To maintain proper ploidy, haploid sex cells must undergo two subsequent meiotic divisions. During meiosis I, homologs pair and remain conjoined until segregation at anaphase.Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex (SC) formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the heterochromatic rDNA while euchromatin is required to pair and segregate autosomal homologies, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from conjunction. Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp (1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescent in situ hybridization (FISH) to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation.Segregation was dependent on the conjunction protein Stromalin in Meiosis while the autosomalspecific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.