Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.
Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.
复制标题
通过高分辨率熔解分析进行灵长类动物基因分型：快速可靠地识别野生狐猴的色觉状态。
DOI：
10.1007/s10329-016-0546-y
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Jacobs RL
中科院分区：
文献类型：
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作者：
Jacobs RL
对遗传多态性的分析可以帮助我们理解灵长类动物的社会性、生态学和行为的种内和种间变异。灵长类视蛋白基因的研究就是最好的例子，因为x连锁视蛋白基因的单核苷酸变异（snv）是色觉变异的基础。对于具有多态三色的灵长类物种，基因分型视蛋白snv一般可以表明个体灵长类是红绿色盲（表示纯合M或纯合L）还是全三色色觉（杂合ML）。鉴于色觉对行为和适应性的潜在影响，表征研究对象的色觉状态在许多灵长类动物领域项目中变得越来越普遍。这类研究传统上涉及基于多步骤测序的方法，既昂贵又耗时。本文提出了一种新的可靠、快速、相对便宜的方法，利用高分辨率熔体分析（HRMA）来表征灵长类动物种群的色视觉。以狐猴为例，我们对代表9个物种的87个狐猴的x连锁视蛋白基因外显子3和/或5的变异进行了表征。我们使用传统的基于测序的方法和我们新颖的基于融化曲线的HRMA协议来评分视蛋白基因型和色觉状态。对于每个物种，不同基因型（纯合子M、纯合子L、杂合子ML）的熔化曲线在熔化温度和/或形状上存在差异。每个样本的融化曲线在不同的重复中是一致的，基因型特异性的融化曲线在不同的DNA来源（血液和粪便）中是一致的。我们发现，一旦基于已知基因型建立了实验室特异性参考曲线，就可以使用HRMA快速可靠地对视蛋白基因型进行评分。虽然这里提出的方案侧重于狐猴视蛋白位点的基因分型，但我们也认为将这种方法应用于灵长类动物种群的各种类型的遗传研究具有更大的潜力。
Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.