Shedlock Andrew M, Janes Daniel E, Edwards Scott V
Department of Organismic and Evolutionary Biology and Museum of Comparative Zoology, Harvard University, Cambridge, MA.
Methods Mol Biol. 2008;422:91-117. doi: 10.1007/978-1-59745-581-7_7.
Phylogenomics research integrating established principles of systematic biology and taking advantage of the wealth of DNA sequences being generated by genome science holds promise for answering long-standing evolutionary questions with orders of magnitude more primary data than in the past. Although it is unrealistic to expect whole-genome initiatives to proceed rapidly for commercially unimportant species such as reptiles, practical approaches utilizing genomic libraries of large-insert clones pave the way for a phylogenomics of species that are nevertheless essential for testing evolutionary hypotheses within a phylogenetic framework. This chapter reviews the case for adopting genome-enabled approaches to evolutionary studies and outlines a program for using bacterial artificial chromosome (BAC) libraries or plasmid libraries as a basis for completing "genome scans" of reptiles. We have used BACs to close a critical gap in the genome database for Reptilia, the sister group of mammals, and present the methodological approaches taken to achieve this as a guideline for designing similar comparative studies. In addition, we provide a detailed step-by-step protocol for BAC-library screening and shotgun sequencing of specific clones containing target genes of evolutionary interest. Taken together, the genome scanning and shotgun sequencing techniques offer complementary diagnostic potential and can substantially increase the scale and power of analyses aimed at testing evolutionary hypotheses for nonmodel species.
系统发育基因组学研究融合了系统生物学的既定原则，并利用基因组科学产生的大量DNA序列，有望以比过去多几个数量级的原始数据回答长期存在的进化问题。尽管期望针对爬行动物等商业上不重要的物种迅速开展全基因组计划是不现实的，但利用大插入片段克隆的基因组文库的实用方法为物种的系统发育基因组学铺平了道路，而这些物种对于在系统发育框架内检验进化假说至关重要。本章回顾了采用基于基因组的方法进行进化研究的理由，并概述了一个以细菌人工染色体（BAC）文库或质粒文库为基础完成爬行动物“基因组扫描”的计划。我们利用BAC填补了哺乳动物的姐妹类群——爬行动物基因组数据库中的一个关键空白，并介绍了为实现这一目标所采用的方法，作为设计类似比较研究的指南。此外，我们提供了一份详细的逐步方案，用于BAC文库筛选和对含有具有进化意义的目标基因的特定克隆进行鸟枪法测序。总之，基因组扫描和鸟枪法测序技术提供了互补的诊断潜力，并且可以大幅提高旨在检验非模式物种进化假说的分析规模和力度。
