Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice(Oryza sativa)

A thermo-insensitive pale green leaf mutant （pgl2） was isolated from T-DNA inserted transgenic lines of rice （Oryza sativa L. subsp, japonica cv. Nipponbare）. Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2gene, an F2 population was constructed by crossing the mutant with Longtefu （Oryza sativa L. subsp, indica）. The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll （Chl） content, while the ratio of Chl a / Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.

A centromere map based on super pan-genome highlights the structure and function of rice centromeres

Rice(Oryza sativa)is a significant crop worldwide with a genome shaped by various evolutionary factors.Rice centromeres are crucial for chromosome segregation,and contain some unreported genes.Due to the diverse and complex centromere region,a comprehensive understanding of rice centromere structure and function at the population level is needed.We constructed a high-quality centromere map based on the rice super pangenome consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice.We showed that rice centromeres have diverse satellite repeat CentO,which vary across chromosomes and subpopulations,reflecting their distinct evolutionary patterns.We also revealed that long terminal repeats(LTRs),especially young Gypsy-type LTRs,are abundant in the peripheral CentO-enriched regions and drive rice centromere expansion and evolution.Furthermore,high-quality genome assembly and complete telomere-to-telomere(T2T)reference genome enable us to obtain more centromeric genome information despite mapping and cloning of centromere genes being challenging.We investigated the association between structural variations and gene expression in the rice centromere.A centromere gene,OsMAB,which positively regulates rice tiller number,was further confirmed by expression quantitative trait loci,haplotype analysis and clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein9 methods.By revealing the new insights into the evolutionary patterns and biological roles of rice centromeres,our finding will facilitate future research on centromere biology and crop improvement.

Wide hybridizations reveal the robustness of functional centromeres in Triticum-Aegilops species complex lines

The Triticum-Aegilops complex groups demonstrated high cross-affinity with each other to overcome the barriers of distant hybridization(Loureiro et al.,2023).Distant hybridization involves two distinct yet closely related events:hybridization and genome doubling.Previous studies have indicated that bursts of transposable elements(TEs)can occur as a consequence or concomitant to hybridization or genome duplication(Parisod et al.,2010).This raises an important scientific question regarding how the TEs-rich centromere region copes with genomic shock(McClintock,1984).The Triticum-Aegilops species complexes,particularly in the F1,So,and subsequent early generations resulting from successive selfcrossing,offer an opportunity to investigate whether the centromere environment undergoes reconstruction and the associated mechanisms that maintain genomic stability.

High temperature increases centromeremediated genome elimination frequency and enhances haploid induction in Arabidopsis

Double haploid production is the most effective way to create true-breeding lines in a single generation.In Arabidopsis,haploid induction via mutation of the centromere-specific histone H3(cenH3)has been shown when the mutant is outcrossed to the wild-type,and the wild-type genome remains in the haploid progeny.However,factors that affect haploid induction are still poorly understood.Here,we report that a mutant of the cenH3 assembly factor Kinetochore Null2(KNL2)can be used as a haploid inducer when pollinated by the wild-type.We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold.We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines,suggesting that haploidinducing lines in crops can be identified in a naturally occurring or chemically induced mutant population,avoiding the generic modification(GM)approach at any stage.Furthermore,a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress,even though it did not induce haploids under standard conditions.Thus,we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

Centromere repositioning and shifts in wheat evolution

The centromere is the region of a chromosome that directs its separation and plays an important role in cell division and reproduction of organisms.Elucidating the dynamics of centromeres is an alternative strategy for exploring the evolution of wheat.Here,we comprehensively analyzed centromeres from the de novoassembled common wheat cultivar Aikang58(AK58),Chinese Spring(CS),and all sequenced diploid and tetraploid ancestors by chromatin immunoprecipitation sequencing,whole-genome bisulfite sequencing,RNA sequencing,assay for transposase-accessible chromatin using sequencing,and comparative genomics.We found that centromere-associated sequences were concentrated during tetraploidization and hexaploidization.Centromeric repeats of wheat(CRWs)have undergone expansion during wheat evolution,with strong interweaving between the A and B subgenomes post tetraploidization.We found that CENH3 prefers to bind with younger CRWs,as directly supported by immunocolocalization on two chromosomes(1A and 2A)of wild emmer wheat with dicentromeric regions,only one of which bound with CENH3.In a comparison of AK58 with CS,obvious centromere repositioning was detected on chromosomes 1B,3D,and 4D.The active centromeres showed a unique combination of lower CG but higher CHH and CHG methylation levels.We also found that centromeric chromatin was more open than pericentromeric chromatin,with higher levels of gene expression but lower gene density.Frequent introgression between tetraploid and hexaploid wheat also had a strong influence on centromere position on the same chromosome.This study also showed that active wheat centromeres were genetically and epigenetically determined.

Maize centromeres:where sequence meets epigenetics

The centromere is a highly organized structure mainly composed of repeat sequences,which make this region extremely difficult for sequencing and other analyses.It plays a conserved role in equal division of chromosomes into daughter cells in both mitosis and meiosis.However,centromere sequences show notable plasticity.In a dicentric chromosome,one of the centromeres can become inactivated with the underlying DNA unchanged.Furthermore,formerly inactive centromeres can regain activity under certain conditions.In addition,neocentromeres without centromeric repeats have been found in a wide spectrum of species.This evidence indicates that epigenetic mechanisms together with centromeric sequences are associated with centromere specification.

Two gap-free reference genomes and a global view of the centromere architecture in rice

Rice(Oryza sativa),a major staple throughout the world and a model system for plant genomics and breeding,was the first crop genome sequenced almost two decades ago.However,reference genomes for all higher organisms to date contain gaps and missing sequences.Here,we report the assembly and analysis of gap-free reference genome sequences for two elite O.sativa xian/indica rice varieties,Zhenshan 97 and Minghui 63,which are being used as a model system for studying heterosis and yield.Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres.We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures.In addition,the similarity of CentO repeats in the same chromosome is higher than across chromosomes,supporting a model of local expansion and homogenization.Both genomes have over 395 non-TE genes located in centromere regions,of which∼41%are actively transcribed.Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes.The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Although the centromeres of some plants have been investlgated prevlously, our knowledge of the wheat centromere Is still very llmlted. To understand the structure and functlon of the wheat centromere, we used two centromeric repeats （RCS1 and CCS1-5ab） to obtain some centromere-assoclated bacterial artificial chromosome （BAC） clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat （Triticum boeoticum Boiss.; 2n=2x=14） BAC library. Southern hybridization results indicated that, of the 32 candidates, there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization （FISH） analysis Indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromerlc regions in hexaplold wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or Dgenome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific （or rich） sequences dispersing on chromosome arms in the BAC clone TbBACS. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation In the period before metaphase.

Dicentric Chromosome Formation and Epigenetics of Centromere Formation in Plants

Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions （called dicentric chromosomes） have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation.

Centromere Epigenetics in Plants

The centromere is an essential chromosome site at which the kinetochore forms and loads proteins needed for faithful segregation during the cell cycle and meiosis（Houben et al., 1999;Cleveland et al.,2003;Ma et al.,2007;Birchler and Han,2009）.Centromere specific sequences such as tandem repeats or transposable elements evolve quickly both within and between the species but have conserved kinetochore proteins（Henikoff and Furuyama,2010）.

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.

Expression of centromere protein-C(CENP-C) in villus tissue of the first-trimester spontaneous abortion and its correlation with chromosome segregation

Objective To investigate the expression of centromere protein-C （CENP-C） in villus tissue of the first-trimester spontaneous abortion （SA） and the correlation study of CENP-C expression with chromosome segregation. Methods Fluorescence in situ hybridization （FISH） and G-banded karyotype analysis were used to detect the numerical chromosome abnormality in 94 villus tissues of women with SA. The participants were separated into case group （n=30） and control group （n--30） according to the results with FISH. The qRT-PCR and Western blotting analysis were used to assess the expression level of CENP-C. Results Forty-eight （51.06%） cases had observed the numerical chromosome abnormality, including 30positive cases and the positive rate was 31.91%. The main types of variation included trisomy 16, 21, 22, X monosomy and triploid. The expression levels of CENP-C mRNA and protein in case group were statistically higher than that in control group （P〈0.05）. Conclusion Expression of CENP-C in the villus tissues of women might be related to SA induced by chromosomal aneuploid.

Ser68 phosphoregulation is essential for CENP-A deposition,centromere function and viability in mice

Centromere identity is defined by nucleosomes containing CENP-A,a histone H3 variant.The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner.We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68.However,a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading.Here,we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability.We also find that mice harboring the corresponding mutations are lethal.Together,these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

An overview of plant centromeres

The centromere is a defining region that mediates chromosome attachment to kinetochore microtubules and proper segregation of the sister chromatids. Intriguingly, satellite DNA and centromeric retrotransposon as major DNA constituents of centromere showed baffling diversification and species-specific. However, the key kinetochore proteins are conserved in both plants and animals, particularly the centromere-specific histone H3-1ike protein （CENH3） in all functional centromeres. Recent studies have highlighted the importance of epigenetic mechanisms in the establishment and maintenance of centromere identity. Here, we review the progress and compendium of research on plant centromere in the light of recent data.

A functional centromere lacking CentO sequences in a newly formed ring chromosome in rice

An awned rice（Oryza sativa） plant carrying a tiny extra chromosome was discovered among the progeny of a telotrisomic line 2nt4L. Fluorescence in situ hybridization（FISH） using chromosome specific BAC clones revealed that this extra chromosome was a ring chromosome derived from part of the long arm of chromosome 4. So the aneuploidy plant was accordingly named as 2nt4L ring. We did not detect any Cent O FISH signals on the ring chromosome, and found only the centromeric probe Centromeric Retrotransposon of Rice（CRR） was co-localized with the centromere-specific histone CENH3 as revealed by sequential FISH after immunodetection. The extra ring chromosome exhibited a unique segregation pattern during meiosis, including no pairing between the ring chromosome and normal chromosome 4during prophase I and pre-separation of sister chromatids at anaphase I.

DNA replication components as regulators of epigenetic inheritance---lesson from fission yeast centromere

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stabil- ity. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

Isolation and Identification of a Functional Centromere Element in the Wild Rice Species Oryza granulata with the GG Genome

The centromere of eukaryotic chromosomes is the crucial locus responsible for sister chromatid cohesion and for correct segregation of chromosomes to daughter cells during cell division. In the structural genomics era, centromeres represent the last frontiers of higher eukaryotic genomes because of their densely methylated, highly repetitive and, heterochromatic DNA （Hall et al., 2004）. Although these functions are conserved among all eukaryotes, centromeric DNA sequences are evolving rapidly （Jiang et al., 2003）.

N^(6)-methyladenosine modification of CENPK mRNA by ZC3H13 promotes cervical cancer stemness and chemoresistance

Background:Stemness and chemoresistance contribute to cervical cancer recurrence and metastasis.In the current study,we determined the relevant players and role of N^(6)-methyladenine(m^(6)A)RNA methylation in cervical cancer progression.Methods:The roles of m^(6)A RNA methylation and centromere protein K(CENPK)in cervical cancer were analyzed using bioinformatics analysis.Methylated RNA immunoprecipitation was adopted to detect m^(6)A modification of CENPK mRNA.Human cervical cancer clinical samples,cell lines,and xenografts were used for analyzing gene expression and function.Immunofluorescence staining and the tumorsphere formation,clonogenic,MTT,and EdU assays were performed to determine cell stemness,chemoresistance,migration,invasion,and proliferation in HeLa and SiHa cells,respectively.Western blot analysis,co-immunoprecipitation,chromatin immunoprecipitation,and luciferase reporter,cycloheximide chase,and cell fractionation assays were performed to elucidate the underlying mechanism.Results:Bioinformatics analysis of public cancer datasets revealed firm links between m^(6)A modification patterns and cervical cancer prognosis,especially through ZC3H13-mediated m^(6)A modification of CENPK mRNA.CENPK expression was elevated in cervical cancer,associated with cancer recurrence,and independently predicts poor patient prognosis[hazard ratio=1.413,95%confidence interval=1.078−1.853,P=0.012].Silencing of CENPK prolonged the overall survival time of cervical cancer-bearing mice and improved the response of cervical cancer tumors to chemotherapy in vivo(P<0.001).We also showed that CENPK was directly bound to SOX6 and disrupted the interactions of CENPK withβ-catenin,which promotedβ-catenin expression and nuclear translocation,facilitated p53 ubiquitination,and led to activation of Wnt/β-catenin signaling,but suppression of the p53 pathway.This dysregulation ultimately enhanced the tumorigenic pathways required for cell stemness,DNA damage repair pathways necessary for cisplatin/carboplatin resistance,epithelial-mesenchymal transition involved in metastasis,and DNA replication that drove tumor cell proliferation.Conclusions:CENPK was shown to have an oncogenic role in cervical cancer and can thus serve as a prognostic indicator and novel target for cervical cancer treatment.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

A NEW METHOD OF IMAGE PROCESSING TECHNIQUE APPLIED TO CHROMOSOME RECOGNITION AND COUNTING

This paper describes a new method based on Four Neighbor Distance Transformation (FNDT) and Equal Diagonal Algorithm (EDA) to extract the medial axis and locate the centromere of the chromosome Compared to the Classical Thinning Algorithm and Four Neighbor Distance Transformation, the new method (FNDT-DEA) is more noise-tolerant, simpler in programming and faster in execution. The FDNTEDA provides the connective and one-pixel thick medial axis representing the length of chromosome. The Crofton directive parameters were used in the algorithm to locate the centromere in some chromosome. The number of chromosomes in a given cell is obtained with calculating either the Euler Number or the clase-curve’s subset. An intergroups classifier has also been designed. The FNDT-DEA gave results quite similar to those given with human assessment. It was concluded that this new method, FNDT-EDA, is appropriate for microcomputer-based analyzing the human chromosomes.