Hairpins involving both inverted and direct repeats are associated with homoplasious indels in non-coding chloroplast DNA of Taraxacum (Lactuceae: Asteraceae).

Sequence variation in 2.2 kb of non-coding regions of the chloroplast genome of eight dandelions (Taraxacum: Lactuceae) from Asia and Europe is interpreted in the light of the phylogenetic signal of base substitutions vs. indels (insertions-deletions). The four non-coding regions displayed a total of approximately 30 structural mutations of which 9 are potentially phylogenetically informative. Insertions, deletions, and an inversion were found that involved consecutive stretches of up to 172 bases. When compared to phylogenetic relationships of the chloroplast genomes based on nucleotide substitutions only, many homoplasious indels (33%) were detected that differed considerably in length and did not comprise simple sequence repeats typically associated with replication slippage. Though many indels in the intergenic spacers were associated with direct repeats, frequently, the variable stretches participated in inverted repeat stabilized hairpins. In each intergenic spacer or intron examined, nucleotide stretches ranging from 30 to 60 bp were able to fold into stabilized secondary structures. When these indels were homoplasious, they always ranked among the most stabilized hairpins in the non-coding regions. The association of higher order structures that involve both classes of repeats and parallel structural mutations in hot spot regions of the chloroplast genome can be used to differentiate among mutations that differ in phylogenetic reliability.     

PCR-based RFLP analysis of an intergenic spacer region in cpDNA of some wild wheat species

Wheat is one of the basic food materials for humans and other animals. Continuing studies into breeding new species, which are suitable for agriculture, are based on the contention that the restricting factors are the effects of the nuclear genes. Such focusing of studies on the nuclear genome leads to restriction on the information on organelle DNAs, although cytoplasmic genetic factors are effective on the morphological, physiological and reproductive features of plants. Restriction fragment length polymorphism analysis (RFLP) of chloroplast DNA (cpDNA) has become a powerful tool for studying phylogenetic relationships. In this study, seven wheat species, belonging to Aegilops and Triticum , were studied. An intergenic spacer region of cpDNA was digested by six different restriction endonuclease enzymes, and fragments so obtained were investigated using the agarose gel electrophoresis technique. Discussion on the phylogenetic relationships among Triticum and Aegilops species is based on the resultant data.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 305–310.     

Phylogenetic analysis of North American Elymus and the monogenomic Triticeae (Poaceae) using three chloroplast DNA data sets.

Although the monogenomic genera of the Triticeae have been analyzed in numerous biosystematic studies, the allopolyploid genera have not been as extensively studied within a phylogenetic framework. We focus on North American species of Elymus, which, under the current genomic system of classification, are almost all allotetraploid, combining the St genome of Pseudoroegneria with the H genome of Hordeum. We analyze new and previously published chloroplast DNA data from Elymus and from most of the monogenomic genera of the Triticeae in an attempt to identify the maternal genome donor of Elymus. We also present a cpDNA phylogeny for the monogenomic genera that includes more data than, and thus builds on, those previously published. The chloroplast DNA data indicate that Pseudoroegneria is the maternal genome donor to all but one of the Elymus individuals. There is little divergence among the Elymus and Pseudoroegneria chloroplast genomes, and as a group, they show little divergence from the rest of the Triticeae. Within the monogenomic Triticeae, the problematic group Thinopyrum is resolved as monophyletic on the chloroplast DNA tree. At the intergeneric level, the data reveal several deeper-level relationships that were not resolved by previous cpDNA trees.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

Amplification of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a significant source of characters for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte and monilophyte species to determine the potential utility of PCR amplification primers for 18 noncoding chloroplast DNA regions that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of five cpDNA regions for resolving relationships in Botrychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnL(UAA)-trnF(GAA) intergenic spacer in this group. To these published data we added sequences of the trnS(GCU)-trnG(UUC) intergenic spacer + the trnG(UUC) intron region, the trnS(GGA)-rpS4 intergenic spacer+rpS4 gene, and the rpL16 intron. Both the trnS(GCU)-trnG(UUC) and rpL16 regions are highly variable in angiosperms and the trnS(GGA)-rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the five sampled regions the trnS(GCU)-trnG(UUC) spacer+trnG(UUC) intron region provided the strongest support for the inferred phylogeny.     

Nucleotide diversity and phylogenetic relationships among <Emphasis Type="Italic">Gladiolus</Emphasis> cultivars and related taxa of family Iridaceae

The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA–trnH and trnL–trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA–trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL–trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.     

Utility of selected non-coding chloroplast DNA sequences for lineage assessment of Musa interspecific hybrids

Single-copy chloroplast loci are used widely to infer phylogenetic relationship at different taxonomic levels among various groups of plants. To test the utility of chloroplast loci and to provide additional data applicable to hybrid evolution in Musa, we sequenced two introns, rpl16 and ndhA, and two intergenic spacers, psaA-ycf3 and petA-psbJ-psbL-psbF and combined these data. Using these four regions, Musa acuminata Colla (A)- and M. balbisiana Colla (B)-containing genomes were clearly distinguished. Some triploid interspecific hybrids contain A-type chloroplasts (the AAB/ABB) while others contain B-type chloroplasts (the BBA/BBB). The chloroplasts of all cultivars in 'Namwa' (BBA) group came from the same wild maternal origin, but the specific parents are still unrevealed. Though, average sequence divergences in each region were little (less than 2%), we propose that petA-psbJ intergenic spacer could be developed for diversity assessment within each genome. This segment contains three single nucleotide polymorphisms (SNPs) and two indels which could distinguish diversity within A genome whereas this same region also contains one SNP and an indel which could categorize B genome. However, an inverted repeat region which could form hairpin structure was detected in this spacer and thus was omitted from the analyses due to their incongruence to other regions. Until thoroughly identified in other members of Musaceae and Zingiberales clade, utility of this inverted repeat as phylogenetic marker in these taxa are cautioned.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Phylogenetic Relationships Among Olea Species, Based on Nucleotide Variation at a Non-Coding Chloroplast DNA Region

Abstract: The purpose of this study was to assess nucleotide variation at a non-coding chloroplast DNA region in Olea species, to evaluate their phylogenetic relationships within the Olea genus and, more particularly, to clarify the relationships between cultivated olive (O. europaea) and the other taxa of section Olea. The analysis was made on an intergenic region between the trnT (UGU) and trnL (UAA) 5' exon, within a large single copy region of the chloroplast genome. Site-specific primers were used to amplify the region by PCR. This sequence analysis was applied to the same array of Olea species as assayed by Lumaret et al. (2000^[16]) using cpDNA RFLPs, thus making it possible to compare phylogenetic relationships analysed at two complementary levels of cpDNA variation. On the 666 bp aligned sequence, 8 different haplotypes were defined, with 9 single nucleotide mutations, a different length of a poly-T region and an indel for O. paniculata. Haplotypes were shared by the species pairs O. europaea-O.laperrinei, O. maroccana-O. cerasiformis, O. capensis-O. lancea and O. africana-O.indica. Phylogenetic analyses of these data distinguished four groups: the species Olea capensis and O. lancea, which both belong to subgenus Ligustroides, the Olea forms from southeast Africa, those from Asia and the taxa of northwest Africa and the Mediterranean Basin, which include olive crop. The results are consistent with those previously found using cpDNA RFLPs, with some minor differences observed within each group. They constitute further evidence to clarify the phylogeny of Olea.     

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.     

Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.     

藻类植物叶绿体基因组研究进展

藻类植物的cpDNA结构复杂,普遍缺失反向重复序列IR,且存在IR的藻类植物种类的cpDNA也有IR变短退化迹象.藻类植物的cpDNA包含的基因一般比高等植物要多,编码能力更强.藻类植物cpDNA全序列的测定方法主要是Fosmid文库构建,配合使用Long-PCR技术.该文对国内外有关藻类植物叶绿体基因组结构、叶绿体编码基因、叶绿体基因组在藻类系统发育中的应用以及藻类植物叶绿体基因组的提取和序列测定方法等进行综述,为藻类植物的系统发育和叶绿体起源以及功能基因组学的研究提供理论依据.     

Chloroplast DNA phylogeny and biogeography of Lepidium (Brassicaceae)

Two intergenic spacers, trnT-trnL and trnL-trnF, and the trnL intron of cpDNA were sequenced to study phylogenetic relationships and biogeography of 73 Lepidium taxa. Insertions/deletions of ≥3 bp (base pairs) provided reliable phylogenetic information whereas indels ≤2 bp, probably originating from slipped-strand mispairing, are prone to parallelism in the context of our phylogenetic framework. For the first time, an hypothesis of the genus Lepidium is proposed based on molecular phylogeny, in contrast to previous classification schemes into sections and greges (the latter category represents groups of related species within a given geographic region), which are based mainly on fruit characters. Only a few of the taxa as delimited in the traditional systems represent monophyletic lineages. The proposed phylogeny would suggest three main lineages, corresponding to (1) sections Lepia and Cardaria, (2) grex Monoplocoidea from Australia, and (3) remaining taxa, representing the bulk of Lepidium species with more or less resolved sublineages that sometimes represent geographical correspondence. The fossil data, easily dispersible mucilaginous seeds, widespread autogamous breeding systems, and low levels of sequence divergence between species from different continents or islands suggest a rapid radiation of Lepidium by long-distance dispersal in the Pliocene/Pleistocene. As a consequence of climatic changes in this geological epoch, arid/semiarid areas were established, providing favorable conditions for the radiation of Lepidium by which the genus attained its worldwide distribution.     

Molecular evidence for a Mediterranean origin of the Macaronesian endemic genus Argyranthemum (Asteraceae)

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.     

The Complete Chloroplast Genome of <Emphasis Type="Italic">Coix lacryma-jobi</Emphasis> and a Comparative Molecular Evolutionary Analysis of Plastomes in Cereals

Graminoid molecular evolution was investigated by chloroplast genome (plastome) scale analyses. A complete plastome from Coix lacryma-jobi (Poaceae) and a draft plastome from Joinvillea plicata (Joinvilleaceae) were sequenced and analyzed. The draft plastome included conserved protein-coding loci routinely analyzed in previous studies plus one additional locus of demonstrated phylogenetic utility. The methodological approach was to directly sequence overlapping amplicons from known plastome regions. Over 100 pairs of amplification and sequencing primers were designed and positioned to flank overlapping 1,200-base pair fragments around the entire plastome. Newly determined sequences were analyzed with published plastomes from representatives of Panicoideae, Ehrhartoideae, and Pooideae. Considerable variation was found for studies within the family and even within Andropogoneae. Readily interpreted mutation patterns were observed, such as small inversions in hairpin-loop regions and indels, which were common in intergenic spacers. Maximum or near-maximum bootstrap support was observed in all analyses resolving relationships between subfamilies. However, the addition of characters from noncoding regions increased the number of parsimony-informative characters and lengthened short internal branches (Andropogoneae), better defining intergeneric relationships. Thus, characters in complete plastomes can be used over a wide scope of phylogenetic studies.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Nuclear and cpDNA sequences combined provide strong inference of higher phylogenetic relationships in the phlox family (Polemoniaceae)

Members of the phlox family (Polemoniaceae) serve as useful models for studying various evolutionary and biological processes. Despite its biological importance, no family-wide phylogenetic estimate based on multiple DNA regions with complete generic sampling is available. Here, we analyze one nuclear and five chloroplast DNA sequence regions (nuclear ITS, chloroplast matK, trnL intron plus trnL-trnF intergeneric spacer, and the trnS-trnG, trnD-trnT, and psbM-trnD intergenic spacers) using parsimony and Bayesian methods, as well as assessments of congruence and long branch attraction, to explore phylogenetic relationships among 84 ingroup species representing all currently recognized Polemoniaceae genera. Relationships inferred from the ITS and concatenated chloroplast regions are similar overall. A combined analysis provides strong support for the monophyly of Polemoniaceae and subfamilies Acanthogilioideae, Cobaeoideae, and Polemonioideae. Relationships among subfamilies, and thus for the precise root of Polemoniaceae, remain poorly supported. Within the largest subfamily, Polemonioideae, four clades corresponding to tribes Polemonieae, Phlocideae, Gilieae, and Loeselieae receive strong support. The monogeneric Polemonieae appears sister to Phlocideae. Relationships within Polemonieae, Phlocideae, and Gilieae are mostly consistent between analyses and data permutations. Many relationships within Loeselieae remain uncertain. Overall, inferred phylogenetic relationships support a higher-level classification for Polemoniaceae proposed in 2000.     

南川绣线菊和细枝绣线菊分子水平研究

对6个野外居群(南川绣线菊和细枝绣线各3个)36个个体进行叶绿体(chloroplast,cp)DNA trnL-trnF片断测序分析.在南川绣线菊中发现了3个单倍型(Ros1-Ros3),在细枝绣线菊中发现了2个单倍型(Myr1-Myr2).两个种的序列联合分析对位排列后得到850 bp,共有9个变异位点,其中一个为碱基插入或缺失,另外8个为碱基置换,变异位点的百分率为0.11.对单倍型的遗传多样性分析表明同一区域亲缘关系相近的单倍型发生于同一居群中,并且存在着明显的分子系统地理学关系.以蔷薇科另两个外属植物Rosa californica 和 Sorbaria sorbifolia为外类群构建这两个种的最大简约(MP)树、最大似然(ML)树及贝叶斯树,结果获得了分辨良好的种间关系树.这表明在分子水平上两个种之间存在明显的差异,这与形态学上的表现是相一致的.遗传多样性分析结果表明了cpDNA trnL-trnF 片段对于绣线菊属的分子地理学研究还是比较有效的,可以通过进一步的大面积采样和分析来揭示植物的遗传结构、冰期避难所等问题.     

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.