Fit in the Discoveries 1 Gene-level, but not chromosome-wide, divergence between a very young 2 house fly proto-Y chromosome and its homologous proto-X chromosome
14 373 III M alleles are found in the reference genome, suggesting that these synonymous variants are not 374 fixed differences between the proto-Y and proto-X. Md-HEATR2 is expressed higher in III M 375 genotypic males than in sex-reversed males (Figure 4A). In the III M genotypic males, the III M 376 (Y-linked) alleles are expressed higher than the X-linked alleles, indicating that the Y-linked 377 alleles are associated with the up-regulation of the gene in III M genotypic males relative to sex-378 reversed males (Figure 4A). The copy of Md-HEATR2 on the III M proto-Y chromosome is 379 therefore up-regulated relative to the proto-X copy, consistent with higher expression of Md-380 HEATR2 in genotypic males.
381 382 Using our Nanopore sequencing reads mapped to the reference genome, we examined 1,273 base 383 pairs upstream of Md-HEATR2 to identify diagnostic sites that could be responsible for 384 regulating the expression differences between the proto-X and proto-Y alleles. We chose that 385 distance because it includes the first variable site we could identify on the scaffold containing 386 Md-HEATR2 in our Nanopore data (i.e., including a larger region would not provide any 387 additional information). We found twelve variable sites with different alleles (SNPs and small 388 indels) between genotypic (III M /III) and sex-reversed (III/III) males (Figure 4B). We next 389 examined whether these sites are located within a potential transcription factor (TF) binding 390 region. We found five TF binding regions predicted upstream of Md-HEATR2 using the 391 'Tfsitescan' tool in the 'object-oriented Transcription Factors Database' (Ghosh 2000). However, 392 none of the twelve variable sites are found within any predicted TF binding regions (Figure 4C).
393 It is possible that the cis-regulatory sequences responsible for differential expression are located 394 outside of the region we were able to investigate, which would be consistent with our failure to 395 identify fixed differences in the exons between the proto-Y and proto-X copies of Md-HEATR2.
396 A long distance would reduce the genetic linkage between the cis-regulatory region and 397 transcribed sequence, allowing for the X-Y differentiation of the regulatory region without 398 differentiation of the transcribed gene. Further work is needed to determine how the differential 399 expression of the proto-X and proto-Y copies of Md-HEATR2 is regulated. 400 401 We hypothesize that the upregulation of the proto-Y copy of Md-HEATR2 is the result of 402 selection for higher expression in males. We think that the cis-regulatory region is under 403 selection, rather than the protein-coding sequence, because we fail to find any protein coding