Parallel selection mapping using artificially selected mice reveals body weight control loci (gene expression)

Understanding how polygenic traits evolve and respond to selection is a major unsolved problem, because challenges exist for identifying genes underlying a complex trait and understanding how multi-locus selection operates in the genome. Here we used artificial selection experiments to study polygenic response to selection. Inbred strains from seven independent long-term selection experiments in mice for extreme bodyweight ("High" lines weigh 77-42g vs. 40-16g in "Controls" lines), were genotyped at 527,572 SNPs to identify genetic variants controlling bodyweight. We identified 67 high-resolution parallel selected regions (PSRs) where multiple High lines share variants rarely found among the Controls. By comparing allele frequencies in one selection experiment against its unselected control, we found classical selective sweep signatures centered on the PSRs. Multiple lines of evidence support two G protein-coupled receptors GPR133 and Prlhr, as positional candidate genes controlling bodyweight. Artificial selection may mimic natural selection in the wild: compared to control loci, we detected reduced heterozygosity in PSRs in wild populations of unusually large mice on islands. Many PSRs overlap loci associated with human height variation, possibly through evolutionary conservation of functional pathways. Our data suggest that parallel selection on complex traits may evoke parallel responses at many genes involved in diverse but relevant pathways. These samples were used to test the enrichment of certain gene functional categories. Mouse tissue samples (muscle and liver) were collected from 44 and 45 days old males from each of the nine out of thirteen lines (5 High lines: BEHi, DUHi, EDHi, MUHi, ROHi and 4 Control lines: BELi, EDLi, MULi and ROLi; n=23 total) at the University of Edinburgh. Frozen tissue samples were stored at -80ºC until use. Tissues were dissociated using TissueLyser II with steel beads (both Qiagen) following manufacturer's instructions. mRNA was extracted and purified using TRIzol Reagent and the PureLink RNA Mini Kit (Life Technologies GmbH, Darmstadt, Germany). cDNA was synthesized using Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Muscle and liver cDNA samples were hybridized on a Mouse GE 4x44k Microarray (Agilent Technologies, Inc., Waldbronn, Germany) according to manufacturer's recommendations. Three technical replicate each was done on pooled cDNA samples from each mouse line.解析多基因性状的演化规律及其对选择的响应机制是尚未解决的核心科学问题，这是因为鉴定复杂性状的因果基因、解析基因组内多基因座选择的作用模式均存在极大挑战。本研究借助人工选择实验探究多基因性状对选择的响应。我们对7项独立开展的小鼠极端体重长期人工选择实验的近交系群体进行基因分型，共检测527572个单核苷酸多态性（Single Nucleotide Polymorphism, SNP），以筛选调控体重的遗传变异；其中高体重选择系个体体重范围为77~42g，对照系个体体重范围为40~16g。本研究共鉴定得到67个高分辨率平行选择区域（Parallel Selected Regions, PSRs），多个高体重选择系在这些区域共享的遗传变异在对照系中极为罕见。通过对比单个选择实验系与其未经过选择的对照系的等位基因频率，我们在平行选择区域附近检测到典型的选择性清除（selective sweep）特征信号。多项证据表明，G蛋白偶联受体（G protein-coupled receptor, GPCR）GPR133与Prlhr可作为调控体重的位置候选基因。人工选择可模拟自然界的自然选择过程：相较于对照基因座，我们在岛屿上体型异常庞大的野生小鼠群体的平行选择区域中，检测到杂合性水平显著降低。大量平行选择区域与人类身高变异相关的基因座存在重叠，这可能源于功能通路的进化保守性。本研究数据表明，针对复杂性状的平行选择可在多条功能多样但相关的通路所涉及的诸多基因中诱发平行响应。本研究使用上述样本开展了特定基因功能类别的富集分析检验。爱丁堡大学的研究人员从13个小鼠品系中的9个品系（含5个高体重选择系：BEHi、DUHi、EDHi、MUHi、ROHi，以及4个对照系：BELi、EDLi、MULi、ROLi）中，分别采集了44日龄与45日龄的雄性个体的肌肉与肝脏组织样本，总样本量n=23。冷冻组织样本保存于-80℃冰箱直至实验使用。按照制造商说明书，使用配备钢珠的TissueLyser II组织破碎仪（均购自凯杰（Qiagen）公司）对组织进行解离处理。使用TRIzol试剂与PureLink RNA迷你试剂盒（赛默飞世尔科技生命科学事业部，德国达姆施塔特）提取并纯化总mRNA。使用Maxima第一链cDNA合成试剂盒（赛默飞世尔科技，德国圣莱昂罗特）合成cDNA。按照制造商推荐的实验流程，将肌肉与肝脏的cDNA样本在小鼠GE 4x44k基因芯片（安捷伦科技有限公司，德国瓦尔德布龙）上进行杂交。每个小鼠品系的混合cDNA样本均开展3次技术重复实验。