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 ability to generate haploids and subsequently induce chromosome doubling significantly accelerates the crop breeding process. Haploids have been induced through the generation of plants from haploid tissues (in situ gynogenesis and androgenesis) and through the selective loss of a parental chromosome set via inter- or intraspecific hybridization. Here, we focus on the mechanisms responsible for this selective chromosome elimination. CENH3, a variant of the centromere-specific histone H3, has been exploited to create an efficient method of haploid induction, and we discuss this approach in some detail. Parallels have been drawn with chromosome-specific elimination, which occurs as a normal part of differentiation and sex determination in many plant and animal systems.

Carbon ion radiotherapy (C-ion RT) offers superior dose conformity in the treatment of deep-seated tumors compared with conventional X-ray therapy. In addition, carbon ion beams have a higher relative biological effectiveness compared with protons or X-ray beams. C-ion RT for the first patient at Gunma University Heavy Ion Medical Center (GHMC) was initiated in March of 2010. The major specifications of the facility were determined based on the experience of clinical treatments at the National Institute of Radiological Sciences (NIRS), with the size and cost being reduced to one-third of those at NIRS. The currently indicated sites of cancer treatment at GHMC are lung, prostate, head and neck, liver, rectum, bone and soft tissue. Between March 2010 and July 2011, a total of 177 patients were treated at GHMC although a total of 100 patients was the design specification during the period in considering the optimal machine performance. In the present article, we introduce the facility set-up of GHMC, including the facility design, treatment planning systems, and clinical preparations.

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana. We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays, although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3.

Wide crossing is one of a number of practical methods that can be used to expand genetic variation in common wheat (Triticum aestivum). However, in crosses between wheat and distantly related species such as maize (Zea mays) and pearl millet (Pennisetum glaucum), non-wheat chromosomes are often eliminated from the hybrid during embryogenesis. In this study, we used pearl millet pollen to pollinate the pistils of a range of plants in the tribe Triticeae, as well as oat. Seven days after pollination, the dynamics of the pearl millet chromosomes in the embryos were observed using in situ hybridization, probing both the pearl millet genomic DNA and its centromere-specific repeats. In embryos from the crosses with oat, all seven of the pearl millet chromosomes were retained. However, in hybrids with the Triticeae species, chromosome elimination occurred during embryogenesis. Pearl millet chromosome showed chromosome rearrangements and non-disjunction together with micronuclei. These rearranged chromosomes and micronuclei derived from the breakage of bridges and retention of acentric fragments in anaphase, respectively. The cause of the chromosome elimination of wheat-pearl millet hybrid is not malfunction of the kinetochores binding to the spindles but the malfunction of the sister chromatids segregation at anaphase especially of chromosome arm.