ASSOCIATION OF MISSENSE VARIANTS IN GDF9 WITH LITTER SIZE IN ENTLEBUCHER MOUNTAIN DOGS R. B. P. Torrecilha* ,† , M. Milanesi †,‡ , M. Gallana § , A.-K. Falbo § , I. M. Reichler ¶ , P. Hug**, V. Jagannathan**, B. B. Trigo †,‡ , S. C. Paulan †,‡ , D. B. Bruno †† , S. D. Garcia †† , N. F. Scaramele ‡ , F. L. Lopes ‡ , G. Dolf**, T. Leeb**, J. S€ olkner ‡‡ , J. F. Garcia* ,†,‡ , A. Pie nkowska-Schelling §, **, C. Schelling § and Y. T. Utsunomiya †,‡ *Department of Preventive Veterinary Medicine and Animal Reproduction, School of Agriculture and Veterinarian Sciences, S~ ao Paulo State University (Unesp), Via de acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, S~ ao Paulo, Brazil. † International Atomic Energy Agency Collaborating Centre on Animal Genomics and Bioinformatics, Cl ovis Pestana, 793, 16050-680, Arac ßatuba, S~ ao Paulo, Brazil. ‡ Department of Support, Production and Animal Health, School of Veterinary Medicine, S~ ao Paulo State University (Unesp), Cl ovis Pestana, 793, 16050-680, Arac ßatuba, S~ ao Paulo, Brazil. § Clinic of Reproductive Medicine, Vetsuisse-Faculty University of Zurich, Eschikon 27, Lindau 8315, Switzerland. ¶ Clinic of Reproductive Medicine, Vetsuisse-Faculty University of Zurich, Winterthurerstr. 260, Z€ urich 8057, Switzerland. **Institute of Genetics, Vetsuisse-Faculty University of Bern, Bremgartenstrasse 109A, Bern 3012, Switzerland. †† Department of Clinics, Surgery and Animal Reproduction, School of Veterinary Medicine, S~ ao Paulo State University (Unesp), Cl ovis Pestana, 793, 16050-680, Arac ßatuba, S~ ao Paulo, Brazil. ‡‡ Division of Livestook Sciences, Department of Sustainable Agriculture System, BOKU - University of Natural Resource and Live Sciences, Gregor-Mendel-Straße 33, 1180, Vienna, Austria. Summary In the past two decades, average litter size (ALS) in Entlebucher Mountain dogs decreased by approximately 0.8 puppies. We conducted a GWAS for ALS using the single-step methodology to take advantage of 1632 pedigree records, 892 phenotypes and 372 genotypes (173 662 markers) for which only 12% of the dogs had both phenotypes and genotypes available. Our analysis revealed associations towards the growth differentiation factor 9 gene (GDF9), which is known to regulate oocyte maturation. The trait heritability was estimated at 43.1%, from which approximately 15% was accountable by the GDF9 locus alone. Therefore, markers flanking GDF9 explained approximately 6.5% of the variance in ALS. Analysis of WGSs revealed two missense substitutions in GDF9, one of which (g.11:21147009G>A) affected a highly conserved nucleotide in vertebrates. The derived allele A was validated in 111 dogs and shown to be associated with decreased ALS (0.75  0.22 puppies per litter). The variant was further predicted to cause a proline to serine substitution. The affected residue was immediately followed by a six-residue deletion that is fixed in the canine species but absent in non-canids. We further confirmed that the deletion is prevalent in the Canidae family by sequencing three species of wild canids. Since canids uniquely ovulate oocytes at the prophase stage of the first meiotic division, requiring maturation in the oviduct, we conjecture that the amino acid substitution and the six-residue deletion of GDF9 may serve as a model for insights into the dynamics of oocyte maturation in canids. Keywords Canis lupus familiaris, multiple ovulation, number of offspring, single nucleotide polymorphism, single-step genomic best linear unbiased prediction Introduction Sennenhund breeds descend from dogs of the pre-Alpine region of Switzerland, which were primarily used for cattle management and the protection of farms. There are four different breeds of Sennenhund dogs: Appenzeller Moun- tain, Bernese Mountain, Greater Swiss Mountain and Entlebucher Mountain (EM) dogs. The latter is the smallest and youngest of the four breeds (NEMDA 2018). The control of inbreeding levels is a major concern in EM dogs, since the breed suffered two important historical popula- tion bottlenecks: (i) one in its formation about 90 years ago, which relied on a small number of founders; and (ii) another during World War II, when its development was put on hold. Inbreeding in the EM population has Address for correspondence Y. T. Utsunomiya, Department of Support, Production and Animal Health, School of Veterinary Medicine, S~ ao Paulo State University (Unesp), Cl  ovis Pestana, 793, 16050-680, Arac ßatuba, S~ ao Paulo, Brazil. E-mail: ytutsunomiya@gmail.com Accepted for publication 06 November 2019 doi: 10.1111/age.12882 1 © 2019 Stichting International Foundation for Animal Genetics