Hepatic transcriptome analysis for differential gene expression between steers of divergent feed efficiency phenotypes in three Canadian beef breeds

The objective of this study was to identify differentially expressed genes in the liver of steers with divergent Residual Feed Intake (RFI). Methods:In total 50 purebred Angus, 48 purebred Charolais and 158 Kinsella Composite breed steers were tested for individual feed intake using the GrowSafe system for an average period of 70 to 73 days. During the feedlot test animals were fed at ad libitum with a finishing diet composed of 75% barley grain, 20% barley silage and 5% rumensin pellet. Body weight of each animal was measured at an interval of 28days and ADG for each animal was obtained from a linear regression of serial body weight (BW) measurements (Kgs) on time (days). MWT was calculated as midpoint BW0.75, where midpoint BW was computed as the sum of initial BW of the animal and the product of its ADG multiplied by half the number test days. DMI of each animal was calculated as the average daily feed intake of the animals for the time during the feedlot test (days). The expected DMI for each animal was predicted using the regression intercept and regression coefficients of ADG and MWT on actual DMI, and RFI was computed as the difference between the standardized daily DMI and the expected DMI. At the end of the test, animals were slaughtered and liver tissue was collected immediately after slaughter separately bagged in plastic bags, labeled and flash frozen in liquid nitrogen. The frozen samples were transferred to the laboratory on ice and stored at -80C until RNA extraction. From the frozen samples, 20 samples from each breed were selected for total RNA extraction including six samples with extreme high and six samples extreme low-RFI phenotypes for each breed. Complementary DNA (cDNA) libraries were constructed for each of the 60 animals and consequently single end sequenced using Illumina Hiseq 2500 sequencer. The raw reads were aligned and mapped to the bovine reference genome UMD 3.1 using Tophat2 aligner with default alignment parameters. Thereafter, reads aligned uniquely to each annotated transcript of gene in the bovine genome were counted using HTSeq-count package with default parameters. Read counts from HTSeq-count, sample information (sample id, sire, feed efficiency group and sequencing mode) and gene annotation table from ENSEMBL Biomart were used for differential expression analysis within each breed using the edgeR package in R for the six extreme low-RFI and six extreme high-RFI samples. Results: At a false discovery rate of 0.05 and fold change > 2 , we identified 72, 41 and 175 differentially expressed genes for Angus, Charolais and KC RFI divergent steers respectively. Overall design: RNA-seq of Angus, Charolais and Kinsella Composite male liver with either low or high Residual Feed Intake (RFI).本研究旨在鉴定剩余采食量（Residual Feed Intake, RFI）存在差异的阉牛肝脏组织中的差异表达基因。方法：本研究共纳入50头纯种安格斯牛、48头纯种夏洛莱牛以及158头金塞拉复合品种阉牛，采用GrowSafe系统对其进行平均70至73天的个体采食量测定。试验期间，试验动物以自由采食模式饲喂，日粮由75%大麦谷物、20%大麦青贮料及5%瘤胃素颗粒组成。每28天测量一次试验动物的体重，通过对连续体重（单位：千克）测量值按时间（单位：天）进行线性回归，得到每头动物的平均日增重（Average Daily Gain, ADG）。代谢体重（Metabolic Body Weight, MWT）的计算公式为中点体重的0.75次方，其中中点体重为试验动物初始体重与平均日增重乘以试验天数一半的乘积之和。每头动物的干物质采食量（Dry Matter Intake, DMI）以育肥试验期间的日均采食量计算得到。利用实际干物质采食量对平均日增重和代谢体重进行回归，通过回归截距与回归系数预测每头动物的预期干物质采食量，剩余采食量（RFI）为标准化日均干物质采食量与预期干物质采食量的差值。试验结束后屠宰试验动物，于屠宰后立即采集肝脏组织，将样本分别装入塑料袋并标记，随后置于液氮中快速冷冻。冷冻样本以冰袋转运至实验室，并于-80℃条件下保存直至RNA提取。从冷冻样本中，每个品种选取20份样本进行总RNA提取，其中每个品种各选取6份高RFI表型样本与6份低RFI表型样本。为60头动物分别构建互补DNA（cDNA）文库，随后采用Illumina Hiseq 2500测序仪进行单端测序。使用Tophat2比对工具以默认参数将原始reads比对至牛参考基因组UMD 3.1。随后，使用HTSeq-count工具包以默认参数，统计比对至牛基因组中每个已注释基因转录本的reads计数。利用HTSeq-count得到的reads计数、样本信息（样本ID、父本、饲料效率分组及测序模式）以及来自ENSEMBL Biomart的基因注释表，使用R语言中的edgeR工具包对每个品种的6份低RFI与6份高RFI样本进行品种内差异表达分析。结果：在错误发现率（False Discovery Rate, FDR）为0.05且折叠变化（Fold Change, FC）>2的条件下，分别在安格斯牛、夏洛莱牛以及金塞拉复合品种阉牛中鉴定出72、41和175个差异表达基因。总体设计：对具有低或高剩余采食量（RFI）表型的安格斯牛、夏洛莱牛及金塞拉复合品种阉牛肝脏组织进行RNA测序。