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.

Differential Expressing of Centromere Protein CenpG in Breast Cancer

Using indirect immunofluorescence (IIF), an anti-centromere protein CenpG-serum was verified. Western blot of the protein extracts of 31 samples of breast cancer tissues and their normal (not cancerous ) tissues a little far away from them in the same individuals showed that, in the majority of the tests (71%), centromere protein CenpG over expressed in breast cancer tissues. And moreover, a kind of protein component whose molecular weight is 43 kd, and which can be recognized by anti-CenpG serum was found in two of the cancer samples. The results suggested that CenpG (together whith it, there may be other relative components),which has been found and named recently, may be related to cancer,and its differential expressing is probably related to malignant cell proliferation.

Human chromosome pellicle antibody recognizing centromere protein-C(CENP-C),the main component of the kinetochore

Recently the antichromosome antisera from several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes. In order to identify the pellicle components, we used these antichromosome antisera to screen a human embryonic cDNA library. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C), a human centromere autoantigen. This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

Cytological identification of an isotetrasomic in rice and its application to centromere mapping

The aneuploid with isochromosome or telochromosome is ideal material for exploring the position of centromere in lingkage map. FOr obtaining these aneuploids in rice, the primary trisomics from triplo-1 to triplo-12 and the aneuploids derived from a triploid of indica rice variety Zhongxian 3037 were carefully investigated. From the offsprings of triplo-10, a primary trisomic of chromosome 10 of the variety, an isotetrasomic "triplo-10-1" was obtained. Cytological investigation revealed that a pair of extra isochromosomes of triplo-10-1 were come from the short arm of chromosome 10. In the offsprings of the isotetrasomic, a secondary trisomic "triplo-10-2" 5 in which the extra- chromosome was an isochromosome derived from the short arm of chromosome 10, was identified. With the isotetrasomic, secondary trisomic, primary trisomic and diploid of variety Zhongxian 3037, different molecular markers were used for exploring the position of the centromere of chromosome 10. Based on the DNA dosage effect, it was verified that the molecular markers G1125, G333 and L169 were located on the short arm, G1084 and other 16 available molecular markers were on the long arm of chromosome 10. So the centromere of chromosome 10 was located somewhere betweenG1125 and G1084 according to the RFLP linkage map given by Kurata et al[1]. The distance from G1125 to G1084 was about 3.2cM.

High-quality Gossypium hirsutum and Gossypium barbadense genome assemblies reveal the landscape and evolution of centromeres

Centromere positioning and organization are crucial for genome evolution;however,research on centro-mere biology is largely influenced by the quality of available genome assemblies.Here,we combined Oxford Nanopore and Pacific Biosciences technologies to de novo assemble two high-quality reference genomes for Gossypium hirsutum(TM-1)and Gossypium barbadense(3-79).Compared with previously published reference genomes,our assemblies show substantial improvements,with the contig N50 improved by 4.6-fold and 5.6-fold,respectively,and thus represent the most complete cotton genomes to date.These high-quality reference genomes enable us to characterize 14 and 5 complete centromeric regions for G.hirsutum and G.barbadense,respectively.Our data revealed that the centromeres of allotetraploid cotton are occupied by members of the centromeric repeat for maize(CRM)and Tekay long terminal repeat families,and the CRM family reshapes the centromere structure of the At subgenome after polyploidization.These two intertwined families have driven the convergent evolution of centromeres between the two subgenomes,ensuring centromere function and genome stability.In addition,the reposi-tioning and high sequence divergence of centromeres between G.hirsutum and G.barbadense have contributed to speciation and centromere diversity.This study sheds light on centromere evolution in a sig-nificant crop and provides an alternative approach for exploring the evolution of polyploid plants.

Unveiling the distinctive traits of functional rye centromeres:minisatellites,retrotransposons,and R-loop formation

Centromeres play a vital role in cellular division by facilitating kinetochore assembly and spindle attachments.Despite their conserved functionality,centromeric DNA sequences exhibit rapid evolution,presenting diverse sizes and compositions across species.The functional significance of rye centromeric DNA sequences,particularly in centromere identity,remains unclear.In this study,we comprehensively characterized the sequence composition and organization of rye centromeres.Our findings revealed that these centromeres are primarily composed of long terminal repeat retrotransposons(LTR-RTs)and interspersed minisatellites.We systematically classified LTR-RTs into five categories,highlighting the prevalence of younger CRS1,CRS2,and CRS3 of CRSs(centromeric retrotransposons of Secale cereale)were primarily located in the core centromeres and exhibited a higher association with CENH3 nucleosomes.The minisatellites,mainly derived from retrotransposons,along with CRSs,played a pivotal role in establishing functional centromeres in rye.Additionally,we observed the formation of R-loops at specific regions of CRS1,CRS2,and CRS3,with both rye pericentromeres and centromeres exhibiting enrichment in R-loops.Notably,these R-loops selectively formed at binding regions of the CENH3 nucleosome in rye centromeres,suggesting a potential role in mediating the precise loading of CENH3 to centromeres and contributing to centromere specification.Our work provides insights into the DNA sequence composition,distribution,and potential function of R-loops in rye centromeres.This knowledge contributes valuable information to understanding the genetics and epigenetics of rye centromeres,offering implications for the development of synthetic centromeres in future plant modifications and beyond.

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.

Non-B-form DNA is associated with centromere stability in newly-formed polyploid wheat

Non-B-form DNA differs from the classic B-DNA double helix structure and plays a crucial regulatory role in replication and transcription.However,the role of non-B-form DNA in centromeres,especially in polyploid wheat,remains elusive.Here,we systematically analyzed seven non-B-form DNA motif profiles(A-phased DNA repeat,direct repeat,G-quadruplex,inverted repeat,mirror repeat,short tandem repeat,and Z-DNA)in hexaploid wheat.We found that three of these non-B-form DNA motifs were enriched at centromeric regions,especially at the CENH3-binding sites,suggesting that non-B-form DNA may create a favorable loading environment for the CENH3 nucleosome.To investigate the dynamics of centromeric non-B form DNA during the alloploidization process,we analyzed DNA secondary structure using CENH3 ChIP-seq data from newly formed allotetraploid wheat and its two diploid ancestors.We found that newly formed allotetraploid wheat formed more non-B-form DNA in centromeric regions compared with their parents,suggesting that non-B-form DNA is related to the localization of the centromeric regions in newly formed wheat.Furthermore,non-B-form DNA enriched in the centromeric regions was found to preferentially form on young LTR retrotransposons,explaining CENH3's tendency to bind to younger LTR.Collectively,our study describes the landscape of non-B-form DNA in the wheat genome,and sheds light on its potential role in the evolution of polyploid centromeres.

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.

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.

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.

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.

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.

Recent advances in plant centromere biology

The centromere, which is one of the essential parts of a chromosome, controls kinetochore formation and chromosome segregation during mitosis and meiosis. While centromere function is conserved in eukaryotes, the centromeric DNA sequences evolve rapidly and have few similarities among species. The histone H3 variant CENH3(CENP-A in human), which mostly exists in centromeric nucleosomes, is a universal active centromere mark in eukaryotes and plays an essential role in centromere identity determination. The relationship between centromeric DNA sequences and centromere identity determination is one of the intriguing questions in studying centromere formation. Due to the discoveries in the past decades, including "neocentromeres" and "centromere inactivation", it is now believed that the centromere identity is determined by epigenetic mechanisms. This review will present recent progress in plant centromere biology.

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.