Abstract Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes in a classical model system, the silkworm Bombyx mori . Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic chromosome spreads. We show that meiotic pairing is robust in both males and female meiosis. Additionally, we show that meiotic bivalent formation in B. mori males is highly similar to bivalent formation in C. elegans , with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole and microtubule recruitment independent of the centromere-specifying factor CENP-A. Author’s Summary Meiosis is the specialized cell division occurring exclusively in ovaries and testes to produce egg and sperm cells, respectively. The accurate distribution of chromosomes (the genetic material) during this process is essential to prevent infertility/sterility and developmental disorders in offspring. As researchers are specifically unable to study the mechanisms regulating meiosis in depth in humans, identifying broadly conserved aspects of meiotic chromosome segregation is essential for making accurate inferences about human biology. Here, we use a sophisticated chromosome painting approach called Oligopaints to visualize and study chromosomes during meiosis in the silkworm, Bombyx mori . We illustrate that Oligopaints are highly specific in B. mori and demonstrate how Oligopaints can be used to study the dynamics of meiotic chromosomes in diverse species.

Oligopaint DNA FISH as a tool for investigating meiotic chromosome dynamics in the silkworm,Bombyx mori