Table_7_Analysis of the Progeny of Sibling Matings Reveals Regulatory Variation Impacting the Transcriptome of Immune Cells in Commercial Chickens.xlsx

There is increasing recognition that the underlying genetic variation contributing to complex traits influences transcriptional regulation and can be detected at a population level as expression quantitative trait loci. At the level of an individual, allelic variation in transcriptional regulation of individual genes can be detected by measuring allele-specific expression in RNAseq data. We reasoned that extreme variants in gene expression could be identified by analysis of inbred progeny with shared grandparents. Commercial chickens have been intensively selected for production traits. Selection is associated with large blocks of linkage disequilibrium with considerable potential for co-selection of closely linked “hitch-hiker alleles” affecting traits unrelated to the feature being selected, such as immune function, with potential impact on the productivity and welfare of the animals. To test this hypothesis that there is extreme allelic variation in immune-associated genes we sequenced a founder population of commercial broiler and layer birds. These birds clearly segregated genetically based upon breed type. Each genome contained numerous candidate null mutations, protein-coding variants predicted to be deleterious and extensive non-coding polymorphism. We mated selected broiler-layer pairs then generated cohorts of F2 birds by sibling mating of the F1 generation. Despite the predicted prevalence of deleterious coding variation in the genomic sequence of the founders, clear detrimental impacts of inbreeding on survival and post-hatch development were detected in only one F2 sibship of 15. There was no effect on circulating leukocyte populations in hatchlings. In selected F2 sibships we performed RNAseq analysis of the spleen and isolated bone marrow-derived macrophages (with and without lipopolysaccharide stimulation). The results confirm the predicted emergence of very large differences in expression of individual genes and sets of genes. Network analysis of the results identified clusters of co-expressed genes that vary between individuals and suggested the existence of trans-acting variation in the expression in macrophages of the interferon response factor family that distinguishes the parental broiler and layer birds and influences the global response to lipopolysaccharide. This study shows that the impact of inbreeding on immune cell gene expression can be substantial at the transcriptional level, and potentially opens a route to accelerate selection using specific alleles known to be associated with desirable expression levels.

随着对影响复杂性状的潜在遗传变异认识的日益深入，人们认识到这些变异会影响转录调控，且能在种群水平上作为表达数量性状位点被检测到。在个体层面，通过测量RNA测序数据中的等位基因特异性表达，可以检测到单个基因转录调控中的等位基因变异。我们推测，通过分析具有共同祖先的近交后代，可以识别出基因表达中的极端变异。商业鸡经过长期的人工选择以培育生产性状。这种选择与大量连锁不平衡的连锁块相关联，具有选择紧密连锁的“搭便车等位基因”的潜力，这些等位基因会影响与所选特征无关的性状，如免疫功能，从而可能影响动物的生产力和福利。为了验证免疫相关基因中存在极端等位基因变异的假设，我们对商业肉鸡和蛋鸡的创始人种群进行了测序。这些鸟在品种类型上表现出明显的遗传分离。每个基因组都包含大量候选无效突变、预测为有害的蛋白质编码变异和广泛的非编码多态性。我们选择了肉鸡-蛋鸡配对，并通过F1代的兄妹交配产生了F2鸟群。尽管预测创始人种群的基因组序列中存在有害编码变异的普遍性，但在仅一个由15个F2同胞组成的群体中，我们发现了近交对生存和孵化后发育的明显有害影响。对孵化雏鸡的循环白细胞种群没有影响。在选择的F2同胞群体中，我们对脾脏和分离的骨髓来源的巨噬细胞（带和不带脂多糖刺激）进行了RNA测序分析。结果证实了预测的个体基因和基因集表达差异的巨大出现。结果的网络分析确定了个体间共表达基因的簇，并暗示了干扰素反应因子家族成员在巨噬细胞表达中的跨作用变异的存在，这些家族成员区分了亲代肉鸡和蛋鸡，并影响了全局对脂多糖的反应。这项研究表明，近交对免疫细胞基因表达的转录水平影响可能非常显著，并可能为加速使用与期望表达水平相关联的特定等位基因的选择提供了一条途径。