Structural variants in Mongolian originated ruminant: role in adaptation of extreme-environment

15 Background: Mongolian cattle (MC) is one of an ancient livestock breeds with good economic traits such as adaptation to Mongolian Plateau 16 extreme low temperature in winter, resistant to pathogenic organisms infection and high quality meat. To reveal the molecular mechanism 17 underlying these, the whole genome sequencing and comparative transcriptome sequencing of MC were performed. 18 Results ： By genome sequencing and structure variation analysis, 8 genes related to pathogenic organisms infection, including 4 members of 19 bata-defensins gene family ( LAP, DEFB1, DEFB2 , and DEFB5 ), 2 members of interferon (IFN) gene family ( IFNW1 and IFNT2 ) and 2 genes 20 coding for BoLA proteins(Mongolian_cattle_21532 and Mongolian_cattle_19448) were found in MC genome inversion region. By 21 transcriptome-sequencing, it was elucidated that 8 genes (F ATP, FABP, PEPCK, SCP-X, ADIPO, FABP1, SCD-1, APO ) related to PPAR α 22 pathway, 23 genes involved in oxidative phosphorylation and 10 P450 genes ( CYP7B1, CYP4V2, CYP3A5, CYP11A1, CYP2C18, CYP2B6, 23 CYP7A1, CYP2R1, CYP2E1, CYP27B1 ) were significantly up-regulated in winter MC transcriptomes, comparative to summer MC 24 transcriptomes. 25 Conclusions: Here, we characterized 41 genes implicated in fatty acid metabolism were up-regulated in winter MC transcriptomes. These genes 26 probably account for the MC adaptation to extreme low temperature. At the same time, 8 genes in MC genome inversion region were discovered. 27 And these genes gave clue to the MC resistant to pathogenic organisms infection. In sum, our revealed genes are of important for us to 28 understand the molecular mechanisms of MC to adapt to their environments and valuable for cattle breeding.


Background
Environmental stress has been a major driving force in the evolution of living organisms (Parsons 2005). Environmental stress may affect 34 species abundance and boundaries, hence affecting biodiversity at all levels, of genes, genomes, individuals, population, species, communities 35 and biota (Nevo 1998). 36 The Mongolian Plateau (MP) is a highland of the Eurasian continent lying between 87°40′-122°15′N and 37°46′-53°08′E. The varieties of 37 climate and geographical condition in the MP endowed this ancient plateau with high biodiversity. Nevertheless, the harsh environmental 38 condition of this plateau is a big challenge for all organisms lived here. And the animals live here provide optimal models for investigation of 39 how psychrophiles adaptation to its' extreme cold. Polar bears are typical mammals uniquely adapted to life in the High Arctic. Population fox is another mammal able to survive in Arctic harsh conditions. Several evolutionary adaptations such as insulating and camouflage colored 44 fur, a compact body, and reduced metabolism during starvation or extremely cold weather enable Arctic foxes such capacity (Audet et al. 2002). 45 Transcriptome sequencing of Arctic foxes and their close relative, red foxes, revealed that several genes involved in various metabolic and 46 molecular processes such as energy metabolism, cardiac gene regulation, apoptosis and blood coagulation were under positive selection in both 47 species, whereas four of genes are under positive selection only in Arctic foxes transcriptome, two of which are fat metabolism genes (Kumar et 48 al. 2015). In addition to extreme cold, hypoxia and drought are the other two stresses may affect animals living in MP. The Qinghai-Tibetan 49 Plateau is known for its extreme environment with low atmospheric oxygen pressure, cold climate, and limited resources (Wu et al. 2001). And and Tibetan mastiff (Gou et al. 2014) gave the insights of these extreme high altitude mammals how to adapt to Tibetan hypoxic and extreme 52 cold environments. Drought is another stress with which animals living in MP must be confronted. A few works were reported of mammals lived 53 in desert environments how to adapt to drought. Genomes of sheep in contrasting environments were sequenced and further comparative 54 analyses were preformed. It was revealed that to adapt to desert environments, 11 genes which localized in three pathways, the arachidonic acid 55 metabolism pathway (ANXA6, GPX3, GPX7, and PTGS2), the renin-angiotensin system pathway (CPA3, CPVL, and ECE1) and the oxytocin 56 signaling pathway (CALM2, CACNA2D1, KCNJ5, and COX2) were positively selected in the Taklimakan desert group sheep. All of these 57 positively selective genes were functionally related to regulating water retention and reabsorption in renal cells and blood vessels in the kidney 58 (Yang et al. 2016). Camelus are uniquely evolved to fit desert harsh environments. Genome sequencing and comparative transcriptome 59 sequencing revealed Na + /K + -ATPase, the epithelial Na + channel (ENaC), aquaporin family genes (AQP1, AQP2 and AQP3) were involved in 60 water and salt reabsorption which are evolved for camel adaptation to desert extreme drought environments (Wu et al 2014   were carefully collected and quickly put into liquid nitrogen. In lab, the total RNA was extracted from above tissues according to the instrument 86 of RNA extraction kits (Qiagen). Transcriptome sequencing was performed on IlluminaHiseq 2000 system.  Table 1 and2). Using 17-mer analyzing, the size of genome was 95 estimated to be 3.2Gb (Supplementary Table 3, Supplementary Fig. 1). The total size of the assembled MC was ~ 2.68Gb which close to the 96 2.66Gb and 2.65Gb of sequences of yak and cattle genome (UMD3.1), respectively, with scaffold N50 of 1.58Mb and contig N50 of 27.16kb  and horse also revealed they diverged about 3 Mya (Fig. 1b). We found population size changes of Mongolia and their domestic ruminants are similar along the history. Interestingly, all of them decreased to the low about ~0.1 Mya which is fitly overlap the peak of atmospheric surface 108 air temperature distribution, suggesting the substantial population decreasing maybe resulted from the temperature sharply increasing (Fig. 1b).

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We also totally identified 749 inversion variants (IVs) in MC-cattle based on the whole genome alignment results. This is challenging 123 because these inverted regions contain many genes, thus we mainly do enrichment analysis for all the genes in the vicinity of breakpoints of  Mo_sheep_14980 of MS and Mongolian_cattle_14394 of MC are LOC781948, which is interferon Omega-1 (Type I interferon). Studies showed 134 IFNT2 had antiviral activity in different cell lines and antiluteolytic effect in cyclic ewes (Boue et al. 2000). In general, these two IFNs mainly 135 perform antiviral activity to strengthen the immune system. 136 We also found two genes encoding major histocompatibitly complex (MHC) class II protein complex (Mongolian_cattle_21532 and    Fig. 6-9). in Mongolian cattle skin in winter ( Fig. 4a and 4b). Based on the data, we speculated that the synthesis and metabolism of fatty acids in 160 Mongolian cattle skin were greater than normal buffalo, and between Mongolian cattle, the signaling was also more strong in winter, indicating 161 its connection to the cold adaption of Mongolian cattle. We also found oxidative phosphorylation pathway was enriched by DEGs. Although in S-W group, no DEGs were enriched in this pathway, 166 we did found 23 DEGs in B-S and 20 B-W enriched in this pathway (Fig. 5a). And most of these DEGs were up-regulated expressed in S and W 167 Mongolian cattle, respectively, suggesting a stronger oxidative phosphorylation process in Mongolian cattle than in other cattle breeds.

PI3K-Akt signaling pathway
We also noticed the downstream part of the PI3K-Akt signaling pathway (Fig. 5B). PI3K-Akt transduction pathway is directly related to cellular 173 quiescence, proliferation, cancer, and longevity. And some studies pointed out that the inhibition of PI3K pathway could indirectly decreases the which may indicate in winter, the energy production was more prosperous.  178 Besides, we also found a totally differentially expressed pathway called complement and coagulation cascades pathway between the skin tissues 179 and muscle tissues, which is connected to the immunocompetence of the species. On skin vs skin group, there was no DEGs enriched in this 180 pathway between B-S group, while 23 and 13 DEGs in B-W and S-W group were significantly enriched, respectively, and all of these DEGs 181 were up-regulated in W state Mongolian cattle ( Fig.6A and 6B). On muscle vs muscle group, no DEGs in S-W group were enriched, while 21 182 and 25 DEGs in B-S and B-W group were significantly enriched in this pathway, respectively, and all of these DEGs (except C3AR1) were 183 up-regulated in B cattle, which is different from the results of skin group (Fig. 6and 6b), suggesting a different immune state in skin and muscle 184 tissues in these two kinds of cattle. Among the up-regulated genes, most encode proteins likecoagulation factors, complement components and 185 other proteins related to immunity. Kininogen-1 (KNG1), also known as alpha-2-thiol proteinase inhibitor, Williams-Fitzgerald-Flaujeac factor 186 or the HMWK-kallikrein factor is a protein that in humans is encoded by the KNG1 gene. Kininogen-1 is the precursor protein to 187 high-molecular-weight kininogen (HMWK), low-molecular-weight kininogen (LMWK), and bradykinin. Bradykinin is a potent 188 endothelium-dependent vasodilator, leading to a drop in blood pressure. It also causes contraction of non-vascular smooth muscle in the 189 bronchus and gut, increases vascular permeability and is also involved in the mechanism of pain (Mutschler et al.1997   transcriptomes. Since all of these genes implicated in fatty acid metabolism, we proposed that similar to cold tolerant pig breeds, MC in cold 217 adaptation could convert the WAT into beige adipocytes, where heats were generated by metabolism of fatty acids and glucose to sustain body 218 core temperature. Unlike polar region climates, where all the year is extreme cold, in summer, the temperature in MP, especially in semiarid 219 regions such as Ordos steppe, is high. Therefore, precious detection of such temperature changes also critical for MC fitting for their 220 environments. Genome structure variation analysis indicated SD area in MC enriched in a GO term called olfactory receptor. The main function 221 of olfactory receptor is perception of chemical and climate factors. Therefor, genome structure variation in MC olfactory receptor probably 222 underlying the molecular mechanism of MC how to preciously detect the temperature change.  It's well characterized that as an antimicrobial peptide, LAP (lingual antimicrobial peptide) is involved in the innate system and has versatile 230 antimicrobial activities ). The investigation of cDNA from cattle mammary gland showed that many members of beta-defensins 231 gene family were expressed, which implied these robust antimicrobial peptides playing important roles for cattle resistant to mastitis. 2 of them 232 belong to interferon (IFN) gene family. In our data, they are IFNW1 and IFNT2. Interferon is known for its' anti-virus activities. In this case, 12, 13-dihydroxy-9Z-octade-cenoic acid (12,13-diHOME) can induct the fatty acid uptake into BAT and skeletal muscle (Lynes et al. 2017). In 248 MC, no BAT were observed, which indicated that in response to extreme cold weather in winter, MC firstly need brown WAT into beige 249 adipocyte, then uptake fatty acid into these nearly formed adipocyte to generate heats. The prerequisite of these two processes is the existence of 250 large amounts of unsaturated fat acid. The indirect evidence is that in winter MC transcriptomes, 10 P450 genes were significantly up-regulated, 251 which implied these up-regulated genes may involved in the oxidation of unsaturated fat acid into oxylipins.

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MC is a valuable cattle breed. Nevertheless, so far, few research works performed on this important livestock. By genome sequencing 253 association with comparative transcriptome sequencing, we revealed the molecular mechanisms of MC how to adaptation to MP harsh 254 environments as well as resistant to pathogenic organisms. Undoubtedly, these uncovered genes will become cherished genetic resources for 255 cattle breeding.    Availability of data and material: All data generated or analyzed during this study are included in this manuscript.

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Competing interests: The authors declare that they have no competing interests.

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