Comparison between indicine and taurine cattle DNA methylation reveals epigenetic variation associated to differences in morphological adaptive traits.

EPIGENETICS 2023, VOL. 18, NO. 1, 2163363 https://doi.org/10.1080/15592294.2022.2163363 RESEARCH PAPER Comparison between indicine and taurine cattle DNA methylation reveals epigenetic variation associated to differences in morphological adaptive traits E. Capraa#, B. Lazzaria#, M. Milanesib,c,d, G. P. Nogueirab, J. f. Garciab,c, Y.T. Utsunomiyab, P. Ajmone-Marsan and A. Stellaa e , a Institute of Agricultural Biology and Biotechnology, National Research Council IBBA CNR, Lodi, Italy; bSchool of Veterinary Medicine, Araçatuba, Department of Production and Animal Health, São Paulo State University (Unesp), Araçatuba, Brazil; cInternational Atomic Energy Agency, Collaborating Centre on Animal Genomics and Bioinformatics, Araçatuba, Brazil; dDepartment for Innovation in Biological, AgroFood and Forest Systems (DIBAF), University of Tuscia, Viterbo, Italy; eDepartment of Animal Science, Food and Nutrition – DIANA, and Nutrigenomics and Proteomics Research Center – PRONUTRIGEN, Università Cattolica del Sacro Cuore, Piacenza, Italy ABSTRACT Indicine and taurine subspecies present distinct morphological traits as a consequence of envir onmental adaptation and artificial selection. Although the two subspecies have been character ized and compared at genome-wide level and at specific loci, their epigenetic diversity has not yet been explored. In this work, Reduced Representation Bisulphite Sequencing (RRBS) profiling of the taurine Angus (A) and indicine Nellore (N) cattle breeds was applied to identify methylation differences between the two subspecies. Genotyping by sequencing (GBS) of the same animals was performed to detect single nucleotide polymorphisms (SNPs) at cytosines in CpG dinucleo tides and remove them from the differential methylation analysis. A total of 660,845 methylated cytosines were identified within the CpG context (CpGs) across the 10 animals sequenced (5 N and 5 A). A total of 25,765 of these were differentially methylated (DMCs). Most DMCs clustered in CpG stretches nearby genes involved in cellular and anatomical structure morphogenesis. Also, sequences flanking DMC were enriched in SNPs compared to all other CpGs, either methylated or unmethylated in the two subspecies. Our data suggest a contribution of epigenetics to the regulation and divergence of anatomical morphogenesis in the two subspecies relevant for cattle evolution and sub-species differentiation and adaptation. Introduction Modern day cattle belong either to the taurine or the indicine sub-species, which derive from inde pendent domestication events. European taurine breeds are characterized by excellent carcass and meat quality or high milk production potential, but are poorly adapted to harsh environments [1,2]. Indicine breeds are more adapted to tropical wet/dry semi-arid, arid and hot environments and to parasites, and possess distinct morphological traits such as a hump, large ears, and excess skin [3,4]. Although the two cattle subspecies have been deeply characterized for genetic differences at the genome-wide level and at specific loci, phenotypic differences between them can only partially be explained by genomic variants [5–8]. The non- ARTICLE HISTORY Received 04 October 2022 Revised 30 November 2022 Accepted 21 December 2022 KEYWORDS Methylation; bovine; evolution; adaptation; breed; cattle; epigenetic; species genetic proportion of phenotypic variation has been defined as phenotypic plasticity and can in part be attributed to epigenetic variation, standing at a cross-road between genetic and environmental variance [9,10]. This effect was largely described in asexually reproducing invertebrates and in some vertebrates [11]. Epigenetic variation was also pro posed as a mechanism triggered by animal domes tication able to shape phenotypic features of domesticated animals in very short time scales [12]. Literature data on dogs and grey wolves reported specific differences in methylation pat terns between the two species, suggesting that epi genetic mechanisms might play an important role in early steps of domestication [13]. Epigenetic variation has a higher plasticity than genetic varia tion and can cope better with environmental CONTACT P. Ajmone-Marsan Department of Animal Science, Food and Nutrition – DIANA, and Nutrigenomics and Proteomics Research Center – PRONUTRIGEN, Università Cattolica del Sacro Cuore, Piacenza, Italy # co-first author Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2022.2163363 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 E. CAPRA ET AL. fluctuations [14]. However, the contribution of epigenetic variation in explaining the missing her itability of phenotypic traits is not well under stood, because transgenerational transmission, persistence over time and stabilization within the livestock genome are still to be explored [14]. In farming, behaviour, diet, stress, and environ mental variation have a strong impact on the ani mal epigenome [15,16]. In pigs and sheep, the impact has been reported to persist along multiple generations [17,18]. Recently, we reported gen ome-wide DNA methylation changes in blood samples from indicine (Nellore) and taurine (Angus) breeds under heat stress [19]. After a stressful period, Nellore showed methylation changes in genes related to cellular defence and stress response, whereas Angus (A) response was less focused. The overall methylation profiles in Nellore (N) and A animals showed remarkable diversity between the two subspecies that was independent of the environmental challenge and presumably related to their origin, breed charac teristics, and polymorphisms at CpG islands [19]. In pigs, epigenome-wide muscle profiling has been reported to show important differences across breeds, probably as a result of long-term selection for quantitative traits, involving a very high num ber of genes [20]. While in the previous investiga tion [19], differential methylated regions (DMRs) were identified comparing individual breeds across environmental conditions, here we use Reduced Representation Bisulphite Sequencing (RRBS) data from N and A animals, to identify cytosines and regions differentially methylated between breeds and to investigate the potential role played by epigenetics in subspecies domestication. The same animals have also been fully sequenced to distinguish differential methylation signals from polymorphisms at CpG dinucleotides, to focus on epigenetic differences while getting rid of the bias generated by genetic differences existing between the two subspecies. Materials and methods Animal sampling A total of 5 N and 5 A healthy you (...truncated)