Genotypic Sex Determination Protocol

As phenotypic sex cannot be assessed prior to stage G43, we genotyped all sampled individuals to assess their genotypic sex using three markers with Y-diagnostic alleles (namely Dmrt1-1, Dmrt1-2, Dmrt1-5) (primer sequences from [52 (link)]; Supplementary Table S1). After an overnight treatment at 56 °C with tissue lysis buffer ATL and 20% proteinase K (Qiagen), PCR reactions were performed in a total volume of 10 µL, including 3 µL of extracted DNA, 2.22 µL of Milli-Q water, 3 µL of Qiagen Multiplex Master Mix, 0.14 to 0.3 µL of labeled forward primer, and 0.14 to 0.3 µL of unlabeled reverse primer (in total 1.78 µL of primer mix). PCRs were conducted on Perkin Elmer 2700 machines using the following thermal profile: 15 min of Hot Start Taq polymerase activation at 95 °C, followed by 35 cycles including denaturation at 94 °C for 30 s, annealing at 55 °C for 1.5 min, and elongation at 72 °C for 1 min, ending the PCR with a final elongation of 30 min at 60 °C. PCR products were then analyzed on an automated ABI Prism 3100 sequencer (Applied Biosystems, Foster City, CA, USA) and alleles were scored using GENEMAPPER v. 4.0 (Applied Biosystems).