Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

Abstract

Background: High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.

Results: We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.

Conclusions: Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Figure 1

Agarose gel analysis of high quality genomic DNA isolated from plant nuclei. For each gel one microliter of genomic DNA was loaded onto a 0.8% agarose gel; the left panel is uncut lambda DNA and the right panel is the genomic DNA sample. A, Genomic DNA isolated using Protocol A: 50 ng lambda DNA, Arabidopsis thaliana (At) and 100 ng lambda DNA, Genlisea aurea (Ga). B, Genomic DNA isolated using Protocol B: 60 ng lambda DNA, Brachypodium distachyon (Bd), 60 ng lambda DNA, Spirodela polyrhiza (Sp), 125 ng lambda, Lemna gibba (Lg).

Figure 2

Nuclear preparation of Genlisea aurea DNA reduces the percentage of chloroplast and mitochondrial genomes in short read sequencing experiments. Genlisea aurea chloroplast genome (blue) is 150 kb, mitochondria genome (red) is 500 kb and nuclear genome (green) is 60,000 kb.