|MILLER, MARISA - University Of Minnesota
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/6/2017
Publication Date: 7/28/2017
Citation: Miller, M.M., Liberatore, K.L., Kianian, S.F. 2017. Optimization and comparative analysis of plant organellar DNA enrichment methods suitable for next generation sequencing. Journal of Visualized Experiments. https://doi.org/10.3791/55528.
Interpretive Summary: We aim to improve understanding of how mitochondria (the "powerhouse of the cell") and chloroplasts (involved in photosynthesis) contribute to plant development, stress response, and overall fitness. Most genome sequencing studies focus on the nuclear genome content as the majority of genes are located in the nucleus. However, additional genetic information is housed in the mitochondria and chloroplast (organellar DNA). We present the optimization of two methods to separate nuclear and organellar DNA from plant leaves, discuss major advantages and disadvantages of each approach, and assess the suitability of resulting DNA from each method for genome sequencing.
Technical Abstract: 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 abundance of sub-types may change through development or when under stress (sub-stoichiometric shifting). Next-generation sequencing technologies are required to obtain a deeper understanding of organellar genome structure and function. Traditional organellar sequencing studies use a number of different methods to obtain organellar DNA: (1) use a large amount of starting tissue, which is homogenized and subjected to differential centrifugation and/or gradient purification, (2) use a smaller amount of tissue (if seeds, material, or space is limited) in the same process as (1) followed by whole genome amplification to obtain sufficient DNA, and (3) sequence total genomic DNA and parse out organellar reads using bioinformatics analysis. All of these methods have inherent challenges and tradeoffs. In (1) it may be difficult to obtain such as large amount of starting tissue, in (2) whole genome amplification could introduce 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 lower sequencing costs and reduce sequence complexity for bioinformatics analyses. Here we compare a traditional differential centrifugation method with an alternative fourth method, a recently adapted methyl-pulldown approach, to fractionate total genomic DNA from nuclear and organellar fractions. Both methods yield sufficient DNA for next generation sequencing that is highly enriched for organellar sequences, albeit in different ratios of mitochondria and chloroplast. We present the optimization of these methods for wheat leaf tissues and discuss major advantages and disadvantages of each approach in the context of (1) required sample input, (2) protocol ease, and (3) downstream application.