Characterizing the Molecular Basis of Supergene Mimicry in Butterflies

描述(申请人提供)：性别限制多态在动物中广泛存在，包括各种人类特征和疾病，但我们对任何生物体中的性别限制多态缺乏普遍的功能了解。燕尾蝴蝶Papilio Polytes中的“超基因”模仿是性别限制多态的一个特别显著的例子，也是一种服从功能特征的例子。虽然许多理论工作已经探索了表基因模仿的进化动力学，但其分子和发育基础几乎还没有被探索。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，提供同类研究中最全面的一项研究，来研究P.Polytes中超基因模仿的功能基础。通过这样做，这项工作将极大地扩展性别决定途径的已知作用，并产生对性分化、二型性和性别限制性多态的分子和细胞原因的一般见解。具体地说，我们将：目标1：利用全面的关联作图方法结合结构变异检测，从特定基因和替代拟态等位基因的突变来源两个方面确定超基因拟态的遗传基础。我们将使用基因组富集法和DNA测序来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，我们将根据有针对性的参考集合收集这些数据，并检查多态和翅膀模式表型之间的统计关联，以在精细的尺度上表征超基因模仿的功能遗传学基础。目的2：研究翅膀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来检查与翅膀模式相关的表达模式，分析在我们的关键时间间隔内，具有不同拟态基因的雄性和雌性前翼、后翼和非翅膀组织中所有基因的表达。然后，我们将继续使用原位杂交，以检查从qRT-PCR时间序列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标。目的3：从功能上测试与模拟多态有关的基因和推测的分子机制，并研究其对配偶选择行为的潜在多效性影响。我们将使用RNAi在翅膀发育过程中下调表达，从功能上测试我们的候选拟态基因(S)。此外，我们将通过对所有拟态基因的大样本蝴蝶进行偏好测试，对表明拟态超基因对配偶选择行为具有多效性的初步数据进行后续研究。

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.