RIG: Genome Size and Karyotype Evolution in Esociform Fishes

这项研究的目的是确定在观察到的团齿目鱼类基因组大小和核型变异中起作用的因素，该目鱼类包括广为人知的北方狗鱼。经典的细胞遗传学研究表明，这类鱼的基因组特征具有广泛的多样性。这种可变性，加上对该群体进化关系的新共识，使其成为在染色体和基因组水平上关于进化的潜在机制和后果的新想法的理想试验场。该项目将产生的知识、数据和材料将为更广泛地研究鱼类染色体和基因组进化的模式和节奏提供基础，鱼类是物种最丰富的脊椎动物家族。这项研究有两个具体目标：a)确定Esociform Umbra属基因组扩展的特征；b)制作Esocias及其近亲的分子细胞遗传学研究所需的方案和试剂。从长远来看，这些协议可能适用于其他表现出独特基因组组织特征的谱系。这三个种中的两个物种的基因组组成的DNA大约是在其他Esociform属中观察到的DNA的两倍。鉴于最近人们对生物进化和基因组大小之间的假设关系重新产生了兴趣，确定促成基因组扩张的因素具有重要意义。初步数据表明，一些转座元件(TE)群体的扩增是导致Umbra基因组扩张的主要力量。目前的项目旨在使用几条互补的证据线来检验这一假设。Southern和斑点杂交实验将揭示不同基因组成分对基因组扩增的相对贡献。将使用以聚合酶链式反应为目标的克隆实验的深度测序和系统发育分析来表征具有代表性的Esociform基因组中TE种群的多样性和相对年龄。最后，荧光原位杂交观察将有助于确定基因组大小变化和核型排列之间任何假定关系的特征。另一个有趣的方面是染色体稳定性的明显变化，这是该群体核型多样性的基础。具体来说，经典的细胞遗传学研究和化石证据表明，在6000多万年的时间里，esox属成员的核型几乎没有变化；而在另一个极端，esociform的不同种群显示出不同的核型。总体而言，二倍体染色体数目在22到78之间。核型和核型稳定性的这种可变性突出了研究胚胎细胞中的染色体进化以揭示形成基因组变异的机制的特征的价值。该项目通过开发和应用染色体特异的DNA探针，启动了确定脊椎动物染色体进化模式和速度的任务。这项工作还将有助于确定细胞遗传学数据在鱼类系统发育中的用途。该项目将研究在染色体和基因组规模上塑造遗传变异性的过程。这项研究的重点是一种已知在染色体和基因组特征上具有广泛而有趣的多样性的鱼类。从这项工作中获得的洞察力将使人们更好地理解产生某些遗传多样性的机制，这些多样性是生命的特征，也是生物进化的燃料。这项研究将有助于促进初级研究人员的专业发展，他是科学界代表性较低的群体的成员，也是一名研究生，他将获得一套目前在美国进化生物学家中尚未广泛使用的科学工具的实践经验。由于研究人员与一家自然历史博物馆的关系以及他对其教育活动的参与，该项目的结果将迅速和方便地提供给普通公众。

The goal of this research is to determine factors that played a role in shaping the observed variation in genome size and karyotype among the fishes of the order Esociformes, a lineage that includes the widely recognized Northern Pike. Classical cytogenetic studies have demonstrated extensive diversity in the genomic characteristics of the species in this group of fishes. This variability, coupled with emerging consensus on the evolutionary relationships of the group makes it an ideal testing ground for emerging ideas on the underlying mechanisms and consequences of evolution at the chromosome and genome levels. The knowledge, data and materials that this project will generate will provide the foundation for broader investigations of the mode and tempo of chromosome and genome evolution in fishes, the most species-rich vertebrate lineage. The research has two specific goals: a) to determine the characteristics of the expansion of the genome in the esociform genus Umbra; and b) to produce protocols and reagents necessary for molecular cytogenetic studies of esociforms and their closest relatives. In the long term, these protocols may be applied to other lineages that exhibit unique features of genomic organization. The genome of two of the three species of Umbra comprise approximately two times as much DNA as those observed in other esociform genera. Determining the factors that contribute to this genome expansion is significant in light of the recently renewed interest in hypothesized relationships between organismal evolution and genome size. Preliminary data point to the amplification of some transposable element (TE) populations as a primary force leading to genome expansion in Umbra. The current project is designed to test that hypothesis using several complementary lines of evidence. Southern and dot blot experiments will reveal the relative contributions of different genomic components to genome expansion. Deep sequencing of PCR-targeted cloning experiments and phylogenetic analyses will be used to characterize the diversity and relative age of TE populations in representative esociform genomes. Finally, fluorescence in situ hybridization observations will help define features of any putative relationships between changes in genome size and karyotypic arrangement. Another interesting aspect of genomic variation among esociforms is the apparent change in chromosomal stability that underlies the diversity of karyotypes in the group. Specifically, classical cytogenetic studies and fossil evidence indicate that the karyotypes of members of the genus Esox have remained virtually unchanged for more than 60 million years; while at the other extreme, different populations of the esociform Dallia pectoralis show divergent karyotypes. Overall, diploid chromosome numbers among esociforms range from 22 to 78. This variability in karyotype and karyotypic stability highlights the value of the study of chromosome evolution in esociforms to uncover characteristics of the mechanisms that shape genome variation. This project initiates the task of determining the mode and rate of chromosomal evolution in esociforms through the development and application of chromosome specific DNA probes. This effort will also help determine the utility of cytogenetic data in fish phylogenetics.This project will examine processes that shape genetic variability at the scale of chromosomes and genomes. The research focuses on one group of fishes that is known to possess extensive and interesting diversity in their chromosomal and genomic characteristics. The insights gained from this work will lead to increased understanding of the mechanisms that are responsible for generating some of the genetic diversity that characterizes life and that constitutes the fuel for biological evolution. This research will help foster the professional development of a beginning investigator who is a member of a group underrepresented in science and a graduate student, who will gain hands-on experience with a set of scientific tools that currently are not in widespread use among evolutionary biologists in the U.S. Thanks to the investigator's affiliation with a natural history museum and his participation in its educational activities, the project's results will be made promptly and conveniently available to the general public.