Abstract
BackgroundIn conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously.ResultsFirst, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately.ConclusionA simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.
Highlights
The majority of plant progenies inherit their plastid and mitochondrial DNA from the maternal parent, and in recent decades, plastid and mitochondrial genomes have been used widely in studies on diversity and evolution
We developed a method that can enrich plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) simultaneously
Because the method can only increase the proportion of ptDNA and mtDNA in a DNA sample, we named it Partial Concentration Extraction (PCE)
Summary
The majority of plant progenies inherit their plastid and mitochondrial DNA from the maternal parent, and in recent decades, plastid and mitochondrial genomes have been used widely in studies on diversity and evolution. Researchers purify ptDNA and mtDNA from green leaves and etiolated seedlings separately employing densitygradient ultracentrifugation (CsCl, sucrose, or percol) [1,2,3,4]. This demanding protocol is unsuitable for wide use for plant plastid and mitochondrial genome sequencing. High-throughput sequencing platforms have been used to capture sequence data from many individual PCR amplifications that cover the entire plastid or mitochondrial genome [5,6] Because this method requires a reference sequence, it can be used only for a few species; it is time consuming. We describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously
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