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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 (Corallium rubrum)

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posted on 2017-02-21, 15:01 authored by M. Pratlong, A. Haguenauer, S. Chenesseau, K. Brener, G. Mitta, E. Toulza, M. Bonabaud, S. Rialle, D. Aurelle, P. Pontarotti
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 (Corallium rubrum, 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.

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