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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Research Foundation  

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

DOI： 10.3389/fpls.2015.00912

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The low frequency of meiotic recombination in chromosomal regions other than hotspots is a general obstacle to efficient breeding. A number of active genes are present in recombination-repressed centromeric regions in higher eukaryotes, suggesting that suppression of meiotic recombination prevents shuffling of genes within a centromeric region. In this study, by using an inter-subspecific cross of Oryza sativa L., we show that modification of inactive chromatin states by either genetic or chemical inhibition of chromatin modifying proteins induced changes in both the position of meiotic recombination and, unexpectedly, the pattern of segregation distortion of parental alleles. Antisense knockdown of rice homologues of DECREASE IN DNA METHYLATION1, which is required for the maintenance of heterochromatin in Arabidopsis thaliana, induced a recombination hotspot in a centromeric region accompanied by a steep increase in the proportion of heterozygotes. Our results describe a previously undocumented phenomenon in which artificial chromatin modification could be used to change the pattern of segregation distortion in rice and open up novel possibilities for efficient crop breeding.

DOI： 10.1007/s11032-015-0299-0

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Language：English   Publisher：TUBITAK SCIENTIFIC & TECHNICAL RESEARCH COUNCIL TURKEY  

Breeding based on doubled-haploid approaches has recently become a common tool for accelerating crop improvement in many plant species. However, many plant species do not have a reliable method for haploid induction. A promising new approach involving centromere engineering has recently been proposed to overcome this limitation. Here we provide a perspective of this novel method for the production of haploid plants, which was originally described in the model plant species Arabidopsis thaliana. Centromeres, known to be critical for the accurate distribution of chromosomes in every cell division, have now become a novel target for crop improvement by enabling doubled-haploid technologies.

DOI： 10.3906/tar-1405-137

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japorticus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofiuorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species. Published by Elsevier B.V.

DOI： 10.1016/j.gene.2014.01.034

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In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers
(ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization
and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution. © 2013 The Authors The Plant Journal © 2013 John Wiley &amp
Sons Ltd.

DOI： 10.1111/tpj.12269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Tobacco (Nicotiana tabacum) is an amphidiploid species (2n = 4x = 48, genome constitution SSTT) derived from a natural hybrid between Nicotiana sylvestris (2n = 2x = 24, SS) and Nicotiana tomentosiformis (2n = 2x = 24, TT). Genomic in situ hybridization (GISH), using the genomic DNA from these ancestral species as probes, revealed the chromosomal origins (S or T) and the occurrence of intergenomic translocations in N. tabacum. Fluorescence in situ hybridization (FISH) was also used to distinguish between chromosomes. However, the use of repetitive DNA sequences as probes for FISH analysis is limited by an inability to identify all chromosomes. In addition to this limitation, the occurrence of chromosomal tertiary constrictions can easily lead to the misclassification of chromosomes. To overcome these issues, immunostaining with anti-N. tabacum centromere-specific histone H3 antibody was carried out to determine the centromere position of each chromosome, followed by FISH analysis with ten distinct repetitive DNA probes. This approach allowed us to identify 22 of the 24 chromosome pairs in N. tabacum and revealed novel intergenomic chromosome rearrangements and B-chromosome-like minichromosomes. Hence, the combination of immunostaining with FISH and GISH is critical to accurately karyotype tobacco.

DOI： 10.1007/s10577-013-9363-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A.thaliana artificial ring chromosome1), originated from a centromeric edge of the long arm of chromosome2, but its size (2.85Mb) is much smaller than that of the original chromosome (26.3Mb). Although AtARC1 contains only a short centromere domain consisting of 180bp repeats approximately 250kb in length, compared with the 3Mb domain on the original chromosome2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.

DOI： 10.1111/tpj.12128

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and alpha-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immunoprecipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR.

DOI： 10.1371/journal.pone.0051315

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements. Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next generation.

DOI： 10.1105/tpc.112.105270

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.

DOI： 10.1007/s00299-011-1198-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

DOI： 10.1007/s10577-011-9247-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A dicentric ring minichromosome (mini delta) was identified in transgenic Arabidopsis thaliana and added to a wild type as a supernumerary chromosome. This line is relatively stable and has been maintained for generations, notwithstanding its ring and dicentric structure. To determine the mechanism for stable transmission of mini delta, the structure and behavior of two new types of ring minichromosomes (mini delta 1 and mini delta 1-1) derived from mini delta were investigated. Fluorescence in situ hybridization analysis revealed that mini delta 1 is dicentric just like mini delta, whereas mini delta 1-1 is monocentric. The estimated sizes of mini delta 1 and mini delta 1-1 were 3.8 similar to 5.0 and 1.7 Mb, respectively. The sizes of the two centromeres on mini delta 1 were identical (ca. 270 kb) and similar to that of mini delta 1-1 (ca. 250 kb). Mini delta 1 was relatively stable during mitosis and meiosis, as is mini delta, whereas mini delta 1-1 was unstable during mitosis, and the number of minichromosomes per cell varied. This possibly resulted from misdivision caused by a short centromere on monocentric mini delta 1-1. Transmission through the female was quite limited for all three ring minichromosomes (0-3.2%), whereas that through the male was relatively high (15.4-27.3%) compared with that of other supernumerary chromosomes in Arabidopsis. Ring structure without telomeres itself seems not to limit the female transmission.

DOI： 10.1007/s10577-011-9250-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.

DOI： 10.1007/s10577-011-9219-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

DOI： 10.1371/journal.pgen.1002121

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Microgametogenesis in angiosperms results in two structurally and functionally different cells, one generative cell, which subsequently forms the sperm cells, and the vegetative cell. We analysed the chromatin properties of both types of nuclei after first and second pollen mitosis in rye (Secale cereale). The condensed chromatin of generative nuclei is ear-marked by an enhanced level of histone H3K4/K9 dimethylation and H3K9 acetylation. The less condensed vegetative nuclei are RNA polymerase II positive. Trimethylation of H3K27 is not involved in transcriptional downregulation of genes located in generative nuclei as H3K27me3 was exclusively detected in the vegetative nuclei. The global level of DNA methylation does not differ between both types of pollen nuclei. In rye, unlike in Arabidopsis thaliana (Ingouff et al. Curr Biol 17:1032-1037 2007; Schoft et al. EMBO Rep 10:1015-1021 2009), centromeric histone H3 is not excluded from the chromatin of the vegetative nucleus and the condensation degree of centromeric and subtelomeric regions did not differ between the generative and vegetative nuclei. Differences between rye and A. thaliana data suggest that the chromatin organization in mature nuclei of pollen grains is not universal across angiosperms.

DOI： 10.1007/s10577-011-9207-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability. Copyright (C) 2011 S. Karger AG, Basel

DOI： 10.1159/000327713

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Two partially reconstructed karyotypes (RK1 and RK2) of Arabidopsis thaliana have been established from a transformant, in which four structurally changed chromosomes (alpha, beta, gamma, and delta) were involved. Both karyotypes are composed of 12 chromosomes, 2n - 1 '' + 3 '' + 4 '' + 5 '' + alpha '' + gamma '' - 12 for RK1 and 2n 3 '' + 4 '' + 5 '' + alpha + beta '' + gamma '' - 12 for RK2, and these chromosome constitutions were relatively stable at least for three generations. Pairing at meiosis was limited to the homologues ( 1, 3, 4, 5, alpha, beta, or gamma), and no pairing occurred among non- homologous chromosomes in both karyotypes. For minichromosome a (mini alpha), precocious separation at metaphase I was frequently observed in RK2, as found for other minichromosomes, but was rare in RK1. This stable paring of mini alpha was possibly caused by duplication of the terminal tip of chromosome 1 that is characteristic of RK1.

DOI： 10.1007/s00412-010-0259-8

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The centromere as a kinetochore assembly site is fundamental to the partitioning of genetic material during cell division. In order to determine the functional centromeres of soybean, we characterized the soybean centromere-specific histone H3 (GmCENH3) protein and developed an antibody against the N-terminal end. Using this antibody, we cloned centromere-associated DNA sequences by chromatin immunoprecipitation. Our analyses indicate that soybean centromeres are composed of two distinct satellite repeats (GmCent-1 and GmCent-4) and retrotransposon-related sequences (GmCR). The possible allopolyploid origin of the soybean genome is discussed in view of the centromeric satellite sequences present.

DOI： 10.1007/s10577-010-9119-x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The centromere is a region utilized for spindle attachment on a eukaryotic chromosome and essential for accurate chromatid segregation. In most eukaryotes, centromeres have specific DNA sequences and are capable of assembling specific proteins to form a complex called the kinetochore. Among these proteins, centromeric histone H3 (CENH3) is one of the most fundamental, since CENH3s have been found in all investigated functional centromeres and recruits other centromeric proteins. In this study, the localization of alien CENH3s were analyzed in Arabidopsis and tobacco-cultured cells to determine the interaction between species-specific centromeric DNA and CENH3. Results showed that CENH3 of Arabidopsis and tobacco were localized on centromeres in the tobacco-cultured cells, unlike the case with CENH3 of rice and Luzula. In addition to these CENH3s, CENH3 of Luzula was partially localized in the Arabidopsis cultured cells. These data suggest that only evolutionally close CENH3s are able to target centromeres in alien species. Furthermore, the ability to target alien centromeres of histone fold domains was investigated using amino-terminal deleted CENH3s.

DOI： 10.1007/s10577-009-9108-0

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Centromeres play an important role in chromosome transmission in eukaryotes and comprise specific DNA and proteins that form complexes called kinetochores. In tobacco, although a centromere-specific histone H3 (NtCENH3) and centromeric DNA sequence (Nt2-7) have been identified, no other kinetochore components have been determined. In this study, we isolated and characterized cDNAs encoding two centromeric proteins CENP-C and MIS12 from Nicotiana tabaccum. Two CENP-C homologues, NtCENP-C-1 and -2, isolated from N. tabaccum were similar to CENP-C from N. sylvestris and N. tomentosiformis, respectively. Similarly, two Mis12 homologues, NtMIS12-1 and -2, in N. tabaccum were shown to originate from N. sylvestris and N. tomentosiformis, respectively. Both respective homologues for CENP-C and Mis12 were expressed at the same level. This indicates that in a tetraploid species, N. tabaccum, two ancestral genes encoding the centromeric proteins participate equally in the functioning of centromeres.

DOI： 10.1007/s10577-009-9064-8

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Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody.

DOI： 10.1007/s00412-008-0193-1

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Language：English   Publisher：THE SOCIETY OF CHROMOSOME RESEARCH  

Centromeres are functional chromosomal loci involved in an overall process that delivers sister chromatids equally into daughter cells during mitosis and meiosis. A complex of centromeric DNA and proteins is formed at the primary constriction point, and is referred to as the kinetochore. Since this function is essential for all eukaryotes, the process and centromeric proteins involved are conserved among eukaryotes. On the other hand, the DNA components and structures of centromeres are highly variable among eukaryotes. In the last decade, a plethora of investigations concerning plant centromeric components including centromeric DNA and proteins have been conducted which have shed light on these enigmatic chromosomal regions and associated functions. In this review, an overview of plant centromeric components including centromeric proteins (CENP-C, centromere specific histone H3 variants, and MIS12) and centromeric DNA and structures of plant centromeres (Arabidopsis, rice, and Luzula) are presented.

DOI： 10.11352/scr.12.5

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The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.

DOI： 10.1007/s00412-007-0102-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS  

Centromeres are difficult to map even in species where genetic resolution is excellent. Here we show that functions between repeats provide reliable single-copy markets for recombinant inbred mapping within centromeres and pericentromeric heterochromatin. Repeat function mapping was combined will anti-CENH3-mediated ChIP to provide a definitive map position for maize centromere 8.

DOI： 10.1534/genetics.106.060467

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Centromeres are sites of spindle attachment for chromosome segregation. During meiosis, recombination is absent at centromeres and surrounding regions. To understand the molecular basis for recombination suppression, we have comprehensively annotated the 3.5-Mb region that spans a fully sequenced rice centromere. Although transcriptional analysis showed that the 750-kb CENH3-containing core is relatively deficient in genes, the recombination-free region differs little in gene density from flanking regions that recombine. Likewise, the density of transposable elements is similar between the recombination-free region and flanking regions. We also measured levels of histone H4 acetylation and histone H3 methylation at 176 genes within the 3.5-Mb span. Active genes showed enrichment of H4 acetylation and H3K4 dimethylation as expected, including genes within the core. Our inability to detect sequence or histone modification features that distinguish recombination-free regions from flanking regions that recombine suggest that recombination suppression is an epigenetic feature of centromeres maintained by the assembly of CENH3-containing nucleosomes within the core. CENH3-containing centrochromatin does not appear to be distinguished by a unique combination of H3 and H4 modifications. Rather, the varied distribution of histone modifications might reflect the composition and abundance of sequence elements that inhabit centromeric DNA.

DOI： 10.1105/tpc.105.037945

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Although holocentric species are scattered throughout the plant and animal kingdoms, only holocentric chromosomes of the nematode worm Caenorhabditis elegans have been analyzed with centromeric protein markers. In an effort to determine the holocentric structure in plants, we investigated the snowy woodrush Luzula nivea. From the young roots, a cDNA encoding a putative centromere-specific histone H3 (LnCENH3) was successfully isolated based on sequence similarity among plant CENH3s. The deduced amino acid sequence was then used to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea. These data from L. nivea are compared with those from C. elegans, and common features of holocentric chromosomes are discussed.

DOI： 10.1105/tpc.105.032961

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The centromeric retrotransposon (CR) family in the grass species is one of few Ty3-gypsy groups of retroelements that preferentially transpose into highly specialized chromosomal domains. It has been demonstrated in both rice and maize that CRR (CR of rice) and CRM (CR of maize) elements are intermingled with centromeric satellite DNA and are highly concentrated within cytologically defined centromeres. We collected all of the CRR elements from rice chromosomes 1, 4, 8, and 10 that have been sequenced to high quality. Phylogenetic analysis revealed that the CRR elements are structurally diverged into four subfamilies, including two autonomous subfamilies (CRR1 and CRR2) and two nonautonomous subfamilies (noaCRR1 and noaCRR2). The CRR1/CRR2 elements contain all characteristic protein domains required for retrotransposition. In contrast, the noaCRR elements have different structures, containing only a gag or gag-pro domain or no open reading frames. The CRR and noaCRR elements share substantial sequence similarity in regions required for DNA replication and for recognition by integrase during retrotransposition. These data, coupled with the presence of young noaCRR elements in the rice genome and similar chromosomal distribution patterns between noaCRR1 and CRR1/CRR2 elements, suggest that the noaCRR elements were likely mobilized through the retrotransposition machinery from the autonomous CRR elements. Mechanisms of the targeting specificity of the CRR elements, as well as their role in centromere function, are discussed.

DOI： 10.1093/molbev/msi069

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Centromere-specific histone H3 (CENH3) has been used to detect active centromeres, and to analyse the DNA sequences closely associated with the centromere, because they localize only in active centromeres and bind directly to the DNA. In maize and rice, the centromeric retrotransposons (CR) are shown to be closely associated with their own CENH3 whereas no such association was found in Arabidopsis thaliana. In this study, this sort of association was investigated in sugarcane. Two expressed sequence tag groups encoding putative sugarcane CENH3 (SoCENH3) were found in a sugarcane-expressed sequence tag database. Their deduced amino acid sequences were similar to these of the CENH3s in rice and maize. An antibody against rice CENH3 seemed to crossreact with the SoCENH3s, and stained sugarcane centromeres. A set of immunoprecipitation tests was conducted with the antibody and chromatin from the sugarcane genome to reveal CENH3-associated DNA sequences in sugarcane. Centromeric tandem repeats (SCEN) and centromeric retrotransposons of sugarcane (CRS) were significantly precipitated with the antibody, meaning these repeats are directly interacting with CENH3 in sugarcane centromeres.

DOI： 10.1007/s10577-005-0847-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from similar to300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize centromeres.

DOI： 10.1105/tpc.018937

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Centromeres are the last frontiers of complex eukaryotic genomes, consisting of highly repetitive sequences that resist mapping, cloning and sequencing. The centromere of rice Chromosome 8 (Cen8) has an unusually low abundance of highly repetitive satellite DNA, which allowed us to determine its sequence. A region of similar to750 kb in Cen8 binds rice CENH3, the centromere-specific H3 histone. CENH3 binding is contained within a larger region that has abundant dimethylation of histone H3 at Lys9 (H3-Lys9), consistent with Cen8 being embedded in heterochromatin. Fourteen predicted and at least four active genes are interspersed in Cen8, along with CENH3 binding sites. The retrotransposons located in and outside of the CENH3 binding domain have similar ages and structural dynamics. These results suggest that Cen8 may represent an intermediate stage in the evolution of centromeres from genic regions, as in human neocentromeres, to fully mature centromeres that accumulate megabases of homogeneous satellite arrays.

DOI： 10.1038/ng1289

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC AM  

The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinctochore complex in Arabidopsis.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC AM  

We sequenced two maize bacterial artificial chromosome (BAC) clones anchored by the centromere-specific satellite repeat CentC. The two BACs, consisting of similar to200 kb of cytologically defined centromeric DNA, are composed exclusively of satellite sequences and retrotransposons that can be classified as centromere specific or noncentromere specific on the basis of their distribution in the maize genome. Sequence analysis suggests that the original maize sequences were composed of CentC arrays that were expanded by retrotransposon invasions. Seven centromere-specific retrotransposons of maize (CRM) were found in BAC 16H10. The CRM elements inserted randomly into either CentC monomers or other retrotransposons. Sequence comparisons of the long terminal repeats (LTRs) of individual CRM elements indicated that these elements transposed within the last 1.22 million years. We observed that all of the previously reported centromere-specific retrotransposons in rice and barley, which belong to the same family as the CRM elements, also recently transposed with the oldest element having transposed similar to3.8 million years ago. Highly conserved sequence motifs were found in the LTRs of the centromere-specific retrotransposons in the grass species, suggesting that the LTRs may be important for the centromere specificity of this retrotransposon family.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs), and a variety of other repeats. One particularly well-conserved CR element, CRM, occurs primarily as complete and uninterrupted elements and is interspersed thoroughly with CentC at the light microscopic level. To determine if these major centromeric DNAs are part of the functional centromere/kinetochore complex, we generated antiserum to maize centromeric histone H3 (CENH3). CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle and colocalizes with the kinetochore protein CENPC in meiotic cells. Chromatin immunoprecipitation demonstrates that CentC and CRM interact specifically with CENH3, whereas knob repeats and Tekay retroelements do not. Approximately 38 and 33% of CentC and CRM are precipitated in the chromatin immunoprecipitation assay, consistent with data showing that much, but not all, of CENH3 colocalizes with CentC.

DOI： 10.1105/tpc.006106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Tetrad analysis is a genetic method that can locate genes and centromeres on a linkage map with a high degree of precision. Despite its effectiveness and accuracy, application of this method is generally limited to fungi, algae and mosses. Here we demonstrate a new method of tetrad analysis that is applicable to other organisms. This combines tetrad analysis with fluorescence in situ hybridization (FISH), and is thus referred to as tetrad-FISH analysis. We demonstrate the effectiveness of this method using tetrads of rye, Secale cereale. The rye strain JNK contains interstitial heterochromatin in a region of Chromosome 2R. We have previously cloned the tandemly repeated sequence forming this heterochromatin in the plasmid pScJNK. We performed FISH using pScJNK as the probe on tetrads obtained from heterozygotes for the heterochromatin region. The frequency of tetrads demonstrating positive signals in two cells that are diagonally opposite one another must correspond to the frequency of recombination in the interval between the heterochromatin and the centromere. Comparison between the results of tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions. We discuss the effectiveness of tetrad-FISH analysis, particularly for the localization of functional centromeres in linkage maps.

DOI： 10.1007/s00438-001-0634-5

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

DOI： 10.1023/A:1016739719421

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of > 50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.

DOI： 10.1046/j.1365-313X.2000.00827.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL RESEARCH COUNCIL CANADA  

Tandem repetitive Afa-family sequences of 340 bp are known to occur in wheat and related species of tribe Triticeae. We isolated six and three Afa-family sequences from Leymus racemosus and Psathyrostachys juncea, respectively, both of which are perennial species. The sequences account for 0.5% and 0.2% of L. racemosus and P. juncea genomes, respectively, and using in situ hybridization were located in subtelomeric and interstitial regions of L. racemosus chromosomes. These sequences are clustered with those of Elymus trachycaulus in the phylogenetic tree. Our findings indicate that the Afa-family sequences have been amplified at least twice in the lineage of L. racemosus, P. juncea, and E. trachycaulus.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

When chromosomes are broken, the breakpoints become highly unstable and acquire the ability to fuse-with other broken ends. The breakpoints are, however, eventually stabilized, and, therefore, the broken chromosomes are transmitted to the daughter cells without further morphological change. This phenomenon, known as "healing of breakpoints", involves the addition of repetitive telomere sequences at the breakpoints by telomerase, the enzyme that normally synthesizes the telomere sequence at normal chromosome terminals. In many higher organisms, however, this property has not been well investigated. In this study, we examined the telomere sequences in wheat deletion lines with breakpoints on chromosome 1B. Lines that had breakpoints around the nucleolar organizer region were first selected on the basis of cytological observations, and the precise breakpoints were determined by mapping a fragment of rDNA and RFLP markers. In three lines - in addition to one previously reported - the DNA fragments encompassing the breakpoints were amplified by PCR using primers located in the rDNA and in telomere sequences. The DNA sequences provide insight into the properties of the telomerase activity at the breakpoints. The telomere sequences initiated from 2- to 4-nucleotide motifs in the original ribosomal DNA sequence which are also found in the repeat unit characteristic of telomere sequences. No specific sequences or structures were observed at or around the breakpoints. At all of the four breakpoints investigated, the newly synthesized telomere sequences contained considerable numbers of atypical telomere sequence units, particularly TTAGGG, which is the common unit of mammalian telomere sequences. Based on these results, we discuss the ability of plant telomerase to initiate the de novo synthesis of telomere sequences at internal breakpoints.

DOI： 10.1007/s004380051150

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Two kinds of tandem repetitive sequences were isolated from Leymus racemosus (Lam.) Tzvelev. One of them was classified in the 350-bp family originally isolated from Secale. The other was a novel repetitive sequence family, named 'TaiI family', which consisted of a repeat unit of 570 bp. Fluorescence in-situ hybridization of the chromosomes of L. racemosus indicated that both families were located in subtelomeric heterochromatin and that the 350-bp family and TaiI family occupied different heterochromatin regions. In addition, even homologous chromosomes did not show the same patterns of TaiI and 350-bp families. The combination of these two families of repetitive sequences, together with Afa-family sequences and rDNAs, helps to identify the ten homologous chromosome pairs of L. racemosus. From these data, we proposed a karyotype of L. racemosus and compared it with other karyotypes already reported.

DOI： 10.1023/A:1009285311247

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Among cultivated rye, Secale cereale L., collected in Japan, we found an extra heterochromatin on the long-arm interstitial region of chromosome 2R. This extra heterochromatin was polymorphic in the population. The plants with the extra heterochromatin showed a specific DNA fragment of 1.2 kb in digests prepared with the restriction enzyme DraI. The fragment was cloned and used as a probe for fluorescent in-situ hybridization (FISH). The clone, pScJNK1, showed a hybridization signal at the extra heterochromatic region. The segregation of the number of signals corresponded to the number of the extra heterochromatin of the 2R chromosome, indicating that the sequence might construct the heterochromatin. Southern hybridization using the clone as a probe showed a ladder pattern, suggesting that the sequence was a tandem repeat. Three sequences homologous to pScJNK1 were isolated; these were 1192-1232 bp, 44.7-45.9% in GC content, highly homologous (> 93%) with each other, and did not show any significant homology to other sequences in a DNA database. Slot blot hybridization using pScJNK1 as a probe indicated that there were about 4000 copies of the sequence in the haploid genome carrying the extra heterochromatin, whereas less than 20 copies existed in the genome without the heterochromatin. Southern hybridization using MspI and HapII indicated that all of the second cytosine nucleotides in CCGG sites in the sequence were methylated in the extra heterochromatin.

DOI： 10.1023/A:1009226612818

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The Afa-family sequences in wheat-related species, Triticeae, are tandem repetitive sequences of 340 bp. All the analyzed Triticeae species carried the sequences in their genomes, though the copy numbers varied about 100-fold among the species. The nucleotide fragments amplified by PCR were cloned and sequenced, and their behavior in the evolution of Triticeae was analyzed by the neighbor-joining (NJ) method. The sequences in genomes with many copies of this family clustered at independent branches of the phylogenic tree, whereas the sequences in genomes with a few copies did not. This may suggest that Afa-family sequences had amplified several times in the evolution of Triticeae, each using a limited number of different master copies. In addition, the sequences of the A and B genomes of hexaploid common wheat indicated that the Afa-family sequences had not evolved in a concerted manner between the genomes. Furthermore, the sequences of each chromosome of the D genome of this species indicated that the sequences had amplified on all over the D-genome chromosomes in a short period.

DOI： 10.1007/PL00006375

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：National Research Council of Canada  

The H genome specific repetitive sequence of Elymus trachycaulus, pEt2, consists of three units of a 337-339 bp repeat aligned in tandem. The sequence is homologous to Afa-family sequences that are widely distributed in the genomes of Triticeae (Gramineae) species.

DOI： 10.1139/gen-41-1-134

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Tandem repetitive sequences consisting of 140-bp repetitive units were cloned from sugar cane genomic DNA and designated the SCEN family. In situ hybridization revealed that they were located on the centromeric region of almost all of the chromosomes of sugar cane. The 140-bp sequence included three CENP-B box-like sequences. Phylogenetic analysis of the members of the SCEN family revealed that the sequences had 75% homology with each other, on average, and that the sequences could not be further classified into smaller subfamilies. The copy number of the sequence was estimated to be 2.6 x 105 per haploid sugar cane genome and, therefore, 4.6 x 103 or 630 kb per chromosome on average.

DOI： 10.1023/A:1009270824142

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

The Afa-family repetitive sequences were isolated from barley (Hordeum vulgare, 2n = 14) and cloned as pHvA14. This sequence distinguished each barley chromosome by in situ hybridization. Double color fluorescence in situ hybridization using pHvA14 and 5S rDNA or HvRT-family sequence (subtelomeric sequence of barley) allocated individual barley chromosomes showing a specific pattern. of pHvA14 to chromosome 1H to 7H. As the case of the D genome chromosomes of Aegilops squarrosa and common wheat (Triticum aestivum) hybridized by its Afa-family sequences, the signals of pHvA14 in barley chromosomes tended to appear in the distal regions that do not carry many chromosome band markers. In the telomeric regions these signals always placed in more proximal portions than those of HvRT-family. Based on the distribution patterns of Afa-family sequences in the chromosomes of barley and D genome chromosomes of wheat,we discuss a possible mechanism of amplification of the repetitive sequences during the evolution of Triticeae. In addition, we show here that HvRT-family also could be used to distinguish individual barley chromosomes from the patterns of in situ hybridization.

DOI： 10.1266/ggs.72.303

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Canadian Science Publishing  

DOI： 10.1139/g95-063

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