Florida Museum of Natural History, University of Florida, Gainesville, FL, USA.
Division of Plant Sciences, University of Missouri, Columbia, MO, USA.
Methods Mol Biol. 2023;2545:475-490. doi: 10.1007/978-1-0716-2561-3_25.
The genetic consequences following polyploidy (i.e., whole-genome duplication; WGD) vary greatly across organisms and through time since polyploidization. At the gene level in allopolyploids, changes include loss/retention of both parental gene copies, function/expression divergence between the two parental copies, and silencing of one parental copy. Functional studies of genes with different retention patterns contribute to a better understanding of the genetic factors underlying the success of polyploids. Most research on gene functions to date focuses on a few well-established genetic models or crops. However, many species that best exemplify the polyploidy process are nongenetic models; the lack of an efficient genome editing system hinders functional studies in these systems. In this chapter, we discuss the considerations of developing CRISPR, a robust and efficient genome editing system, in polyploid plants that are not genetic models. We use diploid and polyploid Tragopogon (Asteraceae) as examples of a well-studied evolutionary model system for which abundant genetic and genomic resources are lacking. Using this system, we provide our protocols for sgRNA design, plasmid construction, a useful protoplast transient assay, and a plant transformation method we developed for this system. We also provide suggestions for possible modifications to these protocols to help promote successful application to other non-models. With the rapid applications of CRISPR in plant sciences, the broad adaptation of CRISPR in studies of the evolutionary significance of WGD holds enormous potential. We hope our studies and methods developed for polyploid Tragopogon will provide a guideline for establishing a CRISPR system in other nongenetic model polyploids of evolutionary or other interest.
多倍体（即全基因组加倍；WGD）后遗传后果在不同生物和多倍体化后时间跨度内变化很大。在异源多倍体中，在基因水平上的变化包括双亲基因副本的丢失/保留、两个亲本副本之间的功能/表达分化以及一个亲本副本的沉默。对具有不同保留模式的基因的功能研究有助于更好地理解多倍体成功的遗传因素。迄今为止，大多数关于基因功能的研究都集中在少数成熟的遗传模型或作物上。然而，许多最好地体现多倍体化过程的物种不是遗传模型；缺乏有效的基因组编辑系统阻碍了这些系统中的功能研究。在本章中，我们讨论了在不是遗传模型的多倍体植物中开发 CRISPR（一种强大而高效的基因组编辑系统）的考虑因素。我们使用二倍体和多倍体蒲公英（菊科）作为一个经过充分研究的进化模型系统的例子，该系统缺乏丰富的遗传和基因组资源。使用该系统，我们提供了 sgRNA 设计、质粒构建、有用的原生质体瞬时测定和我们为该系统开发的植物转化方法的协议。我们还提供了对这些协议进行可能修改的建议，以帮助促进其在其他非模型中的成功应用。随着 CRISPR 在植物科学中的快速应用，CRISPR 在研究 WGD 的进化意义方面的广泛适应性具有巨大的潜力。我们希望我们为多倍体蒲公英开发的研究和方法将为在其他具有进化或其他兴趣的非遗传模型多倍体中建立 CRISPR 系统提供指导。
