In North American gray wolves, black coat color is dominantly inherited via a 3 base pair coding deletion in the canine beta defensin 3 (CBD103) gene. This 3 base pair deletion, called the KB allele, was introduced through hybridization with dogs and subsequently underwent a selective sweep that increased its frequency in wild wolves. Despite apparent positive selection, KBB wolves have lower fitness than wolves with the KyB genotype, even though the 2 genotypes show no observable differences in black coat color. Thus, the KB allele is thought to have pleiotropic effects on as-yet unknown phenotypes. Given the role of skin-expressed CBD103 in innate immunity, we hypothesized that the KB allele influences the keratinocyte gene expression response to TLR3 pathway stimulation and/or infection by canine distemper virus (CDV). To test this hypothesis, we developed a panel of primary epidermal keratinocyte cell cultures from 24 wild North American gray wolves of both Kyy and KyB genotypes. In addition, we generated an immortalized Kyy line and used CRISPR/Cas9 editing to produce a KyB line on the same genetic background. We assessed the transcriptome-wide responses of wolf keratinocytes to the TLR3 agonist polyinosinic:polycytidylic acid (polyI:C), and to live CDV. K locus genotype did not predict the transcriptional response to either challenge, suggesting that variation in the gene expression response does not explain pleiotropic effects of the KB allele on fitness. This study supports the feasibility of using cell culture methods to investigate the phenotypic effects of naturally occurring genetic variation in wild mammals.

中文翻译：

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灰狼中的 K 基因座效应：TLR3 信号转导和对犬瘟热病毒的基因表达反应的实验评估


在北美灰狼中，黑色毛色主要是通过犬β防御素3（CBD103）基因中的3个碱基对编码缺失遗传的。这种 3 碱基对缺失被称为 KB 等位基因，是通过与狗杂交引入的，随后经过选择性扫描，增加了其在野狼中的频率。尽管存在明显的正选择，KBB 狼的适应性低于具有 KyB 基因型的狼，尽管这 2 个基因型在黑色皮毛颜色上没有显示出明显的差异。因此，KB 等位基因被认为对迄今未知的表型具有多效性作用。鉴于皮肤表达的 CBD103 在先天免疫中的作用，我们假设 KB 等位基因影响角质形成细胞基因表达对 TLR3 通路刺激和/或犬瘟热病毒 (CDV) 感染的反应。为了检验这一假设，我们开发了一组来自 24 只 Kyy 和 KyB 基因型野生北美灰狼的原代表皮角质形成细胞培养物。此外，我们还生成了永生化的 Kyy 系，并使用 CRISPR/Cas9 编辑产生了具有相同遗传背景的 KyB 系。我们评估了狼角质形成细胞对 TLR3 激动剂聚肌苷:聚胞苷酸 (polyI:C) 和活 CDV 的全转录组反应。 K 位点基因型不能预测对任一挑战的转录反应，这表明基因表达反应的变化不能解释 KB 等位基因对适应性的多效性影响。这项研究支持使用细胞培养方法研究野生哺乳动物中自然发生的遗传变异的表型效应的可行性。