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ASAP: amplification, sequencing & annotation of plastomes.
BMC Genomics. 2005 Dec 07; 6:176.BG

Abstract

BACKGROUND

Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA) is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate approximately 1 - 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera.

RESULTS

100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and comparative genomics studies.

CONCLUSION

This simple, inexpensive method now allows immediate access to plastid sequence, increasing experimental throughput and serving generally as a universal platform for plastid genome characterization. The method applies well to whole genome studies and speeds assessment of variability across species, making it a useful tool in plastid structural genomics.

Links

PMC Free PDF

Authors+Show Affiliations

Dhingra A
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. adhingra@ifas.ufl.edu
Folta KM
No affiliation info available

MeSH

Base SequenceChloroplastsComputational BiologyDNA PrimersDNA, ChloroplastDNA, PlantFragariaGene DeletionGene LibraryGenes, PlantGenomeGenome, PlantMolecular Sequence DataPhylogenyPlastidsPolymerase Chain ReactionPolymorphism, GeneticSequence Analysis, DNASpecies SpecificityTobacco

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16336644

Citation

Dhingra, Amit, and Kevin M. Folta. "ASAP: Amplification, Sequencing & Annotation of Plastomes." BMC Genomics, vol. 6, 2005, p. 176.
Dhingra A, Folta KM. ASAP: amplification, sequencing & annotation of plastomes. BMC Genomics. 2005;6:176.
Dhingra, A., & Folta, K. M. (2005). ASAP: amplification, sequencing & annotation of plastomes. BMC Genomics, 6, 176.
Dhingra A, Folta KM. ASAP: Amplification, Sequencing & Annotation of Plastomes. BMC Genomics. 2005 Dec 7;6:176. PubMed PMID: 16336644.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - ASAP: amplification, sequencing & annotation of plastomes. AU - Dhingra,Amit, AU - Folta,Kevin M, Y1 - 2005/12/07/ PY - 2005/08/02/received PY - 2005/12/07/accepted PY - 2005/12/13/pubmed PY - 2006/4/14/medline PY - 2005/12/13/entrez SP - 176 EP - 176 JF - BMC genomics JO - BMC Genomics VL - 6 N2 - BACKGROUND: Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA) is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate approximately 1 - 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. RESULTS: 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and comparative genomics studies. CONCLUSION: This simple, inexpensive method now allows immediate access to plastid sequence, increasing experimental throughput and serving generally as a universal platform for plastid genome characterization. The method applies well to whole genome studies and speeds assessment of variability across species, making it a useful tool in plastid structural genomics. SN - 1471-2164 UR - https://www.unboundmedicine.com/medline/citation/16336644/ASAP:_amplification_sequencing_&_annotation_of_plastomes_ DB - PRIME DP - Unbound Medicine ER -