Detection and integrated analysis of lncRNA and mRNA relevant to plateau adaptation of Yak

Abstract Known as the ‘ship of the plateau’, through thousands of years evolution and cruelty environments selection containing low oxygen and strong ultraviolet radiation, yaks have adapted plateau environments and supplied important goods and materials for the people in the Qinghai‐Tibet Plateau. This study aimed to identify differentially expressed (DE) genes and novel long non‐coding RNAs (lncRNAs) of yaks for the Plateau adaptation and their underlying co‐expression and regulatory network. We carried out RNA‐seq analysis for cerebral and cerebellar tissue specimens of Bos taurus, Bos grunniens × Bos Taurus and B. grunniens. Furthermore, 12,072 pseudo lncRNAs were predicted using three software. In total, 4,257 significant DE transcripts were identified using the Ballgown R package (p < .01), of which 1,021 were protein‐coding genes, 14 were known lncRNAs, and 661 were novel lncRNAs. Using WGCNA, a co‐expression network of DE mRNAs and lncRNAs comprising 5 modules was generated to determine functional associations clusters. This study reveals a valuable sub‐network comprising 8 hub genes, one known lncRNA and 5 novel lncRNAs in the major module. These hub genes are associated with blood pressure regulation, generation of reactive oxygen species and metabolism. The analysis of co‐expressed genes thus provides a basis for the regulatory mechanisms in PA in Yaks and for the detection of additional genes between cross‐breed and parent populations.

In the Qinghai-Tibet Plateau, the Yak (Bos grunniens) is a prominent livestock species to the Tibet region (Hu et al., 2012;Qiu et al., 2015). Yaks are called the 'ship of the plateau' and are highly adaptable to low the atmospheric pressure, hypoxia and high ultraviolet radiation in the plateau (Hu et al., 2012;Jincheng et al., 2014;Zhang et al., 2016). To assess the genetic advantages of yaks, numerous studies have reported associations of SNPs and copy numbers variations with plateau adaptation (PA; Hu et al., 2012;Medugorac et al., 2017;Qiu et al., 2015;Zhang et al., 2019); however, the underlying gene expression, regulation and the functions of PA remain unclear, especially the mR-NA-lncRNA co-expression network. The brain is a key tissue as a nerve centre and is responsible for physiological response to adaptation in extreme environments (Marshall-Goebel, Damani, & Bershad, 2019). Most of the cattle (Bos Taurus) died from intracranial haemorrhage after exposure to high altitudes of over 3,000 m, but the Yak survived very well and were highly useful to the people in the plateau (Gerald, Han, & Long, 2003). These evidences indicated the brain is an important and complex system for mammal adaptation of plateau environments. In the 2016, Wang et al. used four organs of Yak and Cattle to provide insights into high-altitude adaptation . But because of the large gap and differences in genetics between B. grunniens and B. taurus, whatever chosen any one reference genomes the noise of population was always unable to distinguish. Actually, as the cross-breed of Yak (B. grunniens) and Cattle (B. taurus), Zang Yakow presents moderate PA. With the help of expression trend in three breeds, it will improve the accuracy to detect candidate genes and predict the co-expression network involved in PA Wu et al., 2018).
In this study, we performed RNA-seq analysis for three breeds: Leiwuqi Yak (B. grunniens), Zang Yakow (B. grunniens × B. taurus) and Sanjiang Cattle (B. taurus). The differences in the PA potentials among these three breeds were determined through high-throughput transcriptome sequencing of cerebral and cerebellar tissues from animals to identify DE genes and predict novel lncRNAs. Furthermore, we constructed a network of DE mRNAs and lncRNAs to assess their interactions and the role of these interactions.

| Sample collection and RNA extraction
All animals had a similar health status and were approximately 4.5 years old and females. Eighteen samples (the cerebrum and cerebellum came from 3 Leiwuqi Yak, 3 Zang Yakow and 3 Sanjiang Cattle) were harvested in the Leiwuqi region (Leiwuqi Yak and Zang Yakow) and Wenchuan region (Sanjiang Cattle), Sichuan, China. The brain cortex of all samples was collected after slaughter while considering animal welfare in October 2018.
Three individuals of the same breed were randomly selected as replicates in a herd of >50 individuals. The Leiwuqi Yak was considered the M group (living average 4,450 m altitude); Sanjiang Cattle was considered H group (living average 1,325 m altitude); Zang Yakow was considered Z group (living average 3,200 m altitude). All clean tissue samples were divided into 1-2 cm 3 sections and snap-frozen in liquid nitrogen. Using TRIzol ® Plus RNA Purification Kit (Invitrogen) in accordance with the manufacturer's instructions, total RNA was extracted from brain tissue.
Ribosomal RNA (rRNA) was eliminated before sequencing, using the Epicentre Ribo-zero TM rRNA Removal Kit in accordance with the manufacturer's instructions.

| RNA sequencing and transcript assembly
After eliminating rRNA, cDNA sequencing libraries of total coding RNAs and ncRNAs were generated using the mRNA-seq sam-  (Kim, Langmead, & Salzberg, 2015). After sorting and conversion to bam with Samtools (version 1.9), we used StringTie (version 1.2.3; Pertea et al., 2015) to assemble transcripts for each sample.
All transcripts were merged with the 'StringTie-merge' function of StringTie with the corresponding sample file names, and output fields were generated in Ballgown format (Frazee et al., 2015).

| Identification of lncRNAs
After pre-processing the RNA-seq data, all transcripts in the merged transcript annotation file were identified using four filters to predict potential transcript. First, transcripts ≤200 nt were eliminated. Second, based on the relative position among transcript and known genes, we used gffcompare to mark the transcript type. Those transcripts with class code 'u' (unknown intergenic), 'x' (exonic overlap on the opposite strand), 'i' (fully contained within a reference intron), 'j' (multi-exon with at least one junction match) and 'o' (other same strand overlap with reference exons) were retained. Third, transcripts with low expression levels (FPKM ≤ 1) were filtered out. Fourth, transcripts more than 1 exon were retained (Cloutier et al., 2013;Fan et al., 2019). The FASTA format data were extracted using the getfasta function of bedtools from the reference genome (Quinlan & Hall, 2010). Whole remaining transcripts were considered new transcripts. Thereafter, three methods were used to predict the potential protein-coding potential of these new transcripts. These three methods are the Coding Potential Calculator (CPC2; Kang et al., 2017), the Coding-Non-Coding-Index (CNCI; Sun et al., 2013)

and the Coding Potential
Assessing Tools (CPAT; Wang et al., 2013). The intersection of these three predicted lncRNAs was considered to comprise the new lncRNAs for downstream analyses.

| DE mRNAs and lncRNAs among M and H groups
New lncRNAs, known mRNAs and known lncRNAs were identified from the reference genome in accordance with the annotation file and through identification of new lncRNAs. The Fragment PerKilobase Million (FPKM) metric was used as the unit of measurement to estimate transcript abundance, using R package Ballgown, which is an integrated tool for statistical analysis of assembled transcriptomes. The group was considered a covariate factor, and different tissues (cerebellum and cerebrum) were considered to adjust the variate factors in the Ballgown model for DE analysis. The thresholds |log2FoldChange|>1 and p < .01 were assigned for DE genes, using the Ballgown package.
GO enrichment analysis was conducted to cluster genes in accordance with the biological process, cellular component and molecular function. Candidate DE genes were analysed using R package "clusterProfiler" with database "org.Bt.eg.db" (R Development Core Team, 2016;Yu, Wang, Han, & He, 2012) for statistical analysis and visualization of functional profiles of genes and gene clusters.

| Co-expression network for DE RNA and PA
The expression levels of these significant DE genes among the whole three populations (Yak, Yakow, and Sanjiang Cattle) were determined to construct a co-expression network using the weighted gene coexpression network analysis (WGCNA) R package (Langfelder & Horvath, 2008). The WGCNA method is a systems biology method for describing the correlation patterns among genes across microarray samples. It can be used for clusters (modules) of highly correlated genes, for summarizing such clusters using the module eigengene or an intramodular hub gene, for relating modules to one another and to external sample traits (using eigengene network methodology), and for calculating module membership measures (Langfelder & Horvath, 2008). The soft power was set from 1 to 30 lops to optimize the association model. Among all genes and lncRNAs, pairwise Pearson's correlations were determined to generate an adjacency matrix. The topological overlap measure (TOM) was estimated via the optimum model using the adjacency matrix.  et al., 2003). Major hub genes in the largest module were selected to enrich with correlated known lncRNAs and novel lncRNAs.

| qPCR validation
RNA samples, which were extracted from the same sources and at the same concentration as library preparation, were reversetranscribed to cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa, Dalian China) according to the manufacturer's instructions. Four hub genes in the most related module of the co-expression network were selected to validate RNA-seq result. Primers were designed using Primeprimer 5.0 (Table S2). As a reference gene, cattle GAPDH was used to adjust relative CT value. Relative quantifications of genes were calculated by the ∆CT method (Rao, Huang, Zhou, & Lin, 2013).

| Sample collection and RNA sequencing
Each sample was totally mapped at an average mapping ratio of 91.57% relative to the reference genome. Of these, 84.22% were F I G U R E 2 The screening and enrichment of Different Expression genes in the Yak and Cattle group. The DE genes were filtered between Yak and Cattle with 2 times fold change (a). The 14 GO terms of all subontologies were significant enriched with whole DE genes (b) uniquely mapped reads and the remaining 2.98% were multiple mapped reads (Table S1).
A summary pipeline of the experimental workflow, bioinformatics and statistical analysis is displayed ( Figure S1). In total, 101,835 transcripts were assembled in the merged assembled file of 18 samples, of which there were 32,667 transcripts expressed in all 18 samples. All transcript expression levels in the 18 samples were presented ( Figure S2) with log2 (FPKM + 1) values. Furthermore, we determined the distribution of transcripts per known gene ( Figure S3). There were 3,755 genes with only one transcript, and the ratio of total genes was 24.16%, indicating that most genes had multiple transcripts. We designed a smoothened colour density representation ( Figure S4) to verify the repeatability and compare the similarities and differences in the expression levels between the cerebrum and cerebellum in each sample. were multi-exon matched, 10.32% were fully contained within introns, 3.98% overlapped with opposite sites, and 1.78% overlapped with exons ( Figure 1b). Comparisons between the FPKM distribution of novel lncRNAs and known lncRNAs are presented in Figure 1c.

| Analysis of DE genes
To identify DE genes between Yak (M) and Sanjiang Cattle (H), we filtered out those transcripts of no variational expression in the linear model. In total, 4,257 significant (p < .01) genes were identified using Ballgown R package with species as covariate factors and tissues as adjustable variate factors. After removed |log2foldchange|<1, there were 2,577 different expression (DE) genes, and 867 of DE genes were upregulated and 1,710 were downregulated (Figure 2a). Of these, 1,021 were protein-coding genes, 14 were known lncRNAs, and 661 were novel lncRNAs. The DE genes were identified in accordance with the top 5 GO terms (Figure 2b). There was few DE genes between the cerebrum and cerebellum in all three groups ( Figure S5) that indicated the differential express of these two tissues. We also compared the DE genes between the Yakow and Sanjiang Cattle ( Figure S6). There were 8,517 DE genes.

| Co-expression network
The R square of the filter criteria was set to 0.8 to optimize and select the soft power of the prediction model ( Figure S7). We also used the scaleFreePLot function of WGCNA to verify the relative levels between the whole estimated network and scale-free network ( Figure S8 and Supplementary file lnc_known_mRNA_node.txt). The associated F I G U R E 5 Sub-network with most enrichment genes and lncRNAs. The top 10 enrichment hub genes (Blue Diamond) in whole network, top 5 known lncRNAs (Green Circle) and top 5 novel lncRNAs (Red Triangle) were selected to build sub-network. The more enrichment line indicated more related regulation of expression F I G U R E 6 RT-qPCR validation with four hub genes expression in Cerebrum. The RT-qPCR result of MRPL49 (a), IL34 (b), ERAP1 (c) and PINK1 (d) was used to verify RNA-seq expression result expression genes were clustered into 6 groups (the grey module was the mismatched module), and each group (excluding the grey module) contained more than 50 genes (Figure 3). Based on the TOM, the correlation of genes in the all modules was visualized (Figure 4; Figure S9) via Cytoscape. The most of genes in the largest module (turquoise) were enriched into 6 GO terms ( Figure S10). Comparison with previous study, there are four important pathways related to PA (Qi et al., 2019;Song et al., 2019;Xiong et al., 2015). We used KEGG pathway database to match our DEGs to these four pathways ( Figure S11).
In total, 979 edges (genes and lncRNAs) were associated with the most significant terms of the largest related module. We selected the top 10 hub genes, 5 known lncRNAs and 5 novel lncRNAs to elucidate the underlying regulatory functional network based on the hub genes ( Figure 5). All lncRNAs and mRNAs expression of whole network were used to calculate Neighbour-Joining tree (NJtree) and principle components analysis (PCA) to present population structure ( Figures S12 and S13). And the variance explained proportion of top two PCs was more than 70% ( Figure S14).

| qPCR validation
The related expression levels of random selected 4 hub genes

| Genetic characteristics of Yak, Yakow and Cattle
Yak and Cattle were estimated to have diverged approximately 4.9 million years ago; therefore, the Yakow cross-breed between Yak and Cattle presented a male sterile phenotype, which was gradually restored with a back-cross between female Yakow and Yak after several generations (Xi et al., 2012). In the 2016, Wang et al. used four organs of Yak and Cattle to provide insights into high-altitude adaptation . This manuscript is the first time to detect Plateau Adaptation genes from Yak, Yakow and Cattle population. The reads unique mapping ratio (Table S1) of Yak was the highest, followed by that were Yakow and Cattle, thus indicating the associations of their genetic backgrounds relationship with the reference genome.

| Co-expression network
In the WGCNA, the clustered genes in one module had the same expression trend among all groups. The 10 hub genes in turquoise module were enriched in macrophage colony-stimulating factor receptor binding and atom of oxygen terms. Of these hub genes, ERAP1 is a key gene significantly contributing to the regulation of blood pressure in mammals (Hisatsune et al., 2015). Oxygen stress in the endoplasmic reticulum is decreased upon a reduction in ERAP1 levels, and blood pressure is decreased to maintain a balance (Goto, Ogawa, Hattori, & Tsujimoto, 2011). IL34, GRAMP3, PINK1 and MRPL49 are reportedly associated with the regulation of the generation of reactive oxygen species and metabolism (Geisler et al., 2010;Jung, Seo, Lee, Lee, & Roe, 2015;Radaeva & Simbirtsev, 2019;Wang et al., 2012;Xiao et al., 2017).
It was reported in 2012 that keratinocytes and neurons are the main sources of IL34 and that IL34 specifically directs the differentiation of myeloid cells in the skin epidermis and central nervous system . It is both a cytokines and a vasoactive peptides, and high expression levels can reduce the rate of complications (myocardial infarction, acute cerebrovascular events and transient ischaemic attacks; Radaeva & Simbirtsev, 2019). IL34 was highly expressed in the Yak group but its expression was very low in the Cattle group according to RNA-seq and qPCR results. In 2018, Lan sequenced Jinchuan Yak samples and found that PINK1 was enriched in the Parkinson's disease pathway (Lan et al., 2018).
The PINK1 expression levels of Yak were significantly higher than those of Yakow and Cattle. Sarkar Soma used gene expression to reveal that MRPL49 is associated with the hypoxia due to high-altitude acclimatization (Soma, 2012). In 2017, Mota and Guimaraes reported that MICAL1 affected age at the first calving in the Nellore cattle (Braeutigam et al., 2014). Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas the suppression of TLE1 expression in otherwise healthy neurons induces cell death (Dastidar, Narayanan, Stifani, & D'Mello, 2012). This maybe the key reason for the intracranial haemorrhage observed after exposing Sanjiang cattle to a high altitude of over 3,500 m.

| CON CLUS ION
The present results indicate that the PA is regulated by numerous genes and lncRNAs, which comprise an underlying network. The functions of these hub genes are associated with the regulation of blood pressure, generation of reactive oxygen species and metabolism. The analysis of co-expressed genes with the PA provides a basis to understand the regulatory mechanisms in the complex PA of Yak.

COMPE TING INTERE S TS
The authors declare that they have no competing interests.

E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
The protocol for Animal Care and Use was approved by the Animal Ethics and Welfare Association of the Southwest Minzu University

DATA AVA I L A B I L I T Y
Whole raw RNA sequence data (Fastq format) of 2 tissues of each 9 animals used in this study were submitted to the National Center for Biotechnology Information Sequence Read Archive (SRA) with Accession Number GSE13 2452.