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Centromeres are indispensable functional units of chromosomes. The evolutionary mechanisms underlying the rapid evolution of centromeric repeats, especially those following polyploidy, remain unknown. In this study, we isolated centromeric sequences of Brassica nigra, a model diploid progenitor (B genome) of the allopolyploid species B. juncea (AB genome) and B. carinata (BC genome) by chromatin immunoprecipitation of nucleosomes containing the centromere-specific histone CENH3. Sequence analysis detected no centromeric satellite DNAs, and most B. nigra centromeric repeats were found to originate from Tyl/copia-class retrotransposons. In cytological analyses, six of the seven analyzed repeat clusters had no FISH signals in A or C genomes of the related diploid species B. rapa and B. oleracea. Notably, five repeat clusters had FISH signals in both A and B subgenomes in the tetraploid B. juncea. In the tetraploid B. carinata, only CL23 displayed three pairs of signals in terminal or interstitial regions of the C-derived chromosome, and no evidence of colonization of CLs onto C-subgenome centromeres was found in B. carinata. This observation suggests that centromeric repeats spread and proliferated between genomes after polyploidization. CL3 and CRB are likely ancient centromeric sequences arising prior to the divergence of diploid Brassica which have detected signals across the genus. And in allotetraploids B. juncea and B. carinata, the FISH signal intensity of CL3 and CRB differed among subgenomes. We discussed possible mechanisms for centromeric repeat divergence during Brassica speciation and polyploid evolution, thus providing insights into centromeric repeat establishment and targeting.

Simple and consistent production of haploid is always an appealing pursuit for both crop breeders and researchers. Although diverse strategies have been developed to produce haploids over the past decades, most of them are applicable in only a limited number of plant species. In 2010, Ravi and Chan reported that haploid Arabidopsis thaliana plants can be efficiently induced through the introduction of a single genetic alteration in centromere histone H3 (CENH3). Subsequent studies demonstrated that haploids can be efficiently induced either through genetic engineering of CENH3 N-terminal tail or histone fold domain or by replacing CENH3 with an ortholog. The mutation of a pollen-specific phospholipase gene, MATRILINEAL (MTL) has been revealed to trigger the haploid induction (HI) in maize, which present another promising HI approach by the editing of MTL in plant. Here, we review the progress of the CENH3-medialed HI and propose a revised centromere-size model by suggesting a competitive loading process between wild-type and mutant CENH3 during HI. This model can explain both the findings of HI failure when wild-type and mutant CENH3 genes are coexpressed and the alien centromere loading of CENH3 in stable hybrids. In addition, we review the current understanding of MTL-mediated HI in plant. The conservation of CENH3 and MTL in plants indicates wide potential application for HI. We discuss the utility and potential of these two methods in crops by comparing their mechanisms and applications to date in plants. This review will promote the study and application of both CENH3- and MTL-mediated haploid induction in plants.

By putting heterologous genomic regulatory systems into contact, chromosome addition lines derived from interspecific or intergeneric crosses allow the investigation of transcriptional regulation in new genomic environments. Here, we report the transcriptional and epigenetic adaptation of stably inherited alien maize chromosomes in two oat–maize addition (OMA) lines. We found that the majority of maize genes displayed maize-specific transcription in the oat genomic environment. Nevertheless, a quarter of the expressed genes encoded by the two maize chromosomes were differentially expressed genes (DEGs). Notably, highly conserved orthologs were more severely differentially expressed in OMAs than less conserved orthologs. Additionally, syntenic genes and highly abundant genes were over-represented among DEGs. Gene suppression was more common than activation among the DEGs; however, the genes in the former maize pericentromere, which expanded to become the new centromere in OMAs, were activated. Histone modifications (H3K4me3, H3K9ac and H3K27me3) were consistent with these transcriptome results. We expect that cis regulation is responsible for unchanged expression in OMA versus maize; and trans regulation is the predominant mechanism behind DEGs. The genome interaction identified here reveals the important consequences of interspecific/intergeneric crosses and potential mechanisms of plant evolution when genomic environments interact.

DNA2 is a nuclease/helicase that is involved in Okazaki fragment maturation, replication fork processing, and end resection of DNA double‐strand breaks. Similar such helicase activity for resolving secondary structures and structure‐specific nuclease activity are needed during DNA replication to process the chromosome‐specific higher order repeat units present in the centromeres of human chromosomes. Here, we show that DNA2 binds preferentially to centromeric DNA. The nuclease and helicase activities of DNA2 are both essential for resolution of DNA structural obstacles to facilitate DNA replication fork movement. Loss of DNA2‐mediated clean‐up mechanisms impairs centromeric DNA replication and CENP‐A deposition, leading to activation of the ATR DNA damage checkpoints at centromeric DNA regions and late‐S/G2 cell cycle arrest. Cells that escape arrest show impaired metaphase plate formation and abnormal chromosomal segregation. Furthermore, the DNA2 inhibitor C5 mimics DNA2 knockout and synergistically kills cancer cells when combined with an ATR inhibitor. These findings provide mechanistic insights into how DNA2 supports replication of centromeric DNA and give further insights into new therapeutic strategies.

CENH3 is a centromere-specific histone H3 variant and has been used as a marker to identify active centromeres and DNA sequences associated with functional centromere/kinetochore complexes. In this study, up to four distinct CENH3 (BrCENH3) cDNAs were identified in individuals of each of three diploid species of Brassica. Comparison of the BrCENH3 cDNAs implied three related gene families: BrCENH3-A in Brassica rapa (AA), BrCENH3-B in B. nigra (BB), and BrCENH3-C in B. oleracea (CC). Each family encoded a histone fold domain and N-terminal histone tails that vary in length in all three families. The BrCENH3-B cDNAs have a deletion of two exons relative to BrCENH3-A and BrCENH3-C, consistent with the more ancient divergence of the BB genome. Chromatin immunoprecipitation and immunolabeling tests with anti-BrCENH3 antibodies indicated that both centromeric tandem repeats and the centromere-specific retrotransposons of Brassica are directly associated with BrCENH3 proteins. In three allotetraploid species, we find either co-transcription of the BrCENH3 genes of the ancestral diploid species or gene suppression of the BrCENH3 from one ancestor. Although B genome centromeres are occupied by BrCENH3-B in the ancestral species B. nigra, in allotetraploids both BrCENH3-A and BrCENH3-C proteins appear to assemble at these centromeres.

Synthesized allopolyploids are valuable materials for comparative analyses of two or more distinct genomes, such as the expression changes (activation, inactivation or differential expression) of orthologous genes following allopolyploidization. CENH3 is a centromere- specific histone H3 variant and has been regarded as a central component in kinetochore formation and centromere function. In this study, interspecific hybrids of Oryza genus (AA × CC, AA × CCDD) and their backcross progenies were produced, and the genome constitutions were identified as AC, ACC, ACD, AACD, or AA(CD) by Genomic in situ hybridization (GISH). We further cloned and sequenced the CENH3 genes from O. sativa (AA), O. officinalis (CC) and O. latifolia (CCDD). Sequencing of RT-PCR products revealed that CENH3_C2 and CENH3_D, the two CENH3 alleles from O. latifolia, showed polymorphism in several sites, while CENH3_C2 and CENH3_C1 from O. officinalis were different at only two amino acids positions. Moreover, we found that the CENH3 genes from both parents are expressed in interspecific hybrids and their progenies. Specifically, based on our cDNA sequencing data, the ratio of expression level between CENH3_A and CENH3_C1 was approximately 1 in AC and 0.5 in ACC genomes, respectively. As a result, the CENH3 expression patterns shed more light on the inter-coordination between varied centromeric DNA sequences and highly conserved kinetochore protein in synthesized allopolyploids of Oryza genus.

Knowledge about the composition and structure of centromeres is critical for understanding how centromeres perform their functional roles. Here, we report the sequences of one centromere-associated bacterial artificial chromosome clone from a Coix lacryma-jobi library. Two Ty3/gypsy-class retrotransposons, centromeric retrotransposon of C. lacryma-jobi (CRC) and peri-centromeric retrotransposon of C. lacryma-jobi, and a (peri)centromere-specific tandem repeat with a unit length of 153 bp were identified. The CRC is highly homologous to centromere-specific retrotransposons reported in grass species. An 80-bp DNA region in the 153-bp satellite repeat was found to be conserved to centromeric satellite repeats from maize, rice, and pearl millet. Fluorescence in situ hybridization showed that the three repetitive sequences were located in (peri-)centromeric regions of both C. lacryma-jobi and Coix aquatica. However, the 153-bp satellite repeat was only detected on 20 out of the 30 chromosomes in C. aquatica. Immunostaining with an antibody against rice CENH3 indicates that the 153-bp satellite repeat and CRC might be both the major components for functional centromeres, but not all the 153-bp satellite repeats or CRC sequences are associated with CENH3. The evolution of centromeric repeats of C. lacryma-jobi during the polyploidization was discussed.

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-like 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.

Little is known of the dynamics of centromeric DNA in polyploid plants. We report the sequences of two centromere-associated bacterial artificial chromosome clones from a Triticum boeoticum library. Both autonomous and non-autonomous wheat centromeric retrotransposons (CRWs) were identified, both being closely associated with the centromeres of wheat. Fiber-fluorescence in situ hybridization and chromatin immunoprecipitation analysis showed that wheat centromeric retrotransposons (CRWs) represent a dominant component of the wheat centromere and are associated with centromere function. CRW copy number showed variation among different genomes: the D genome chromosomes contained fewer copies than either the A or B genome chromosomes. The frequency of lengthy continuous CRW arrays was higher than that in either rice or maize. The dynamics of CRWs and other retrotransposons at centromeric and pericentromeric regions during diploid speciation and polyploidization of wheat and its related species are discussed.

Background Radish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish. Results We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish. Conclusions Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0480-y) contains supplementary material, which is available to authorized users.

Rice was transformed with either long DNA-segments of random genomic DNA from rice, or centromere-specific DNA sequences from either maize or rice. Despite the repetitive nature of the transgenic DNA sequences, the centromere-specific sequences were inserted largely intact and behave as simple Mendelian units. Between 4 and 5% of bombarded callus clusters were transformed when bombarded with just pCAMBIA 1305.2. Frequency of recovery dropped to 2-3% when BACs with random genomic inserts were co-bombarded with pCAMBIA, and fell to less than 1% when BACs with centromeric DNA inserts and pCAMBIA were co-bombarded. A similar effect was noted on regeneration frequency. Differences in transformation ability, regeneration and behavior of plants transgenic for BACs with random genomic DNA inserts, as compared to those with centromeric DNA inserts, suggests functional differences between these two types of DNA.

The centromere is the chromosomal site for assembly of the kinetochore where spindle fibers attach during cell division. In most multicellular eukaryotes, centromeres are composed of long tracts of satellite repeats that are recalcitrant to sequencing and fine-scale genetic mapping. Here, we report the genomic and genetic characterization of the complete centromere of rice (Oryza sativa) chromosome 3. Using a DNA fiber-fluorescence in situ hybridization approach, we demonstrated that the centromere of chromosome 3 (Cen3) contains ∼441 kb of the centromeric satellite repeat CentO. Cen3 includes an ∼1,881-kb domain associated with the centromeric histone CENH3. This CENH3-associated chromatin domain is embedded within a 3113-kb region that lacks genetic recombination. Extensive transcription was detected within the CENH3 binding domain based on comprehensive annotation of protein-coding genes coupled with empirical measurements of mRNA levels using RT-PCR and massively parallel signature sequencing. Genes

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.

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

The functional centromeres of rice ( Oryza sativa , AA genome) chromosomes contain two key DNA components: the CRR centromeric retrotransposons and a 155-bp satellite repeat, CentO. However, several wild Oryza species lack the CentO repeat. We developed a chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3. Chromatin immunoprecipitation cloning was carried out in the CentO-less species Oryza rhizomatis (CC genome) and Oryza brachyantha (FF genome). Three previously uncharacterized genome-specific satellite repeats, CentO-C1, CentO-C2, and CentO-F, were discovered in the centromeres of these two species. An 80-bp DNA region was found to be conserved in CentO-C1, CentO, and centromeric satellite repeats from maize and pearl millet, species which diverged from rice many millions of years ago. In contrast, the CentO-F repeat shows no sequence similarity to other centromeric repeats but has almost completely replaced other centromeric sequences in O. brachyantha , including the CRR-related sequences that normally constitute a significant fraction of the centromeric DNA in grass species.

The centromere of the maize (Zea mays) B chromosome contains several megabases of a B-specific repeat (ZmBs), a 156-bp satellite repeat (CentC), and centromere-specific retrotransposons (CRM elements). Here, we demonstrate that only a small fraction of the ZmBs repeats interacts with CENH3, the histone H3 variant specific to centromeres. CentC, which marks the CENH3-associated chromatin in maize A centromeres, is restricted to an approximately 700-kb domain within the larger context of the ZmBs repeats. The breakpoints of five B centromere misdivision derivatives are mapped within this domain. In addition, the fraction of this domain remaining after misdivision correlates well with the quantity of CENH3 on the centromere. Thus, the functional boundaries of the B centromere are mapped to a relatively small CentC- and CRM-rich region that is embedded within multimegabase arrays of the ZmBs repeat. Our results demonstrate that the amount of CENH3 at the B centromere can be varied, but with decreasing amounts, the function of the centromere becomes impaired.

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 approximately 300 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.

Background Coix, Sorghum and Zea are closely related plant genera in the subtribe Maydeae. Coix comprises 9–11 species with different ploidy levels (2n = 10, 20, 30, and 40). The exclusively cultivated C. lacryma-jobi L. (2n = 20) is widely used in East and Southeast Asia for food and medicinal applications. Three fertile cytotypes (2n = 10, 20, and 40) have been reported for C. aquatica Roxb. One sterile cytotype (2n = 30) closely related to C. aquatica has been recently found in Guangxi of China. This putative hybrid has been named C. aquatica HG (Hybrid Guangxi). The genome composition and the evolutionary history of C. lacryma-jobi and C. aquatica HG are largely unclear. Results About 76% of the genome of C. lacryma-jobi and 73% of the genome of C. aquatica HG are repetitive DNA sequences as shown by low coverage genome sequencing followed by similarity-based cluster analysis. In addition, long terminal repeat (LTR) retrotransposable elements are dominant repetitive sequences in these two genomes, and the proportions of many repetitive sequences in whole genome varied greatly between the two species, indicating evolutionary divergence of them. We also found that a novel 102 bp variant of centromeric satellite repeat CentX and two other satellites only appeared in C. aquatica HG. The results from FISH analysis with repeat probe cocktails and the data from chromosomes pairing in meiosis metaphase showed that C. lacryma-jobi is likely a diploidized paleotetraploid species and C. aquatica HG is possibly a recently formed hybrid. Furthermore, C. lacryma-jobi and C. aquatica HG shared more co-existing repeat families and higher sequence similarity with Sorghum than with Zea. Conclusions The composition and abundance of repetitive sequences are divergent between the genomes of C. lacryma-jobi and C. aquatica HG. The results from fine karyotyping analysis and chromosome pairing suggested diploidization of C. lacryma-jobi during evolution and C. aquatica HG is a recently formed hybrid. The genome-wide comparison of repetitive sequences indicated that the repeats in Coix were more similar to those in Sorghum than to those in Zea, which is consistent with the phylogenetic relationship reported by previous work. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1025) contains supplementary material, which is available to authorized users.

Rice (Oryza sativa L.) centromeres are composed of 155-bp satellite repeats (CentO), centromere-specific retrotransposon (CRR), and a variety of other repeats. Previous studies have shown that CentO and CRR elements are both parts of the functional centromere/kinetochore complex. In this study, a naturally occurring karyotype rearrangement involving a reciprocal translocation between chromosomes 9 and 11 in an indica rice Zhongxian 3037 has been identified. The recombinant centromere in Chr11L · 9L has two CentO tandem arrays, separated by a long array of 5S rDNAs. Chromatin immunoprecipitation and immunostaining showed that centromere-specific histone H3 (cenH3) variant was bound to the two flanking CentO arrays, but not to the 5S rDNAs residing between the CentO repeats. No obvious difference was detected in H3K4me2 and H3K9ac modification of the 5S rDNAs between the wild type and the mutant. Therefore, the translocation results in a recombinant stable chromosome with interrupted centromeric domains. A lack of cenH3 binding in 5S rDNA sequences residing within the centromeric core suggests that not all centromeric sequences confer centromere identity in rice.

UThese authors contributed equally to this work. SUMMARY The physical map of the hexaploid wheat chromosome 3B was screened using centromeric DNA probes. A 1.1-Mb region showing the highest number of positive bacterial artificial chromosome (BAC) clones was fully sequenced and annotated, revealing that 96% of the DNA consisted of transposable elements, mainly long terminal repeat (LTR) retrotransposons (88%). Estimation of the insertion time of the transposable elements revealed that CRW (also called Cereba) and Quinta are the youngest elements at the centromeres of common wheat (Triticum spp.) and its diploid ancestors, with Quinta being younger than CRW in both diploid and hexaploid wheats. 3 Chromatin immunoprecipitation experiments revealed that both CRW and Quinta families are targeted by the centromere-specific histone H3 variant CENH3. Immuno colocalization of retroelements and CENH3 antibody indicated that a higher proportion of Quinta than CRWs was associated with CENH3, although CRWs were more abundant. Long arrays of satellite repeats were also identified in the wheat centromere regions, but they lost the ability to bind with CENH3. In addition to transposons, two functional genes and one pseudogene were identified. The gene density in the centromere appeared to be between three and four times lower than the average gene density of chromosome 3B. Comparisons with related grasses also indicated a loss of microcollinearity in this region. Finally, comparison of centromeric sequences of Aegilops tauschii (DD), Triticum boeoticum (AA) and hexaploid wheat revealed that the centromeres in both the polyploids and diploids are still undergoing dynamic changes, and that the new CRWs and Quintas may have undertaken the core role in kinetochore formation.

A fluorescencein situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked toGm-6 andPi-5(t) inO. officinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72±2.62 for 24E21 and 54±5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice andO. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice andO. officinalis were in the same BAC clones. The homologous sequences ofGm-6 andPi-5(t) inO. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some homolgous between cultivated rice andO. officinalis. The identification of chromosome 4 ofO. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map inO. officinalis with rice BAC clones.

The centromere of an eukaryotic chromosome can move to a new position during evolution, which may result in a major alteration of the chromosome morphology and karyotype. This centromere repositioning phenomenon has been extensively documented in mammalian species and was implicated to play an important role in mammalian genome evolution. Here we report a centromere repositioning event in plant species. Comparative fluorescence in situ hybridization mapping using common sets of fosmid clones between two pairs of cucumber ( Cucumis sativus L.) and melon ( Cucumis melo L.) chromosomes revealed changes in centromere positions during evolution. Pachytene chromosome analysis revealed that the current centromeres of all four cucumber and melon chromosomes are associated with distinct pericentromeric heterochromatin. Interestingly, inactivation of a centromere in the original centromeric region was associated with a loss or erosion of its affixed pericentromeric heterochromatin. Thus, both centromere activation and inactivation in cucurbit species were associated with a gain/loss of a large amount of pericentromeric heterochromatin.

The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions). No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.

Background Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.). Results In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed. Conclusions Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

The copy number of 5S rDNA and centromeric sequence RCS2 was determined by extended DNA fiber based fluorescence in situ hybridization (Fiber-FISH) in rice (Oryza sativa ssp. indica cv. Guangluai No. 4) genome. In order to determine the copy number, it is necessary to know the basepair number that a given length DNA fiber contains under a microscope. Therefore, the length of two DNA fragments, in which the basepair number had been already known, was measured. The insert sequence of the tested BAC 38D17 was 136 kb and its extended DNA was 56.4 μm long, 2.41 kb/μm on average, while that of the tested BAC 44B4 was 144.5 kb in total and 55.7 μm long, 2.60 kb/μm on average under the microscope. They were very close to the theoretical value of B-DNA in the Watson-Crick DNA model, which is 2.97 kb/μm. According to the average value of basepair number per μm of the two samples mentioned above, that is, 2.51 kb/μm, it could be estimated that the copy number was about 686 for 5S rDNA and 286–1121 for the centromere sequence RCS2.