Establishment and inheritance of minichromosomes from Arabidopsis haploid induction

Minichromosomes are small, sometimes circular, rearranged chromosomes consisting of one centromere and short chromosomal arms formed by treatments that break DNA, including plant transformation. Minichromosomes have the potential to serve as vectors to quickly move valuable genes across a wide range of germplasm, including into adapted crop varieties. To realize this potential, minichromosomes must be reliably generated, easily manipulated, and stably inherited. Here we show a reliable method for minichromosome formation in haploids resulting from CENH3-mediated genome elimination, a process that generates genome instability and karyotypic novelty specifically on one parental genome. First, we identified 2 out of 260 haploids, each containing a single-copy minichromosome originating from centromeric regions of chromosomes 1 and 3, respectively. The chromosome 1 minichromosome we characterized did not pair at meiosis but displayed consistent transmission over nine selfing generations. Next, we demonstrated that CENH3-based haploid induction can produce minichromosomes in a targeted manner. Haploid inducers carrying a selectable pericentromeric marker were used to isolate additional chromosome-specific minichromosomes, which occurred in 3 out of 163 haploids. Our findings document the formation of heritable, rearranged chromosomes, and we provide a method for convenient minichromosome production.