鸭绿色蛋壳表型遗传机制解析

In the evolution of birds, emergence of eggshell color improves bird fertility, but formation mechanism of it is poorly understood. Blue-green is one of eggshell color frequently appearing among birds. Formation mechanism of chicken blue eggshell has been revealed at molecular level by us. However, an exploratory research indicated that the eggshell color in duck is caused by a different mutation. The project aims to identify key mutation (dO) causing duck blue eggshell by candidate gene analysis and linkage mapping. Candidate gene analysis will focus on 5 genes (SLCO1A1、SLCO1A2、SLCO1A3、SLCO1A4、SLCO1A6) mediating transportation of biliverdin (a pigment for coloration of blue eggshell). Expression of these genes in shell gland will be detected using qPCR, and expressed genes will be sequenced. To identify dO, sequence variants will be subjected to analysis of association, selective sweep, identity by descent (IBD) and function. To identify dO, linkage analysis will be also performed. A resource family will be constructed by crossing blue-shelled ducks with white-shelled ducks, and the dO will be mapped to a chromosomal interval using the family and a set of markers covering duck genome. Finally, the dO will be identified by analyzing expression and sequence variants of genes within the interval. This project is useful to elucidate formation mechanism underlying duck blue eggshell, which will add new knowledge to relative field, and facilitate blue-shelled duck breeding by molecular-assisted selection.

在鸟类进化中，蛋壳颜色的出现提高了鸟类的繁殖力，但其形成机制却是一个有待深入研究的领域。蓝绿色是鸟类中普遍存在的一种蛋壳颜色，申请人曾揭示了鸡绿壳形成的分子机制，但对鸭绿壳探索时却发现，该性状并非由相同突变引起。本项目期望通过候选基因和连锁定位两种策略鉴定引起鸭绿壳表型的关键突变（dO）。候选基因策略对5个胆绿素（绿壳着色色素）转运基因（SLCO1A1、SLCO1A2、SLCO1A3、SLCO1A4、SLCO1A6）展开研究。用qPCR检测这些基因在蛋壳腺的表达情况；对表达的基因进行测序，用关联、选择扫荡、IBD和突变功能分析鉴定dO。连锁分析用绿壳鸭与白壳鸭杂交构建性状分离群体，在基因组中选取分子标记对dO进行定位；通过分析定位区段内的基因表达和序列变异以鉴定dO。本项目有助于阐明鸭绿壳表型形成的分子机制，拓宽蛋壳颜色形成机制研究领域的知识面，为绿壳鸭选育提供一种分子检测手段。

鸭绿壳表型是一个主要受基因调控的性状，但目前调控基因（dO）和调控机制均不清楚。在之前的研究中，我们发现鸡绿壳性状由SLCO1B3基因5’端一个逆转录病毒插入引起，但是对鸭SLCO1B3基因研究后却发现该性状并非由相同突变引起。为了鉴定dO，探索鸭绿壳表型形成的分子机制，本研究分析了10个有机阴离子转运多肽（OATP）基因在绿壳和白壳鸭肝脏和蛋壳腺中的表达量；检测了胆绿素转运基因SLCO2B1内错义突变与绿壳表型的关联性及其对蛋白结构的影响；通过简化基因组测序在全基因组水平对鸭绿壳基因（dO）进行了定位；应用转录组测序比较了绿壳和白壳鸭蛋壳腺转录组差异。在10个OATP基因中，只有SLCO2A1、SLCO2B1和SLCO5A1基因在蛋壳腺中表达。通过5’RACE，本研究克隆了SLCO2B1 5’端序列，获得OATP2B1完整蛋白序列；发现了22个同义突变，9个错义突变，2个3’UTR突变。其中，5个变异与绿壳表型显著（P<0.05）关联，1个错义突变引起了OATP2B1跨膜螺旋结构、氨基酸亲水性和主链氢键的改变。定位研究发现了14个基因组水平显著（P<0.001）的SNP，这些SNP分布在12个contig上。转录组测序发现464个差异表达基因。GO注释和富集分析显示，离子跨膜转运是差异表达基因富集的一个重要功能项。本研究对dO进行了初步定位，定位结果对候选基因的选取具有参考价值。OATP结果建议鸭绿壳表型并非由这些基因差异表达引起，而离子转运基因差异表达对胆绿素运输体活性的影响可能与鸭绿壳表型的形成有关。