Miller Marisa E, Liberatore Katie L, Kianian Shahryar F
Cereal Disease Laboratory, United States Department of Agriculture-Agricultural Research Service; Department of Horticultural Science, University of Minnesota.
Cereal Disease Laboratory, United States Department of Agriculture-Agricultural Research Service; Department of Plant Pathology, University of Minnesota.
J Vis Exp. 2017 Jul 28(125):55528. doi: 10.3791/55528.
Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.
植物细胞器基因组包含大量重复元件，这些元件可能会进行配对或重组，以形成复杂结构和/或亚基因组片段。细胞器基因组在给定的细胞或组织类型中也以混合状态存在（异质性），并且大量的亚型可能会在整个发育过程中或受到胁迫时发生变化（亚化学计量移位）。需要新一代测序（NGS）技术来更深入地了解细胞器基因组的结构和功能。传统的测序研究使用几种方法来获取细胞器DNA：（1）如果使用大量起始组织，将其匀浆并进行差速离心和/或梯度纯化。（2）如果使用较少量的组织（即种子、材料或空间有限时），则按照（1）中的相同步骤进行操作，随后进行全基因组扩增以获得足够的DNA。（3）生物信息学分析可用于对总基因组DNA进行测序并解析出细胞器读数。所有这些方法都有其固有的挑战和权衡。在（1）中，可能难以获得如此大量的起始组织；在（2）中，全基因组扩增可能会引入测序偏差；而在（3）中，核基因组和细胞器基因组之间的同源性可能会干扰组装和分析。在具有大核基因组的植物中，富集细胞器DNA以降低测序成本和生物信息学分析的序列复杂性是有利的。在这里，我们将传统的差速离心方法与第四种方法（一种改良的CpG甲基化下拉方法）进行比较，以将总基因组DNA分离为核和细胞器组分。两种方法都能产生足够用于NGS的DNA，这些DNA高度富集细胞器序列，尽管在线粒体和叶绿体中的比例不同。我们展示了针对小麦叶片组织对这些方法的优化，并在样品输入、操作简便性和下游应用的背景下讨论了每种方法的主要优缺点。
