bioRxiv preprint doi: https://doi.org/10.1101/379925; this version posted August 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Whole blood transcriptome analysis reveals footprints of cattle adaptation to sub-arctic 2 conditions 3 Kisun Pokharel1, Melak Weldenegodguad1,2, Ruslan Popov3, Mervi Honkatukia1,4, Hanna Huuki1, 4 Heli Lindeberg5, Jaana Peippo1, Tiina Reilas1, Stepan Zarovnyaev6, and Juha Kantanen1* 5 1Production Systems, Natural Resources Institute Finland (Luke), Myllytie 1, FI-31600, Jokioinen, 6 Finland 7 2Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 8 1627, FI-70311 Kuopio, Finland 9 3Yakutian Research Institute of Agriculture (FGBNU Yakutskij NIISH), 67001 Yakutsk, ul. 10 Bestyzhevo-Marlinskogo 23/1, Russia, The Sakha Republic (Yakutia) 11 4The Nordic Genetic Resources Center (Nordgen), P.O. Box 115, NO-1431 Ås, Norway 12 5Production Systems, Natural Resources Institute Finland (Luke), Halolantie 31A, FI-71750, 13 Maaninka, Finland 14 6GBU Saha Agroplem, 67700 Yakutsk, ul. Ordzhonkidze 20/204, Russia, The Sakha Republic 15 (Yakutia) 16 *Corresponding author 17 Juha Kantanen 18 Phone: +358 (0)29 5326210 19 Email: [email protected] 20 21 Summary 22 Indigenous cattle breeds in northern Eurasia have adapted to harsh climate conditions. The local 23 breeds are important genetic resources with cultural and historical heritages, and therefore, their 24 preservation and genetic characterization are important. In this study, we aim to identify genes and 25 biological processes that are important for their adaptation to the cold and harsh conditions. For this 26 purpose, we profiled the whole-blood transcriptome of two native breeds and one commercial breed 27 using high-throughput RNA sequencing. More than 15,000 genes were identified, of which 2, 89 and 28 162 genes were significantly upregulated exclusively in Northern Finncattle, Yakutian cattle and 29 Holstein cattle, respectively. The functional classification of these significantly differentially bioRxiv preprint doi: https://doi.org/10.1101/379925; this version posted August 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 30 expressed genes identified several biological processes and pathways related to signalling 31 mechanisms, cell differentiation, and host-pathogen interactions that, in general, point towards 32 immunity and disease resistance mechanisms. The gene expression pattern observed in Northern 33 Finncattle was more similar to that of Yakutian cattle, despite sharing similar living conditions as the 34 Holstein cattle included in our study. In conclusion, our study identified unique biological processes 35 in these breeds that help them to adapt and survive in sub-arctic environments. 36 Keywords: adaptation, gene expression, Yakutian cattle, Northern Finncattle, Holstein cattle, blood 37 Running title: Genomics of cattle adaptation to sub-arctic conditions bioRxiv preprint doi: https://doi.org/10.1101/379925; this version posted August 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 38 Introduction 39 As a result of natural and human-led selection, domestic animals have been able to adapt, survive, be 40 productive and reproduce in challenging environments (Mirkena et al., 2010). Animals in the northern 41 hemisphere (Lapland, northern Russia and Siberia) are used for food production and other socio- 42 cultural needs and, thus, have been the basis of human life in the North. These animals may have 43 different biological capacities to adapt to extremes in temperature, daylight and feed availability. 44 However, there has been growing interest towards improving local breeds by introducing genetic 45 material from superior breeds, with a preference towards commercial breeds. Intensive breeding and 46 the replacement of native breeds with commercial breeds may appear to be advantageous at first, but 47 these practices will have long-lasting consequences. In addition to irreversibly losing the unique 48 genetic resources of native breeds, we will also be losing the cultural and historical heritage 49 associated with those local breeds. The signatures of adaptation, as well as the history of formation, 50 that are encoded in the native breeds may also fade away (Gaouar et al., 2015). Therefore, the local 51 breeds must be genetically characterized and preserved. 52 Here, we have studied two native cattle breeds (Northern Finncattle and Yakutian cattle) and one 53 international breed (Holstein cattle). All of these breeds are used for milk and meat production. 54 Previous studies using genetic markers have indicated the genetic distinctiveness among these breeds 55 (Li et al., 2007; Li and Kantanen, 2010). Among the three breeds, Holstein cattle have high economic 56 importance, due to high productivity, and have the shortest adaptation history. Holstein cattle are the 57 most popular dairy breed globally, with an intensive selection programme and high milk production; 58 although they originated in a temperate climate, they have adapted to different parts of the world and 59 can survive in varying climatic conditions. Northern Finncattle and Yakutian cattle have economic, 60 social and cultural values (Kantanen et al., 2015). The Northern Finncattle breed is native to Northern 61 Finland and Finnish Lapland. The breed nearly became extinct during the1970s but is currently 62 maintained (current census is 850 cows) through active in vivo and in vitro conservation activity. The 63 Yakutian cattle are characterized by being purebred aboriginal native cattle. Adult Yakutian cows 64 (current population is approx. 1,000 animals) typically weigh 350-400 kg, and their height is 111 cm, bioRxiv preprint doi: https://doi.org/10.1101/379925; this version posted August 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 65 on average. The animals are well adapted to harsh Siberian conditions, where the temperature falls 66 below -50°C in long winters (Kantanen et al., 2009). 67 High-throughput RNA sequencing (RNA-Seq) has been proven to be an efficient method for studying 68 the regulation of gene expression (Wang et al., 2009). In recent years, RNA-Seq has been applied to 69 domestic animals, with a major focus on productivity traits (Bai et al., 2016; Li et al., 2016; Pokharel 70 et al., 2018; Silva-Vignato et al., 2017). A number of transcriptome studies have been conducted in 71 different Holstein breeds (Bai et al., 2016; Li et al., 2016; Sandri et al., 2015), but to date, there are no 72 reports on gene expression studies in either Yakutian cattle or Northern Finncattle. In this study, we 73 have applied RNA-Seq technology and profiled the whole blood transcriptome of the three 74 aforementioned breeds to characterize their genetic differences. 75 Materials and methods 76 Sample collection 77 Animal handling procedures and sample collection were conducted in accordance with legal 78 regulations that were approved by the Russian authorization board (FS/UVN-03/163733/07.04.2016) 79 and the Animal Experiment Board in Finland (ESAVI/7034/04.10.07.2015). A 2.5-ml sample of 80 blood from each of three Yakutian cattle, three Northern Finncattle and three Holstein cattle cows was 81 collected into a PAXgene® Blood RNA IVD tube (Ref. PreAnalytiX®, Hombrechtikon, Switzerland) 82 during the winter period of 2016 and 2017 and stored at -18°C. The Finnish samples were taken in a 83 slaughterhouse at exsanguination, and the Yakutian samples were collected by jugular venipuncture in 84 the northern Yakutian village Sakkyryr (YC6) and central Yakutia Magaras (YC10 and YC11). All of 85 the animals included in this study were 4- to 8-year-old females, except for one 14-year-old Holstein 86 cow (HC3). 87 RNA extraction 88 The RNA was extracted with the PAXgene® Blood RNA kit (Ref. 762174, Ref. PreAnalytiX®, 89 Hombrechtikon, Switzerland), according to the kit manual, with minor adjustments to the protocol: bioRxiv preprint doi: https://doi.org/10.1101/379925; this version posted August 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 90 the samples were thawed at room temperature overnight, the initial centrifugation time at 5,000 × g 91 was increased to 15 minutes, the initial pellet was resuspended into double the amount of BR1 buffer 92 and divided into two separate reactions, the proteinase K incubation time was extended to 1 hour, and 93 the columns were incubated at room temperature in elution buffer for 10 minutes prior to elution. The 94 concentration and quality of the RNA was measured with a spectrophotometer (NanoDrop ND-1000, 95 Thermo Fisher Scientific, Delaware, USA), and the integrity of the RNA was measured with an 96 Agilent Bioanalyzer 2100 (Agilent, Waldbronn, Germany) using the Agilent 6000 RNA Nano kit 97 (Ref. 5067-1511, Agilent, Waldbronn, Germany). All samples selected for sequencing had an RNA 98 Integrity Number (RIN) value of at least 7. 99 Library preparation and sequencing 100 Library preparation and sequencing tasks were outsourced to the Finnish Functional Genomics Center 101 (FFGC) in Turku, Finland.