Point mutation impairs centromeric CENH3 loading and induces haploid plants

Abstract

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wildtype A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. T he generation and use of haploids is one of the most powerful biotechnological means to accelerate the breeding process of cultivated plants. The advantage of haploid plants for breeders is that homozygosity can be achieved at all loci in a single generation via whole-genome duplication, without the need of selfing or backcrossing over many generations as conventionally required to obtain true-breeding lines. Haploids can be obtained in vitro or in vivo, although many species and genotypes are recalcitrant to these processes (reviewed in ref. 1). Alternatively, substantial changes to centromeric histone H3 (CENH3), such as replacing the hypervariable N-terminal tail of CENH3 with the tail of conventional histone H3 and fusing it to GFP (producing "tailswap-cenh3"), or complementing the cenh3.2-null mutant with homologs from the mustard family CENH3s creates haploid inducer lines in the model plant Arabidopsis thaliana (2-4). Haploidization occurred only when such a haploid inducer was crossed with a wild-type plant. The haploid inducer line proved to be stable upon selfing, suggesting that competition between modified and wild-type centromeres in the developing hybrid embryo results in the inactivation of the centromeres from the inducer parent. Consequently, chromosomes from the inducer parent are lost, and progeny can be recovered that retain only the haploid chromosome set of the wild-type parent.Because CENH3 is almost universal in eukaryotes, this method has the potential to produce haploids in any plant species. To elucidate whether, in addition to the severe conformational change using the CENH3-tailswap (2, 3), nontransgenic-induced minimal mutations in endogenous CENH3 also could affect the centromere function for haploid induction, we screened a population of barley (Hordeum vulgare) produced by ethyl methanesulfonate-induced targeting of local lesions in genomes (TILLING) (5); this diploid species has two functional variants...

Turning rice meiosis into mitosis

“…MiMe combined with genome elimination indeed leads to clonal offspring, as previously demonstrated in Arabidopsis [13]. However, the CENH3 manipulation used in Arabidopsis to provoke genome elimination has not been transferred in other species to date [20,[39][40][41], except in maize but with a lower frequency [42]. The frequency of genome elimination can be genetically controlled in cereals, notably in maize [43][44][45].…”

Restructuring of Holocentric Centromeres During Meiosis in the Plant Rhynchospora pubera

“…The latter is possible, since the removal of CENH3 has been observed in vegetative pollen cells of Arabidopsis thaliana (Schoft et al 2009;Merai et al 2014). Furthermore, the weak CENH3 signals observed in the functional cell suggests that a reduced amount of CENH3 is still sufficient for proper chromosome segregation (Liu et al 2006;Lermontova et al 2011;Karimi-Ashtiyani et al 2015). Figure 8 Model of differential holocentromere organization in the holocentric plant R. pubera.…”

Seeds of doubt: Mendel’s choice of Hieracium to study inheritance, a case of right plant, wrong trait

“…This approach has particular appeal as CENH3 is a conserved protein across all plant species, meaning it can potentially be applied to generate haploid inducers in any plant species. This was recently demonstrated in maize and barley, although in barley, multiple copies of CENH3 complicated the haploid induction (Karimi-Ashtiyani et al 2015; Kelliher et al 2016). …”