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...

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

Holocentric chromosomes occur in a number of independent eukaryotic lineages. They form holokinetic kinetochores along the entire poleward chromatid surfaces, and owing to this alternative chromosome structure, species with holocentric chromosomes cannot use the two-step loss of cohesion during meiosis typical for monocentric chromosomes. Here we show that the plant Luzula elegans maintains a holocentric chromosome architecture and behaviour throughout meiosis, and in contrast to monopolar sister centromere orientation, the unfused holokinetic sister centromeres behave as two distinct functional units during meiosis I, resulting in sister chromatid separation. Homologous non-sister chromatids remain terminally linked after metaphase I, by satellite DNA-enriched chromatin threads, until metaphase II. They then separate at anaphase II. Thus, an inverted sequence of meiotic sister chromatid segregation occurs. This alternative meiotic process is most likely one possible adaptation to handle a holocentric chromosome architecture and behaviour during meiosis.

SUMMARYIn higher plants, the large-scale structure of monocentric chromosomes consists of distinguishable eu-and heterochromatic regions, the proportions and organization of which depend on a species' genome size. To determine whether the same interplay is maintained for holocentric chromosomes, we investigated the distribution of repetitive sequences and epigenetic marks in the woodrush Luzula elegans (3.81 Gbp/1C). Sixtyone per cent of the L. elegans genome is characterized by highly repetitive DNA, with over 30 distinct sequence families encoding an exceptionally high diversity of satellite repeats. Over 33% of the genome is composed of the Angela clade of Ty1/copia LTR retrotransposons, which are uniformly dispersed along the chromosomes, while the satellite repeats occur as bands whose distribution appears to be biased towards the chromosome termini. No satellite showed an almost chromosome-wide distribution pattern as expected for a holocentric chromosome and no typical centromere-associated LTR retrotransposons were found either. No distinguishable large-scale patterns of eu-and heterochromatin-typical epigenetic marks or early/late DNA replicating domains were found along mitotic chromosomes, although super-high-resolution light microscopy revealed distinguishable interspersed units of various chromatin types. Our data suggest a correlation between the centromere and overall genome organization in species with holocentric chromosomes.