Characterizing the Molecular Basis of Supergene Mimicry in Butterflies

描述（由申请人提供）：限性多态性在动物中广泛存在，包括各种人类性状和疾病，但我们缺乏对任何生物体中限性多态性的一般功能性理解。凤蝶（Papilio polytes）的“超基因”模仿是性别限制多态性的一个特别突出的例子，也是一个适合功能表征的例子。虽然许多理论工作已经探索了超基因拟态的进化动力学，但它的分子和发育基础实际上尚未探索。我们建议通过整合基因组学，功能遗传学，分子和发育生物学以及行为学来研究P. polytes超基因拟态的功能基础，提供同类研究中最全面的研究。这样做，这项工作将大大扩展已知的作用的性别决定途径，并产生一般的见解的分子和细胞的原因，性别分化，二型性，和性别限制的多态性。具体而言，我们将：目标1：确定超基因拟态的遗传基础，无论是在特定的基因和替代拟态等位基因的突变起源，使用综合关联映射方法结合结构变异检测。我们将使用基因组富集方法和DNA测序，从每个翅型表型的多个个体中生成目标基因组间隔的高覆盖率序列数据。然后，我们将组装这些数据对一个有针对性的参考大会，并检查多态性和翅膀模式表型之间的统计关联，以表征在一个精细的尺度超基因拟态的功能遗传基础。目的2：研究在整个翅发育过程中候选基因表达的组织特异性模式。我们将通过使用qRT-PCR在整个发育过程中运行一个完整的时间序列，分析具有各种拟态基因型的雄性和雌性的前翅、后翅和非翅组织中关键间隔内所有基因的表达，来检查与翅型相关的表达模式。然后，我们将使用原位杂交进行随访，以检查从qRT-PCR时间序列中确定的关键时间点的表达空间模式，并使用RNA-seq来表征拟态超基因的下游靶标。目标3：功能测试的基因和推测的分子机制负责模仿多态性和研究潜在的多效性对择偶行为的影响。我们将使用RNAi在翅膀发育期间敲低表达来功能性地测试我们的候选拟态基因。此外，我们将跟进的初步数据表明，拟态超基因对择偶行为的多效性影响的偏好测试所有拟态基因型的蝴蝶大样本。

DESCRIPTION (provided by applicant): Sex-limited polymorphism is widespread in animals, including a variety of human traits and diseases, yet we lack a general functional understanding of sex-limited polymorphism in any organism. "Supergene" mimicry in the swallowtail butterfly Papilio polytes stands out as a particularly striking example of sex-limited polymorphism and one that is amenable to functional characterization. While much theoretical work has explored the evolutionary dynamics of supergene mimicry, its molecular and developmental basis is virtually unexplored. We propose to investigate the functional basis of supergene mimicry in P. polytes by integrating genomics, functional genetics, molecular and developmental biology, and behavior, providing the single most comprehensive investigation of its kind. In so doing, this work will greatly expand the known role of the sexual determination pathway, and generate general insights into molecular and cellular causes of sexual differentiation, dimorphism, and sex-limited polymorphism. Specifically, we will: Aim 1: Identify the genetic basis of supergene mimicry, both in terms of the specific gene and the mutational origin of alternative mimicry alleles, using a comprehensive association mapping approach combined with structural variant detection. We will use a genomic enrichment method and DNA sequencing to generate high coverage sequence data for the target genomic interval from multiple individuals of each wing pattern phenotype. We will then assemble these data against a targeted reference assembly and examine statistical associations among polymorphisms and wing pattern phenotype to characterize the functional genetic basis for supergene mimicry at a fine scale. Aim 2: Characterize tissue-specific patterns of candidate gene expression throughout the process of wing development. We will examine wing pattern associated expression patterns by using qRT-PCR to run a complete time series across development, analyzing expression of all genes in our critical interval, on forewings, hindwings, and non-wing tissue, for males and females with various mimicry genotypes. We will then follow-up using in situ hybridization, to examine spatial patterns of expression at critical time points identified from the qRT-PCR time series, and RNA-seq, to characterize the downstream targets of the mimicry supergene. Aim 3: Functionally test the genes and inferred molecular mechanisms responsible for mimetic polymorphism and examine potential pleiotropic effects on mate choice behavior. We will functionally test our candidate mimicry gene(s) using RNAi to knock-down expression during wing development. Furthermore, we will follow-up on preliminary data that suggest the mimicry supergene has pleiotropic effects on mate choice behavior by preference testing large samples of butterflies of all mimicry genotypes.