Prdm9, a Major Determinant of Meiotic Recombination Hotspots, Is Not Functional in Dogs and Their Wild Relatives, Wolves and Coyotes Violeta Mun ˜ oz-Fuentes 1,2 *, Anna Di Rienzo 3 , Carles Vila ` 1 1 Estacio ´ n Biolo ´gia de Don ˜ ana EBD-CSIC, Sevilla, Spain, 2 Department of Population and Conservation Biology, Evolutionary Biology Center, Uppsala University, Uppsala, Sweden, 3 Department of Human Genetics, University of Chicago, Chicago, Illinois, United States of America Abstract Meiotic recombination is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproducing organisms. In humans, 80% of crossovers are estimated to occur at specific areas of the genome called recombination hotspots. Recently, a protein called PRDM9 was identified as a major player in determining the location of genome-wide meiotic recombination hotspots in humans and mice. The origin of this protein seems to be ancient in evolutionary time, as reflected by its fairly conserved structure in lineages that diverged over 700 million years ago. Despite its important role, there are many animal groups in which Prdm9 is absent (e.g. birds, reptiles, amphibians, diptera) and it has been suggested to have disruptive mutations and thus to be a pseudogene in dogs. Because of the dog’s history through domestication and artificial selection, we wanted to confirm the presence of a disrupted Prdm9 gene in dogs and determine whether this was exclusive of this species or whether it also occurred in its wild ancestor, the wolf, and in a close relative, the coyote. We sequenced the region in the dog genome that aligned to the last exon of the human Prdm9, containing the entire zinc finger domain, in 4 dogs, 17 wolves and 2 coyotes. Our results show that the three canid species possess mutations that likely make this gene non functional. Because these mutations are shared across the three species, they must have appeared prior to the split of the wolf and the coyote, millions of years ago, and are not related to domestication. In addition, our results suggest that in these three canid species recombination does not occur at hotspots or hotspot location is controlled through a mechanism yet to be determined. Citation: Mun ˜ oz-Fuentes V, Di Rienzo A, Vila ` C (2011) Prdm9, a Major Determinant of Meiotic Recombination Hotspots, Is Not Functional in Dogs and Their Wild Relatives, Wolves and Coyotes. PLoS ONE 6(11): e25498. doi:10.1371/journal.pone.0025498 Editor: Dean S. Dawson, Oklahoma Medical Research Foundation, United States of America Received June 16, 2011; Accepted September 6, 2011; Published November 10, 2011 Copyright: ß 2011 Mun ˜ oz-Fuentes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by a grant from the ‘‘Programa de Captacio ´ n del Conocimiento para Andalucı ´a’’ (Spain) and the National Institutes of Health (NIH) grant GM083098 (USA). VMF was supported by a European Union Marie Curie fellowship and a Spanish National Research Council (CSIC) JAE postdoctoral position. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: violeta.munoz@ebd.csic.es Introduction Meiotic recombination has been the focus of much attention because it is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproduc- ing organisms, and it may profoundly affect population genetic diversity by unlinking genes located on the same chromosome (e.g. [1,2]; and references therein). In humans, 80% of crossovers are estimated to take place in 10%–20% of the genome sequence, which contain the so-called recombination hotspots [3]. The location of these hotspots was found not to be conserved across closely related species, such as human and chimpanzee [4–6]. The increasing availability of bioinformatic and genomic tools to study recombination have contributed to the recent explosion of literature on this topic in order to understand the fundamentals of how this process takes place and its consequences. A protein called PRDM9 (also known as Meisetz) has been pinpointed as playing a role in the determination of recombination hotspots and its study has recently attracted much interest. However, many questions remain unanswered about its molecular mode of action. PRDM9 was found to be expressed in germ-line cells during meiosis in mice [7] and it was later shown to play an essential role in meiosis and speciation in a number of metazoan species [8]. Most recently, evidence has been provided that it is a determinant of sequence-specific meiotic recombination hotspots in humans and mice [9–12]. The PRDM9 protein in human and mice has three functional domains: (1) an N-terminal KRAB domain typically associated with zinc finger proteins and involved in protein-protein interactions and transcriptional repression; (2) a central SET domain with histone methyl transferase activity (thus capable of trimethylating H3K4 and consequently altering chromatin configuration); and (3) multiple C 2 H 2 zinc finger (ZF) domains in tandem near the C-terminal part of the protein [13– 16] (Fig. 1). The ZF array selectively binds to specific DNA sequences, and amino acid substitutions in the ZFs as well as polymorphism in their number affects the DNA sequences that the protein recognizes [8,9,17]. PRDM9 is a rapidly evolving protein due to the instability derived from the minisatellite structure of the ZF array, thus conferring a capacity for different alleles to quickly emerge, which will bind to a variety of DNA sequences. Multiple studies have suggested that this gene has undergone strong positive selection [8,11,17] and its rapid evolution implies changes in the DNA sequence patterns that different PRDM9 alleles may recognize, with the potential to affect hotspot location PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e25498