Table_6_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.

日益认识到，导致复杂性状的遗传变异影响转录调控，并在种群水平上作为表达数量性状基因座被检测到。在个体层面，单个基因的转录调控中的等位基因变异可以通过测量RNA测序数据中的等位基因特异性表达来检测。我们认为，通过分析具有共同祖父母的近交后代，可以识别基因表达中的极端变异。商业鸡经过对生产性状的密集选择。选择与大量连锁不平衡的连锁块相关联，这可能导致与被选择特征无关的性状（如免疫功能）的“搭便车等位基因”的共选择，从而可能影响动物的生产力和福利。为了验证这种假设，即免疫相关基因中存在极端的等位基因变异，我们对商业肉鸡和蛋鸡的创始人种群进行了测序。这些鸟类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的候选无效突变、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-蛋鸡对进行交配，并通过F1代的兄妹交配产生了F2鸟类群体。尽管预计创始人的基因组序列中存在有害的编码变异，但在仅有的15个F2同胞家族中，我们发现近亲繁殖对存活和孵化后发育的负面影响是明显的。对孵化雏鸡的循环白细胞群体没有影响。在选择的F2同胞家族中，我们对脾脏和分离的骨髓来源的巨噬细胞（有无脂多糖刺激）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极大化。结果的网络分析识别了在不同个体之间变化的共表达基因簇，并暗示了干扰素反应因子家族在巨噬细胞中的表达存在跨作用变异，这种变异区分了亲本肉鸡和蛋鸡，并影响了全局对脂多糖的反应。这项研究表明，近亲繁殖对免疫细胞基因表达的影响在转录水平上可能是显著的，并可能开辟了一条加速使用已知与理想表达水平相关联的特定等位基因的选择路线。