Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

The chloroplast genome arrangement ofLobelia thuliniana (Lobeliaceae): Expansion of the inverted repeat in an ancestor of theCampanulales

A clone-bank ofSac I restriction fragments was constructed from the chloroplast DNA (cpDNA) ofLobelia thuliniana E. B. Knox (Lobeliaceae). These cloned fragments and a set of 106 clones spanning the tobacco chloroplast genome were used as probes to determine the cpDNA restriction fragment arrangement forSac I and six other restriction enzymes (BamH I,EcoR V,Hind III,Nci I,Pst I, andXho I) and the chloroplast genome arrangement ofL. thuliniana relative to tobacco, which has been fully sequenced and is collinear with the hypothesized ancestral genome arrangement of angiosperms. The results confirm and refine our previous understanding of the chloroplast genome arrangement in the large single-copy region (LSC) and reveal (1) a roughly 11 kilobase (kb) expansion of the inverted repeat (IR) into the small single-copy region (SSC) and (2) apparent sequence divergence of the DNA segment inL. thuliniana that corresponds to ORF1901 in tobacco. The expansion of the IR into the SSC is present in all other examined members ofLobeliaceae, Cyphiaceae, andCampanulaceae, which indicates that the IR expansion was an early event in the cpDNA evolution of theCampanulales. The IR expansion into the SSC was not present inSphenoclea, which additionally supports exclusion of this genus from theCampanulaceae.     

The Molecular Basis of Genetic Diversity among Cytoplasms of Triticum and Aegilops. III. Chloroplast Genomes of the M and Modified M Genome-Carrying Species

The restriction fragment patterns of chloroplast DNAs of all M or modified M genome-carrying Aegilops species, and those of common wheat (Triticum aestivum), Ae. umbellulata and Ae. squarrosa as referants, have been analyzed using eight restriction endonucleases, BamHI, EcoRI, HindIII, KpnI, PstI, SalI, SmaI and XhoI. Nine distinctly different chloroplast genomes are evident, and the mutual relatedness among them is estimated based on the number of different restriction fragments. The results lead to the following conclusions. (1) Chloroplast genomes of three Comopyrum species, Ae. comosa, Ae. heldreichii and Ae. uniaristata, are more closely related with each other and are greatly different from those of the Amblyopyrum species, Ae. mutica, and of Ae. umbellulata and Ae. squarrosa. (2) Ae. crassa's chloroplast genome lies at the center of chloroplast genome diversification, whereas those of common wheat, Ae. squarrosa and Ae. uniaristata are three extreme forms lying far from the center. (3) Chloroplast genomes of three 4x species, Ae. biuncialis, Ae. columnaris and Ae. triaristata, arose from Ae. umbellulata, and that of a fourth 4x species, Ae. ventricosa , arose from Ae. squarrosa. The chloroplast origins of two other 4x species, Ae. ovata and Ae. crassa, remain unsolved. (4) The chloroplast genomes of two Ae. mutica strains are identical, even though their cytoplasms exert quite different effects on male fertility, heading date and growth vigor of common wheat.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Comparative analysis of chloroplast DNA in Pyrus species: physical map and gene localization

A physical map of chloroplast DNA (cpDNA) of pear [Pyrus ussuriensis var. hondoensis (Nakai et Kikuchi) Rehder] was constructed using five restriction enzymes, SalI, XhoI, BamHI, SacI and PstI. This information will make it possible to investigate the phylogenetic relationships between Pyrus species. Pear cpDNA was found to be a circular molecule with a total size of about 156 kb in which two inverted repeats of 24.8 kb divide the molecule into small (17 kb) and large (90 kb) single-copy regions. The endonuclease recognition sites in the physical map were determined by single and double digestion of 13 lambda phage clones which covered the entire sequence of the pear cpDNA. Twenty nine genes were localized on the physical map of the pear cpDNA. The structure of pear cpDNA was almost the same in terms of genome size and gene order as that of tobacco cpDNA. RFLP analysis was carried out on cpDNAs from five Pyrus species (Pyrus pyrifolia, Pyrus ussuriensis, Pyrus calleryana, Pyrus elaeagrifolia and Pyrus communis). Two mutations, a recognition-site mutation and a length mutation (deletion), were found only in the cpDNA of P. pyrifolia cultivars. These mutations were localized on the physical map of pear cpDNA. The number of mutations of cpDNA in Pyrus species are small in comparison with those of other angiosperms, suggesting a high degree of genome conservatism in Pyrus species.     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure

Summary The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.     

Structural analysis of chloroplast DNA in Prunus (Rosaceae): evolution, genetic diversity and unequal mutations

In order to understand the evolutionary aspects of the chloroplast DNA (cpDNA) structures in Rosaceous plants, a physical map of peach (Prunus persica cv. Hakuhou) cpDNA was constructed. Fourteen lambda phage clones which covered the entire sequence of the peach cpDNA were digested by restriction enzymes (SalI, XhoI, BamHI, SacI, and PstI) used singly or in combination. The molecular size of peach cpDNA was estimated to be about 152 kb. The gene order and contents were revealed to be equivalent to those of standard type of angiosperms by the localization of 31 genes on the physical map. Eighteen accessions from 14 Prunus species (P. persica, P. mira, P. davidiana, P. cerasis, P. cerasifera, P. domestica, P. insititia, P. spinosa, P. salicina, P. maritima, P. armeniaca, P. mume, P. tomentosa, P. zippeliana, and P. salicifolia) and one interspecific hybrid were used for the structural analysis of cpDNAs. Seventeen mutations (16 recognition site changes and one length mutation) were found in the cpDNA of these 18 accessions by RFLP analysis allowing a classification into 11 genome types. Although the base substitution rate in the recognition site (100p = 0.72) of cpDNA in Prunus was similar to that of other plants, i.e., Triticum–Aegilops, Brassica, and Pisum, it differed from Pyrus (100p = 0.15) in Rosaceae. Seven mutations including one length mutation were densely located within a region of about 9.1 kb which includes psbA and atpA in the left border of a large single-copy region of Prunus cpDNAs. The length mutation was detected only in P. persica and consisted of a 277 bp deletion which occurred in a spacer region between the trnS and trnG genes within the 9.1 kb region. Additional fragment length mutations (insertion/deletion), which were not detected by RFLP analysis, were revealed by PCR and sequence analyses in P. zippeliana and P. salicifolia. All of these length mutations occurred within the 9.1 kb region between psbA and atpA. This region could be an intra-molecular recombinational hotspot in Prunus species.     

Chloroplast genome differences between Paspalum dilatatum Poir and the related species P. notatum Flugge

 Chloroplast DNA (cpDNA) of Paspalum dilatatum and P. notatum was digested singly or in combination with the restriction endonucleases PstI, PvuII, SalI, KpnI and XhoI. Data obtained from filter hybridization experiments with barley and wheat cpDNA probes were used to construct restriction site maps of the chloroplast genomes of the Paspalum species. The cpDNA fragments were ordered into a circular configuration of approximately 139.3 kbp that contained two inverted repeat regions of approximately 23 kbp and a small and large single-copy region of approximately 11 kbp and 83 kbp, respectively. The cpDNA maps showed that P. dilatatum and P. notatum shared a large number of restriction sites with the proportion of shared restriction sites S=0.90. No restriction site differences were detected in the KpnI maps. Eight species-specific restriction site differences that could be used to identify the cytoplasm of each Paspalum species were identified in the PstI, PvuII, SalI, and XhoI cleavage maps. The overall structural organization of the Paspalum cpDNAs is rather similar to those of most cpDNAs from other plants. The results presented in this study will be of value for exploring further phylogenetic relationships within the genus Paspalum. Received: 27 February 1997 / Accepted: 7 March 1997     

The consensus land plant chloroplast gene order is present, with two alterations, in the moss Physcomitrella patens

Summary A restriction endonuclease cleavage site map for the enzymes ClaI and BglII, and a partial map for SacI, has been constructed for the chloroplast genome of the moss Physcomitrella patens (Hedw.) BSG. The plastid chromosome contains approximately 122 kb organized into small (21 kb) and large (82 kb) single-copy regions separated by two copies of a repeat sequence (9.4 kb) oriented in an inverted arrangement. Genes for 17 proteins and 2 ribosomal RNAs have been mapped using heterologous probes from corn, spinach, pea, and petunia. The general order and arrangement of the moss chloroplast genes are similar to the consensus land plant genome typified by that of spinach, with two major exceptions. First, there is an inversion of approximately 20 kb, bordered internally by psbA and atpH, and also containing the genes atpF and atpA. Second, rpl2 and rps19 have been relocated to a different position within the large single-copy region, adjacent to the 20 kb inversion.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

Background

Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.

Methodology/Principal Findings

We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.

Conclusion

Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.     

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae,Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.     

Identification of plastid genotypes in Oenothera subsect. Munzia by restriction fragment length polymorphisms (RFLPs)

 Five discrete plastid genotypes (plastomes), designated I–V and typified by Oenothera Hookeri, biennis, Lamarckiana, parviflora and argillicola respectively, have been previously characterized within the European subsect. Euoenothera. The evolutionarily more-derived plastome types (I, II and V) are generally less tolerant of new hybridization events than the ancestral types (III and IV), and were first identified based on their incompatibility reactions with standard hybrid nuclei. Restriction maps for all five plastomes are available for the enzymes PvuII, SalI, KpnI and PstI (Gordon et al. 1982). The present study employs PvuII and KpnI restriction digests to compare 28 of the 45 species of subsect. Munzia with Euoenothera plastomes I–V. The results of plastome RFLP fingerprinting show uniform divergence of the South American taxa from their European congeners; all share the previously documented 45-kb inversion in the large single-copy region reported by Hachtel et al. (1991). However, at least six new plastome types have evolved within subsect. Munzia, giving rise to small-fragment size differences of 0.1–0.7 kb. In two of these cases (Oe. featherstonei and Oe. longiflora) unique fragments occurred. For Oe. featherstonei the unique KpnI fragment resulted from a novel 2.2 kb insertion, whereas in Oe. longiflora an additional PvuII restriction site has been created. Received: 2 June 1998 / Accepted: 14 July 1998     

Chloroplast genome characterization in the red alga Griffithsia pacifica

Summary It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rhodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes.In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoescht-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.