An <em>EAV-HP</em> Insertion in 5′ Flanking Region of <em>SLCO1B3</em> Causes Blue Eggshell in the Chicken

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F2 chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.目前学界尚未明确鸟类蛋壳着色的遗传决定机制。本研究证实，鸡的蓝壳蛋壳由一段EAV-HP插入序列介导产生，该插入序列可促进鸡输卵管子宫部（壳腺）中SLCO1B3基因的表达。本研究通过对F₂代鸡群进行连锁分析，精细定位了蓝壳蛋基因的遗传图谱位置，并针对精细定位区间内的4个候选基因，分别在蓝壳与非蓝壳母鸡的子宫壳腺中检测其表达水平。结果发现，仅SLCO1B3基因在蓝壳母鸡的子宫组织中表达，而非蓝壳母鸡的子宫组织中无该基因的表达。焦磷酸测序（pyrosequencing）分析结果显示，在杂合子母鸡（O*LC/O*N）的子宫组织中，仅来自蓝壳鸡的SLCO1B3等位基因发生了表达。SLCO1B3基因属于有机阴离子转运多肽（organic anion transporting polypeptide, OATP）家族；该家族蛋白作为膜转运蛋白，已被报道可转运两亲性有机化合物，包括哺乳动物体内的胆汁盐。本研究随后对SLCO1B3的全基因组区域进行了重测序，在SLCO1B3的5'侧翼区域发现了一段EAV-HP插入序列。该插入序列与蓝壳蛋表型紧密关联，且遵循完全孟德尔分离定律。原位杂交（in situ hybridization）实验同样证实，蓝壳蛋表型与子宫壳腺中SLCO1B3的异位表达相关。本研究结果强烈提示，这段EAV-HP插入序列是导致蓝壳蛋表型的致突变位点。研究还在另一个中国蓝壳蛋鸡品种以及一个美国蓝壳蛋鸡品种中发现了该插入序列。此外，我们发现阿劳坎鸡（Araucana）蓝壳群体中的插入位点与中国蓝壳鸡品种存在差异，这表明两大洲的蓝壳鸡群体中该插入序列为独立整合事件，为分子水平的平行进化提供了一个实例。