Discovery and functional analysis of novel candidate genes and variants underlying craniofacial diversification in Cyprinodon pupfishes

项目总结： 颅面畸形是人类最常见的出生缺陷形式，但它们的分子 人们对此仍知之甚少。高度保守的颅面发育途径 在不同的脊椎动物物种之间共享是由适应性进化塑造的，以产生 适应性颅面表型的巨大多样性。对中国的基础调查 这些表型的遗传基础将导致更好的诊断、预防和治疗 人类的先天缺陷。事实上，关于许多人的遗传基础的补充或新信息 模型系统中的人类病理通常可以从自然发生的系统中获得，该系统 表现出相似的不同表型。这些自然系统现在可以用于基因组 和转基因方法，并为“进化”的前向遗传学提供了机会。 在这里，我建议利用我的实验室开发一种新的脊椎动物的丰富经验 自下而上的系统：加勒比海鲤鱼的高度分化的头面部形态。 我们初步的全基因组差异扫描和关联图谱已经确定了这两个 已知的头面部候选基因和与颌骨相关的10个新候选基因 长度变化。不同的基因组区域通常局限于只有一个基因的单个基因。 或者一些上游调控区域或内含子区域的候选变体。我们的预赛 数量性状基因座图谱还表明，这些区域中的一些可以解释高达15%的 实验室饲养的F2代杂交后代的颌长变异。因此，我假设固定的突变在 这些物种控制着已知和新的颅面部的时空表达 鲤鱼高度分化的头面部特征的基因。我提议 在这个非模型系统中研究新的适应性表型的遗传基础 群体基因组学、从头组装基因组学、数量遗传学、 转录学、原位杂交和CRISPR-Cas9基因组编辑。 我们在加勒比海鲤鱼上的初步成功证明了我们的方法和 有扩张的潜力。瓢虫鱼显示出新的头面部特征；持续的基因流动和强大的 选择为精细定位候选变异提供了一个理想的自然“实验” 有了这些特点。我们还在首创原位杂交和CRISPR-Cas9方法 鲤鱼。通过在自然系统中整合候选基因和变体发现来展示 不同的头面部特征和强大的功能分析，拟议的研究将 展示新的非模型系统的可行性和威力，以获得对 人类疾病的发育遗传学。 1

Project Summary: Craniofacial abnormalities are the most common form of human birth defects, but their molecular basis remains poorly understood. Highly conserved craniofacial developmental pathways shared across diverse vertebrate species have been shaped by adaptive evolution to produce a tremendous diversity of adaptive craniofacial phenotypes. Fundamental investigation of the genetic basis of these phenotypes will lead to better diagnosis, prevention, and treatment of human birth defects. Indeed, complementary or new information on the genetic basis of many human pathologies in model systems is often obtainable from naturally occurring systems that display analogous divergent phenotypes. These natural systems are now feasible for genomic and transgenic approaches and provide an opportunity for ‘evolutionary’ forward genetics. Here I propose to leverage my lab’s extensive experience developing a new vertebrate system from the ground up: highly divergent craniofacial morphology in Caribbean pupfishes. Our preliminary genome-wide divergence scans and association mapping have identified both well-known craniofacial candidate genes and ten new candidate genes associated with jaw length variation. Divergent genomic regions are often restricted to a single gene with only one or a few candidate variants in upstream regulatory or intronic regions. Our preliminary quantitative trait locus mapping also indicates that some of these regions explain up to 15% of jaw length variation in lab-reared F2 intercrosses. Thus, I hypothesize that fixed mutations in these species control spatiotemporal expression of both known and novel craniofacial genes underlying highly divergent craniofacial features in pupfishes. I propose to investigate the genetic basis of novel adaptive phenotypes in this non-model system using a combination of population genomics, de novo genome assembly, quantitative genetics, transcriptomics, in situ hybridization, and CRISPR-Cas9 genome editing. Our initial success in Caribbean pupfishes demonstrates the power of our approach and potential for expansion. Pupfish exhibit novel craniofacial traits; ongoing gene flow and strong selection provide an ideal natural ‘experiment’ for fine-mapping candidate variants associated with these traits. We are also pioneering in situ hybridization and CRISPR-Cas9 approaches in pupfishes. By integrating candidate gene and variant discovery in a natural system exhibiting diverse craniofacial features with powerful functional assays, the proposed research will demonstrate the feasibility and power of new non-model systems to gain novel insights into the developmental genetics of human diseases. 1