Dataset Information

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

ABSTRACT:

Background

In 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.

Results

First, 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.

Conclusion

A 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.

SUBMITTER: Hao W

PROVIDER: S-EPMC4177114 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

Hao Wanjun W Fan Shihang S Hua Wei W Wang Hanzhong H

PloS one 20140924 9

<h4>Background</h4>In 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 ...[more]

PMID: 25251391

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Project description:BACKGROUND: The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA) of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA) and plastid DNA (ptDNA) sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence. RESULTS: We sequenced 30 and 420 kilobases (kb) of the mitochondrial and plastid genomes of V. carteri, respectively -- resulting in partial assemblies of these genomes. The mitochondrial genome is the most bloated green-algal mtDNA observed to date: ~61% of the sequence is noncoding, most of which is comprised of short palindromic repeats spread throughout the intergenic and intronic regions. The plastid genome is the largest (>420 kb) and most expanded (>80% noncoding) ptDNA sequence yet discovered, with a myriad of palindromic repeats in the noncoding regions, which have a similar size and secondary structure to those of the mtDNA. We found that 15 kb (~0.01%) of the nuclear genome are homologous to the palindromic elements of the mtDNA, and 50 kb (~0.05%) are homologous to those of the ptDNA. CONCLUSION: Selfish elements in the form of short palindromic repeats have propagated in the V. carteri mtDNA and ptDNA, resulting in the distension of these genomes. Copies of these same repeats are also found in a small fraction of the nucDNA, but appear to be inert in this compartment. We conclude that the palindromic repeats in V. carteri represent a single class of selfish DNA and speculate that the derivation of this element involved the lateral gene transfer of an organelle intron that first appeared in the mitochondrial genome, spreading to the ptDNA through mitochondrion-to-plastid DNA migrations, and eventually arrived in the nucDNA through organelle-to-nucleus DNA transfer events. The overall implications of palindromic repeats on the evolution of chlorophyte organelle genomes are discussed.

Dataset Information

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

Background

Results

Conclusion

Publications

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

Similar Datasets

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