Functional analysis of the Arabidopsis centromere by T-DNA insertion-induced centromere breakage

Two minichromosomes (alpha and delta) in addition to two other aberrant chromosomes (beta and gamma) were found in a transgenic Arabidopsis plant produced by an in planta vacuum infiltration technique. The minichromosomes were successfully separated by successive crossing and selfing and added to wild-type Columbia (Col-0) as a supernumerary chromosome. FISH indicated that both of the two minichromosomes originated from the short arm of chromosome 2. The mini alpha chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini delta lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini delta comprised a ring chromosome carrying two copies of the region from the 180-bp repeat cluster to BAC-F3C11. Both of the 180-bp clusters (each approximately 500 kb in length) were thought to possess normal centromere functions because the centromere-specific histone H3 variant (HTR12) was detected on both clusters. Notwithstanding this dicentric and ring form, mini delta was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome beta also comprised a dicentric-like structure, with one of the two 180-bp repeat sites derived from chromosome 1 and the other from chromosome 2. However, the latter was quite small and failed to bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp array of the chromosome 2 centromere, from the edge of the 180-bp array on the short-arm side, is sufficient to form a functional domain.