Dataset Information

High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.

ABSTRACT:

Background

Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used.

Methodology/principal findings

To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results.

Conclusions/significance

These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

SUBMITTER: Zeng CL

PROVIDER: S-EPMC3504036 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.

Zeng Chang-Li CL Wang Guang-Yong GY Wang Jian-Bo JB Yan Gui-Xin GX Chen Bi-Yun BY Xu Kun K Li Jun J Gao Gui-Zhen GZ Wu Xiao-Ming XM Zhao Bo B Liu Lei L

PloS one 20121121 11

<h4>Background</h4>Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linka ...[more]

PMID: 23185237

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Project description:Moricandia arvensis, a plant species originating from the Mediterranean, has been classified as a rare C3-C4 intermediate species, and it is a possible bridge during the evolutionary process from C3 to C4 plant photosynthesis in the family Brassicaceae. Understanding the genomic structure, gene order, and gene content of chloroplasts (cp) of such species can provide a glimpse into the evolution of photosynthesis. In the present study, we obtained a well-annotated cp genome of M. arvensis using long PacBio and short Illumina reads with a de novo assembly strategy. The M. arvensis cp genome was a quadripartite circular molecule with the length of 153,312 bp, including two inverted repeats (IR) regions of 26,196 bp, divided by a small single copy (SSC) region of 17,786 bp and a large single copy (LSC) region of 83,134 bp. We detected 112 unigenes in this genome, comprising 79 protein-coding genes, 29 tRNAs, and four rRNAs. Forty-nine long repeat sequences and 51 simple sequence repeat (SSR) loci of 15 repeat types were identified. The analysis of Ks (synonymous) and Ka (non-synonymous) substitution rates indicated that the genes associated with "subunits of ATP synthase" (atpB), "subunits of NADH-dehydrogenase" (ndhG and ndhE), and "self-replication" (rps12 and rpl16) showed relatively higher Ka/Ks values than those of the other genes. The gene content, gene order, and LSC/IR/SSC boundaries and adjacent genes of the M. arvensis cp genome were highly conserved compared to those in related C3 species. Our phylogenetic analysis demonstrated that M. arvensis was clustered into a subclade with cultivated Brassica species and Raphanus sativus, indicating that M. arvensis was not involved in an independent evolutionary origin event. These results will open the way for further studies on the evolutionary process from C3 to C4 photosynthesis and hopefully provide guidance for utilizing M. arvensis as a resource for improvinng photosynthesis efficiency in cultivated Brassica species.

Project description:BACKGROUND: The EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit Capsicum genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in Capsicum genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4E, eIF4G and eIF(iso)4G) break the cycle of several RNA viruses without affecting the plant life cycle, which makes these genes potential targets to screen for resistant germplasm. RESULTS: We developed and assayed a cDNA-based EcoTILLING platform with 233 cultivated accessions of the genus Capsicum. High variability in the coding sequences of the eIF4E and eIF(iso)4E genes was detected using the cDNA platform. After sequencing, 36 nucleotide changes were detected in the CDS of eIF4E and 26 in eIF(iso)4E. A total of 21 eIF4E haplotypes and 15 eIF(iso)4E haplotypes were identified. To evaluate the functional relevance of this variability, 31 possible eIF4E/eIF(iso)4E combinations were tested against Potato virus Y. The results showed that five new eIF4E variants (pvr2(10), pvr2(11), pvr2(12), pvr2(13) and pvr2(14)) were related to PVY-resistance responses. CONCLUSIONS: EcoTILLING was optimised in different Capsicum species to detect allelic variants of target genes. This work is the first to use cDNA instead of genomic DNA in EcoTILLING. This approach avoids intronic sequence problems and reduces the number of reactions. A high level of polymorphism has been identified for initiation factors, showing the high genetic variability present in our collection and its potential use for other traits, such as genes related to biotic or abiotic stresses, quality or production. Moreover, the new eIF4E and eIF(iso)4E alleles are an excellent collection for searching for new resistance against other RNA viruses.

Dataset Information

High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.

Background

Methodology/principal findings

Conclusions/significance

Publications

High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.

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