Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

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Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A ... continued below

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McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L. & dePamphilis, Claude W. August 26, 2005.

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Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

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  • Journal Name: Biotechniques; Journal Volume: 41; Journal Issue: 1; Related Information: Journal Publication Date: 07/2006

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  • Report No.: LBNL--59014
  • Grant Number: DE-AC02-05CH11231
  • Office of Scientific & Technical Information Report Number: 901813
  • Archival Resource Key: ark:/67531/metadc877582

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  • August 26, 2005

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  • Sept. 22, 2016, 2:13 a.m.

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  • Sept. 30, 2016, 2:42 p.m.

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McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L. & dePamphilis, Claude W. Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes, article, August 26, 2005; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc877582/: accessed December 17, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.