Crop Phenomics and High-Throughput Phenotyping: Past Decades, Current Challenges, and Future Perspectives.

Editorial Manager(tm) for Developmental Cell Manuscript Draft Manuscript Number: DC-D-06-00013R1 Title: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores Article Type: Research Article Section/Category: Keywords: centromere CID CENP-A kinetochore mitosis aneuploidy Corresponding Author: Dr. Gary H. Karpen, Corresponding Author's Institution: Lawrence Berkeley National Lab/UC Berkeley First Author: Patrick Heun, PhD Order of Authors: Patrick Heun, PhD; Sylvia Erhardt, PhD; Michael D Blower, PhD; Samara Weiss, BA; Andrew D Skora, BA; Gary H Karpen, PhD Manuscript Region of Origin: Abstract: The centromere-specific histone variant CENP-A (CID in Drosophila) is a structural and functional foundation for kinetochore formation and chromosome segregation. Here, we show that overexpressed CID is mislocalized into normally non-centromeric regions in Drosophila tissue culture cells and animals. Analysis of mitoses in living and fixed cells reveals that mitotic delays, anaphase bridges, chromosome fragmentation, and cell and organismal lethality are all direct consequences of CID mislocalization. In addition, proteins that are normally restricted to endogenous kinetochores assemble at a subset of ectopic

Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the early development of Hordeum vulgare × Hordeum bulbosum embryos. The following conclusions regarding the role of the centromere-specific histone H3 variant (CENH3) in the process of chromosome elimination were drawn: (i) centromere inactivity of H. bulbosum chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination in unstable H. vulgare × H. bulbosum hybrids; (ii) centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; (iii) in stable species combinations, cross-species incorporation of CENH3 occurs despite centromere-sequence differences, and not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present in species combinations; and (iv) diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis.

https://www.pnas.org/content/pnas/108/33/E498.full.pdf

Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids

The plant genome is organized into chromosomes that provide the structure for the genetic linkage groups and allow faithful replication, transcription and transmission of the hereditary information. Genome sizes in plants are remarkably diverse, with a 2350-fold range from 63 to 149,000 Mb, divided into n=2 to n= approximately 600 chromosomes. Despite this huge range, structural features of chromosomes like centromeres, telomeres and chromatin packaging are well-conserved. The smallest genomes consist of mostly coding and regulatory DNA sequences present in low copy, along with highly repeated rDNA (rRNA genes and intergenic spacers), centromeric and telomeric repetitive DNA and some transposable elements. The larger genomes have similar numbers of genes, with abundant tandemly repeated sequence motifs, and transposable elements alone represent more than half the DNA present. Chromosomes evolve by fission, fusion, duplication and insertion events, allowing evolution of chromosome size and chromosome number. A combination of sequence analysis, genetic mapping and molecular cytogenetic methods with comparative analysis, all only becoming widely available in the 21st century, is elucidating the exact nature of the chromosome evolution events at all timescales, from the base of the plant kingdom, to intraspecific or hybridization events associated with recent plant breeding. As well as being of fundamental interest, understanding and exploiting evolutionary mechanisms in plant genomes is likely to be a key to crop development for food production.

/pdf/organisation-of-the-plant-genome-in-chromosomes-m714q914k5.pdf

Organisation of the plant genome in chromosomes

This book serves as an introduction to the flourishing field of super-resolution imaging. It is a compiled volume, with different authors for each of its 14 chapters. While not having a strong outline or textbook format, the chapters group into several sections.

Super-Resolution Imaging

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 wild-type 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.

https://www.pnas.org/content/pnas/112/36/11211.full.pdf

Point mutation impairs centromeric CENH3 loading and induces haploid plants

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.

Haploidization via Chromosome Elimination: Means and Mechanisms.

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.

Chromosome elimination by wide hybridization between Triticeae or oat plant and pearl millet: pearl millet chromosome dynamics in hybrid embryo cells

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.

/pdf/centromere-location-in-arabidopsis-is-unaltered-by-extreme-27syed63b5.pdf

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

The organization of centromeric chromatin of diploid barley (Hordeum vulgare) encoding two (α and β) CENH3 variants was analysed by super-resolution microscopy. Antibody staining revealed that both CENH3 variants are organized in distinct but intermingled subdomains in interphase, mitotic and meiotic centromeres. Artificially extended chromatin fibres illustrate that these subdomains are formed by polynucleosome clusters. Thus, a CENH3 variant-specific loading followed by the arrangement into specific intermingling subdomains forming the centromere region appears. The CENH3 composition and transcription vary among different tissues. In young embryos, most interphase centromeres are composed of both CENH3 variants, while in meristematic root cells, a high number of nuclei contain βCENH3 mainly dispersed within the nucleoplasm. A similar distribution and no preferential arrangement of the two CENH3 variants in relationship to the spindle poles suggest that both homologs meet the same function in metaphase cells.

Takayoshi Ishii

Papers

Point mutation impairs centromeric CENH3 loading and induces haploid plants

Haploidization via Chromosome Elimination: Means and Mechanisms.

Chromosome elimination by wide hybridization between Triticeae or oat plant and pearl millet: pearl millet chromosome dynamics in hybrid embryo cells

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

The differential loading of two barley CENH3 variants into distinct centromeric substructures is cell type- and development-specific