Supplementary methods (libraries preparation and analysis of RAD-Sequencing and real-time PCR protocol) from Evidence for a genetic sex determination in Cnidaria, the Mediterranean red coral (<i>Corallium rubrum</i>) M. Pratlong A. Haguenauer S. Chenesseau K. Brener G. Mitta E. Toulza M. Bonabaud S. Rialle D. Aurelle P. Pontarotti 10.6084/m9.figshare.4676104.v1 https://rs.figshare.com/articles/journal_contribution/Supplementary_methods_libraries_preparation_and_analysis_of_RAD-Sequencing_and_real-time_PCR_protocol_from_Evidence_for_a_genetic_sex_determination_in_Cnidaria_the_Mediterranean_red_coral_i_Corallium_rubrum_i_/4676104 Sexual reproduction is widespread among eukaryotes, and the sex-determining processes vary greatly among species. While genetic sex determination (GSD) has been intensively described in bilaterian species, no example has yet been recorded among non-bilaterians. However, the quasi-ubiquitous repartition of GSD among multicellular species suggests that similar evolutionary forces can promote this system, and that these forces could occur also in non-bilaterians. Studying sex determination across the range of Metazoan diversity is indeed important to understand better the evolution of this mechanism and its lability. We tested the existence of sex-linked genes in the gonochoric red coral (<i>Corallium rubrum</i>, Cnidaria) using Restriction site-Associated DNA sequencing (RAD-Seq). We analysed 27 461 single nucleotide polymorphisms (SNPs) in 354 individuals from 12 populations including 53 that were morphologically sexed. We found a strong association between the allele frequencies of 472 SNPs and the sex of individuals, suggesting an XX/XY sex-determination system. This result was confirmed by the identification of 435 male-specific loci. An independent test confirmed that the amplification of these loci enabled us to identify males with absolute certainty. This is the first demonstration of a GSD system among non-bilaterian species and a new example of its convergence in multicellular eukaryotes. 2017-02-21 15:01:32 genetic sex determination Corallium rubrum RAD-sequencing Cnidaria