Organellar genome sequences provide numerous phylogenetic markers andyield 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.

Introduction

Unlike eukaryotic nuclear genomes, organellar genomes occur in high copy number per cell and are of a size more amenable for complete sequencing. Gene orthology is typically clear even across a wide taxonomic range; thus, organellar genes provide a disproportionately large fraction of genes currently used for phylogeny (1). Furthermore, comparisons of organellar genomes can provide insights into the evolutionary transformations from cyanobacteria and proteobacteria into plastids and mitochondria, the functions of these organelles, and the patterns of co-evolution that have occurred with the many nuclear genes whose products function inside of these organelles.

The earliest organelle genome sequences were generated by digesting, cloning, and mapping purified organellar DNA, followed by sequencing small fragments individually from the clone bank (2). With the advent of cost-effective, high-throughput sequencing, genome sequences are being generated more efficiently by shotgun cloning directly from organellar DNA isolations, performing a single sequencing read from each end of a large number of randomly selected clones, then assembling these into a complete genome sequence computationally. There are several possibilities for preparing a template that is acceptable for this process and, for some taxa, these have become simple and reliable protocols (megasun.bch.umontreal.ca/People/lang/FMGP/methods/mtDNA.html). Intact organelles can be isolated, most often by sucrose or Percoll gradient centrifugations (3), and in some cases, the differences in base composition and topology (i.e., circular versus linear DNA) between organellar and nuclear DNAs can be exploited using bis-benzimide or cesium chloride gradients to isolate organellar DNA for sequencing (4). Large quantities of fresh tissue are typically necessary to produce small amounts of organellar DNA (although it is often possible to amplify these small amounts using rolling circle amplification or RCA). Even after enrichment, the low proportion of organellar to nuclear DNA can lead to significant nuclear contamination (>50% of the total DNA) in many species, including those with large nuclear genomes or interfering polyphenolics. Another method is to amplify large sections of organellar DNA by long-PCR between regions for which primers exist, which has been used effectively for many animal mitochondrial DNAs (mtDNAs) and occasionally for plastid DNAs (ptDNAs) as well (5). Jansen et al. (3) review current land plant ptDNA isolation and sequencing methods.

Although these procedures have succeeded for a variety of plastid genomes, many organisms exist for which they are not feasible. It is difficult or impossible to produce significantly enriched organellar DNA from many plants, even with large quantities of fresh tissue. The PCR method (5) eliminates the need for enriched ptDNA, but is only practical if the genome is not highly rearranged or if gene order is known via prior mapping. A set of PCR primers spaced around the entire plastid genome is necessary, and amplification-induced artifacts may occur. Heterotrophic plants often exhibit both rapid sequence divergence and unusual plastid ultrastructure that make these procedures infeasible; accordingly, the complete sequence of only one heterotrophic angiosperm has been published (6). The method we present enables plastid genome sequencing from both parasitic and nonparasitic plants using small amounts of fresh, frozen, or desiccated tissue and should be equally applicable for sequencing mitochondrial genomes.

Materials and Methods

DNA Isolation and Partial Genomic Library Construction

Fresh material from Cuscuta exaltata, Cuscuta obtusiflora (parasitic), and Ipomoea purpurea (autotrophic) was grown from seed. Tissue from Yucca schidigera (autotrophic) was collected and snap-frozen in liquid nitrogen. Nuclear genome sizes of all species were determined by flow cytometry following the protocol described previously (7). One gram tissue from each plant was pulverized to powder by mortar and pestle after being frozen in liquid nitrogen for 20 s. DNA was extracted in 10 mL buffer using a 2× cetyltrimethylammonium bromide (CTAB) procedure (8) with 1% polyethylene glycol (PEG) 8000 in the buffer. After isopropanol precipitation, DNA was spooled out, rinsed with 70% ethanol, and resuspended in 500 µL water. To clean and concentrate the DNA, it was reprecipitated by adding 125 µL 4 M NaCl plus 625 µL 13% PEG 8000 and incubated on ice for 20 min before centrifugation at 13,000× g at 4°C for 15 min. DNA pellets were resuspended in 75 µL water. DNA fragments ranging from 40 to 45 kb were excised from a 0.8% agarose gel using field inversion gel electrophoresis (FIGE).