Characterizing the Molecular Basis of Supergene Mimicry in Butterflies

描述（由申请人提供）：性别限制多态性在动物中广泛存在，包括各种人类特征和疾病，但我们缺乏对任何生物体中性别限制多态性的一般功能理解。“超基因”模仿在凤尾蝶（Papilio polytes）中脱颖而出，成为性别限制多态性的一个特别引人注目的例子，并且可以适应功能表征。虽然许多理论工作已经探索了超基因模仿的进化动力学，但其分子和发育基础实际上尚未被探索。本研究拟从基因组学、功能遗传学、分子生物学、发育生物学、行为学等方面综合研究多形拟南蝽超基因模仿的功能基础，为同类研究提供最全面的研究。这样，这项工作将极大地扩展性别决定途径的已知作用，并对性别分化、二态性和性别限制多态性的分子和细胞原因产生一般性见解。具体来说，我们将：目标1：利用结合结构变异检测的综合关联作图方法，从特定基因和替代模仿等位基因的突变起源两方面，确定超基因模仿的遗传基础。我们将使用基因组富集方法和DNA测序，从每个翼型表型的多个个体中生成目标基因组区间的高覆盖率序列数据。然后，我们将根据目标参考组合收集这些数据，并检查多态性和翼型表型之间的统计关联，以在精细尺度上表征超基因模仿的功能遗传基础。目的2：在整个翅膀发育过程中表征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来检查翅膀模式相关的表达模式，分析所有基因在我们的临界区间内的表达，在前翼，后翼和非翅膀组织上，对于具有各种模仿基因型的雄性和雌性。然后，我们将使用原位杂交技术，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，以及RNA-seq，以表征模仿超基因的下游靶点。目的3：功能测试基因和推测的分子机制负责模仿多态性，并检查对配偶选择行为的潜在多效性影响。我们将使用RNAi对候选模仿基因进行功能测试，以敲除翅膀发育过程中的表达。此外，我们将通过对所有模仿基因型的大样本蝴蝶进行偏好测试，对初步数据进行跟踪，这些数据表明模仿超基因对配偶选择行为具有多效性影响。

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.