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 ;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.

We report a neocentromere event on maize chromosome 3 that occurred due to chromosome breakage. The neocentromere lies on a fragment of the short arm that lacks the primary centromere DNA elements, CentC and CRM. It is transmitted in the genomic background of oat via a new centromere (and kinetochore), as shown by immunolocalization of the oat CENH3 protein. Despite normal transmission of the maize fragment in most progeny, neocentromeres appear to vary in size within the same tissue, as shown by fluorescent measurements. A secondary truncation in one line lowered mitotic transmission to 3% and precipitously reduced the size of the chromosome. The results support the view that neocentromere formation is generally associated with major genomic disturbances such as wide species crosses or deletion of an existing centromere. The data further suggest that new centromeres may undergo a period of instability that is corrected over a period of several generations.

The meiotic drive system on maize abnormal chromosome 10 (Ab10) is contained within a terminal domain of chromatin that extends the long arm of Ab10 to $1.3 times the size of normal chromosome 10L. Ab10 type I (Ab10-I) does not recombine with normal chromosome 10 (N10) over an $32-cM terminal region of the long arm. Comparative RFLP mapping demonstrates that multiple independent rearrangements are responsible for the current organization of Ab10-I, including a set of nested inversions and at least one long supernumerary segment at the end of the chromosome. Four major meiotic drive functions, i.e., the recombination effect, smd3, 180-bp neocentromere activity, and the distal tip function, all map to the distal supernumerary segment. TR-1-mediated neocentromere activity (the fifth known drive function) is nonessential in the type II variant of Ab10 and maps to a central region that may include a second supernumerary insertion. Both neocentromere activity and the recombination effect behave as dominant gain-of-function mutations, consistent with the view that meiotic drive involves new or alien gene products. These and other data suggest that the Ab10 meiotic drive system was initially acquired from a related species and that a complex haplotype evolved around it.