1, Yao Biana,1, Xiaowan Goua, Yuzhu Donga, Sachin Rustgib,2, Bangjiao Zhanga, Chunming Xua, Ning Lia, Bao Qic, Fangpu Hand, Diter von Wettsteinb,2, and Bao Liua,2 aKey Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun 130024, China; bDepartment of Crop and Soil Sciences, School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164; cFaculty of Agronomy, Jilin Agricultural University, Changchun 130118, China; and dInstitute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Contributed by Diter von Wettstein, October 17, 2013 (sent for review September 3, 2013) Polyploidy or whole-genome duplication is recurrent in plant examples of evolutionarily recent successful speciation events via evolution, yet only a small fraction of whole-genome duplica- allopolyploidization, i.e., hybridization and doubling of genomes tions has led to successful speciation. A major challenge in the from Triticum/Aegilops species (11). Speciation of allohexaploid establishment of nascent polyploids is sustained karyotype in- common wheat, Triticum aestivum L., the founder crop for the stability, which compromises fitness. The three putative diploid initial establishment of Middle-Eastern and European agricul- progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes ture, is characterized by two sequential polyploidization events, occurred sympatrically, and their cross-fertilization in different i.e., allotetraploidization and allohexaploidization. The former combinations may have resulted in fertile allotetraploids with var- event involved hybridization of two diploid species, Triticum ious genomic constitutions. However, only SSAA or closely related urartu (genome AA) and a yet-unknown or extinct goat-grass species of the genus Aegilops section Sitopsis (genome SS ∼ BB). genome combinations have led to the speciation of tetraploid This event led to the speciation of allotetraploid emmer wheat, wheats like Triticum turgidum and Triticum timopheevii. We ana- Triticum turgidum ssp. dicoccoides (12). The latter event that lyzed early generations of four newly synthesized allotetraploid GENETICS sh sh m m l l b b involved hybridization of a primitive domesticated form of wheats with genome compositions S S A A ,SS AA, S S DD, T. turgidum (genome BBAA) with a goat-grass species Aegilops fl and AADD by combined uorescence and genomic in situ hybrid- tauschii (genome DD) led to the speciation of common wheat ization-based karyotyping. Results of karyotype analyses showed sh sh m m l l (11). In parallel, allohexaploidization by hybridization of another that although S S A A and S S AA are characterized by imme- allotetraploid wheat, Triticum timopheevii (genome GGAA) (13) diate and persistent karyotype stability, massive aneuploidy and and Einkorn wheat, Triticum monococcum (genome AmAm) has extensive chromosome restructuring are associated with SbSbDD resulted in the speciation of Triticum zhukovskyi (genome and AADD in which parental subgenomes showed markedly dif- GGAAAmAm)(14)(Fig. S1A). Thus, the natural hexaploid ferent propensities for chromosome gain/loss and rearrange- bread wheat has three diploid progenitors (13) (Fig. S1A) that ments. Although compensating aneuploidy and reciprocal have diverged from a common ancestor only about 2.5–4.5 Mya translocation between homeologs prevailed, reproductive fitness (15). Therefore, it is perhaps not surprising that allotetraploid- was substantially compromised due to chromosome instability. ization by hybridization of any two of these three diploid species Strikingly, localized genomic changes in repetitive DNA and can still be accomplished artificially to produce fertile tetraploid copy-number variations in gene homologs occurred in both chro- plants (16, 17) (Fig. S1B). Furthermore, the diploid progenitor mosome stable lines, SshSshAmAm and SlSlAA. Our data demon- species are known to exist sympatrically across several geo- strated that immediate and persistent karyotype stability is graphical areas in the Eastern Mediterranean region and the intrinsic to newly formed allotetraploid wheat with genome com- Near East (13). These features of the diploid progenitor species binations analogous to natural tetraploid wheats. This property, of polyploid wheat raise an intriguing question: Why has only the coupled with rapid gene copy-number variations, may have laid genome combination of SSAA or closely related ones led to the foundation of tetraploid wheat establishment. Significance intergenomic rearrangement | genomic shock | polyploid speciation This paper discusses speciation by allopolyploidy of tetra- olyploidy or whole-genome duplication (WGD) is a driving ploid wheat, which in turn is a milestone event in estab- Pforce in plant and vertebrate evolution (1–5). However, lishing hexaploid bread wheat. The results suggested that recent molecular phylogenetic studies have argued against karyotype stabilization together with variation in copy number a general creative role of WGD in plant evolution (6–8). This of coding genes and localized changes in genomic repeats may incongruence in opinions is long standing; in fact Stebbins in his have contributed to the establishment of tetraploid wheat as seminal work in 1970s stated, “polyploidy has contributed little successful species. These observations are of relevance to the to progressive evolution” (9). Clearly, the two schools of thought plant-breeding community for developing unique wheat cul- tivars by wide-hybridization and chromosomal engineering. regarding the roles of WGD in plant evolution have existed for years, and can converge if we accept the idea that WGDs have Author contributions: S.R., D.v.W., and B.L. designed research; H.Z., Y.B., X.G., B.Z., and occurred frequently in nature but only a small fraction thereof B.Q. performed research; H.Z., Y.B., Y.D., S.R., C.X., N.L., F.H., and B.L. analyzed data; have contributed to successful speciation (7, 10). However, the and H.Z., S.R., D.v.W., and B.L. wrote the paper. genetic and genomic factors determining one of the two funda- The authors declare no conflict of interest. mental fates for a nascent polyploid remained elusive. Freely available online through the PNAS open access option. The Triticum–Aegilops complex includes both diploid and 1H.Z. and Y.B. contributed equally to this work. fi polyploid species with phylogenetically well-de ned organismal 2To whom correspondence may be addressed. E-mail: [email protected], [email protected], or relationships (11). This plant group is therefore suitable to ad- [email protected]. dress the issue raised above. This is especially so because all This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. polyploid species of the Triticum–Aegilops complex represent 1073/pnas.1319598110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319598110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 successful speciation of the two natural allotetraploid wheat by GISH. The procedure enabled characterization of parental species, T. turgidum and T. timopheevii? This question is germane subgenomes and allowed unequivocal discrimination of all to the more general issue as to why only a small fraction of chromosomes in each of the four synthetic wheats (Fig. 1 A–D; WGDs have led to successful speciation in the evolutionary Fig. S2). Chromosome identifications were independently vali- history of angiosperms. dated using three additional FISH probes, namely 5S ribosomal A major challenge for the establishment of newly formed DNA (rDNA), 45S rDNA, and (GAA)n oligonucleotide, where allopolyploids as new species is sustained karyotype instability, in all cases the results were found confirmatory (Fig. S3). Using in particular, whole-chromosome aneuploidy, which, at the or- this robust molecular cytogenetic procedure, we obtained full ganismal level, is associated with compromised cellular metab- karyotypes of a total of 489 individual plants from the four olism and reduced fitness (18). In newly formed allohexaploid synthetic lines: 92 of AT1, 96 of AT2, 205 of AT3 and 96 of AT4 wheat, extensive whole-chromosome aneuploidy was reported (Table S1). The plants of each line were sampled from several (19, 20). However, the immediate chromosomal consequences successive generations (Table S1), and therefore allowed as- associated with formation of allotetraploid wheat have remained sessment of trangenerational chromosome dynamics, i.e., sta- uninvestigated. bility versus instability. Here, we conducted in-depth molecular cytogenetic analyses of karyotype stability using fluorescence and genomic in situ Immediate and Transgenerational Karyotype Stability Showed by Synthetic Allotetraploids with SshSshAmAm and SlSlAA Genomes. Of hybridization (FISH and GISH) techniques and performed lo- sh sh m m cus-specific molecular genetic analysis of gene copy-number the 92 karyotyped AT1 plants (S S A A ), only 2 (2.2%) variations (CNVs) in a set of protein-coding genes using newly plants were numerical aneuploids showing monosomy for a sin- gle chromosome 1Am (2n = 27), whereas the remaining plants synthesized allotetraploid wheats. Different genomic combina- = tions of diploid Triticum/Aegilops species that are parsimoniously were euploids (2n 28), containing full