Humans have a defined set of chromosomes that are essential for our normal growth, development, and reproduction. Carrying too many (or too few) copies of these essential chromosomes is known as aneuploidy, a condition that has been extensively investigated due to its negative impact on human health. Extra, nonessential chromosomes that are derived from the essential set of chromosomes have also been associated with a broad range of serious health issues such as developmental disorders, infertility, and cancer. These supernumerary chromosomes, however, remain mostly understudied, resulting in a large gap in our understanding of their etiology and dynamics. A critical barrier to closing this knowledge gap has been the availability of a robust model system that will allow investigation into the unknown aspects of supernumerary chromosome biology. The broad objective of the proposed research is to overcome this barrier by exploiting the newly established B chromosome system in D. melanogaster to model the etiology and dynamics of supernumerary chromosomes in humans. This work will capitalize on the arsenal of molecular, cellular, and genetic tools available in D. melanogaster to investigate several unknown aspects of supernumerary chromosome biology. To understand the genetic environment that permits supernumerary chromosomes to arise from the essential chromosomes, this study will use ultra-long DNA sequencing technology and advanced bioinformatic assembly methods to construct a map of the B chromosome and reveal the sequence context of where it arose from its original chromosome. The proposed research will also reveal how supernumerary chromosomes affect the stability of the essential chromosomes by applying a combination of cytological and genetic analyses to directly and indirectly assess chromosome-chromosome interactions. Finally, to investigate the cellular machinery required to prevent the transmission of supernumerary chromosomes, classic genetic approaches will be combined with a newly developed assay for imaging chromosome dynamics during female meiosis. Overall, the newly established B chromosome model system is uniquely positioned to deliver significant advances in our understanding of supernumerary chromosome biology that will lead to advances in their prevention and treatment in humans.