Maize centromeres: organization and functional adaptation in the genetic background of oat.

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.

Jin W ，Melo JR ，Nagaki K ，Talbert PB ，Henikoff S ，Dawe RK ，Jiang J ... -  《-》

Centromeric retroelements and satellites interact with maize kinetochore protein CENH3.

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.

Zhong CX ，Marshall JB ，Topp C ，Mroczek R ，Kato A ，Nagaki K ，Birchler JA ，Jiang J ，Dawe RK ... -  《-》

Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic Loci shaped primarily by retrotransposons.

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.

Wolfgruber TK ，Sharma A ，Schneider KL ，Albert PS ，Koo DH ，Shi J ，Gao Z ，Han F ，Lee H ，Xu R ，Allison J ，Birchler JA ，Jiang J ，Dawe RK ，Presting GG ... -  《PLoS Genetics》

Sequences associated with A chromosome centromeres are present throughout the maize B chromosome.

Maize chromosome spreads containing the supernumerary B chromosome were hybridized with probes from various repetitive elements including CentC, CRM, and CentA, which have been localized to centromeric regions on the A chromosomes. Repetitive elements that are enriched or found exclusively near the centromeres of A chromosomes hybridized to many sites distinct from the centromere on the B chromosome. To examine whether these elements recruit kinetochore proteins at locations other than the canonical B centromere, cells were labeled with antibodies against CENH3, a key kinetochore protein. No labeling was detected outside the normal centromere and no evidence of B chromosome holocentromeric activity was observed. This finding suggests that, as in other higher eukaryotes, DNA sequence alone is insufficient to dictate kinetochore location in plants. Additionally, examination of the B centromere region in pachytene chromosomes revealed that the B-specific element ZmBs hybridizes to a much larger region than the site of hybridization of CentC, CRM, and CentA and the labeling by anti-CENH3 antibodies.

Lamb JC ，Kato A ，Birchler JA 《CHROMOSOMA》

[Detection of maize centromeric repeats in the relatives of maize using fluorescence in situ hybridization].

In order to analyze the conservation of maize centromeric satellite DNA (CentC) and centromeric retrotransposon (CRM) in the subspecies and relatives of Zea mays, dual fluorescence in situ hybridization (FISH) was used to detect the existence and distribution of the above two repetitive sequences in Zea mays ssp. mexicana, Z. diploperennis, Z. perennis, Tripsacum dactyloides, Coix lacryma-jobi, and Sorghum bicolor. In Z. mays ssp. mexicana, Z. diploperennis, and Z. perennis, both CentC and CRM probes produced strong or relatively strong signals in the centromeric regions of all chromosomes. There was an obvious variation in the intensity of hybridization signals on different chromosomes, indicating that different centromeres have different amounts of CentC and CRM sequences. In some centromeres, the intensity of CentC signals differed from that of CRM signals and was free from overlapping. In T. dactyloides, only weak CentC and CRM signals were detected in the centromeric regions of most chromosomes, while in C. lacryma-jobi and S. bicolor only relatively strong or strong CRM signals primarily located in the centromeric regions were detected. This result indicates that CentC is highly conserved among the subspecies of Z. mays and the species of Zea, and has high conservation in Tripsacum, a genus that is most closely related to Zea, and CRM is conserved among the species of grass family either closely or distantly related to Zea.

She CW ，Jiang XH ，Song YC ，Liu W ... -  《-》