Angiosperm Chloroplast Genomes Research Articles

Comparative chloroplast genomes provided insights into the evolution and species identification on the Datureae plants.

Generally, chloroplast genomes of angiosperms are always highly conserved but carry a certain number of variation among species. In this study, chloroplast genomes of 13 species from Datureae tribe that are of importance both in ornamental gardening and medicinal usage were studied. In addition, seven chloroplast genomes from Datureae together with two from Solanaceae species retrieved from the National Center for Biotechnology Information (NCBI) were integrated into this study. The chloroplast genomes ranged in size from 154,686 to 155,979 and from 155,497 to 155,919 bp for species of Datura and Brugmansia, respectively. As to Datura and Brugmansia, a total of 128 and 132 genes were identified, in which 83 and 87 protein coding genes were identified, respectively; Furthermore, 37 tRNA genes and 8 rRNA genes were both identified in Datura and Brugmansia. Repeats analysis indicated that the number and type varied among species for Simple sequence repeat (SSR), long repeats, and tandem repeats ranged in number from 53 to 59, 98 to 99, and 22 to 30, respectively. Phylogenetic analysis based on the plastid genomes supported the monophyletic relationship among Datura and Brugmansia and Trompettia, and a refined phylogenic relationships among each individual was resolved. In addition, a species-specific marker was designed based on variation spot that resulted from a comparative analysis of chloroplast genomes and verified as effective maker for identification of D. stramonium and D. stramonium var. inermis. Interestingly, we found that 31 genes were likely to be under positive selection, including genes encoding ATP protein subunits, photosystem protein subunit, ribosome protein subunits, NAD(P)H dehydrogenase complex subunits, and clpP, petB, rbcL, rpoCl, ycf4, and cemA genes. These genes may function as key roles in the adaption to diverse environment during evolution. The diversification of Datureae members was dated back to the late Oligocene periods. These chloroplast genomes are useful genetic resources for taxonomy, phylogeny, and evolution for Datureae.

Chloroplast genomes of four Carex species: Long repetitive sequences trigger dramatic changes in chloroplast genome structure.

The chloroplast genomes of angiosperms usually have a stable circular quadripartite structure that exhibits high consistency in genome size and gene order. As one of the most diverse genera of angiosperms, Carex is of great value for the study of evolutionary relationships and speciation within its genus, but the study of the structure of its chloroplast genome is limited due to its highly expanded and restructured genome with a large number of repeats. In this study, we provided a more detailed account of the chloroplast genomes of Carex using a hybrid assembly of second- and third-generation sequencing and examined structural variation within this genus. The study revealed that chloroplast genomes of four Carex species are significantly longer than that of most angiosperms and are characterized by high sequence rearrangement rates, low GC content and gene density, and increased repetitive sequences. The location of chloroplast genome structural variation in the species of Carex studied is closely related to the positions of long repeat sequences; this genus provides a typical example of chloroplast structural variation and expansion caused by long repeats. Phylogenetic relationships constructed based on the chloroplast protein-coding genes support the latest taxonomic system of Carex, while revealing that structural variation in the chloroplast genome of Carex may have some phylogenetic significance. Moreover, this study demonstrated a hybrid assembly approach based on long and short reads to analyze complex chloroplast genome assembly and also provided an important reference for the analysis of structural rearrangements of chloroplast genomes in other taxa.

The Complete Chloroplast Genome of Endangered Species Stemona parviflora: Insight into the Phylogenetic Relationship and Conservation Implications.

Stemona parviflora is an endangered species, narrowly endemic to Hainan and Southwest Guangdong. The taxonomic classification of S. parviflora remains controversial. Moreover, studying endangered species is helpful for current management and conservation. In this study, the first complete chloroplast genome of S. parviflora was assembled and compared with other Stemona species. The chloroplast genome size of S. parviflora was 154,552 bp, consisting of 87 protein-coding genes, 38 tRNA genes, 8 rRNA genes, and one pseudogene. The ψycf1 gene was lost in the cp genome of S. sessilifolia, but it was detected in four other species of Stemona. The inverted repeats (IR) regions have a relatively lower length variation compared with the large single copy (LSC) and small single copy (SSC) regions. Long repeat sequences and simple sequence repeat (SSR) were detected, and most SSR were distributed in the LSC region. Codon usage bias analyses revealed that the RSCU value of the genus Stemona has almost no difference. As with most angiosperm chloroplast genomes, protein-coding regions were more conservative than the inter-gene spacer. Seven genes (atpI, ccsA, cemA, matK, ndhA, petA, and rpoC1) were detected under positive selection in different Stemona species, which may result from adaptive evolution to different habitats. Phylogenetic analyses show the Stemona cluster in two main groups; S. parviflora were closest to S. tuberosa. A highly suitable region of S. parviflora was simulated by Maxent in this study; it is worth noting that the whole territory of Taiwan has changed to a low fitness area and below in the 2050 s, which may not be suitable for the introduction and cultivation of S. parviflora. In addition, limited by the dispersal capacity of S. parviflora, it is necessary to carry out artificial grafts to expand the survival areas of S. parviflora. Our results provide valuable information on characteristics of the chloroplast genome, phylogenetic relationships, and potential distribution range of the endangered species S. parviflora.

The chloroplast genome sequencing and phylogenetic analysis of Ulmus bergmanniana

In this study, the whole chloroplast genome of Ulmus bergmanniana was sequenced, assembled, annotated and characterized for the first time. The results showed that the total length of U. bergmanniana chloroplast genome was 160,060 bp and the GC content was 35.52%, which had the typical circular double-stranded tetrad structure of angiosperm chloroplast genome. It includes a large single-copy region (LSC), a small single-copy region (SSC), and a pair of inverted repeat regions (IRa/IRb), with lengths of 88,161 bp, 19,029 bp, and 26,435 bp, respectively. A total of 131 genes were annotated, including 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Based on the chloroplast genome sequences of 14 plants in U. bergmanniana and NCBI database, the phylogenetic tree was constructed, and the phylogenetic position of U. bergmanniana in Ulmaceae was determined. The results will provide a theoretical basis for molecular identification and resource development and utilization of U. bergmanniana.

Comparative Analyses of Euonymus Chloroplast Genomes: Genetic Structure, Screening for Loci With Suitable Polymorphism, Positive Selection Genes, and Phylogenetic Relationships Within Celastrineae.

In this study, we assembled and annotated the chloroplast (cp) genome of the Euonymus species Euonymus fortunei, Euonymus phellomanus, and Euonymus maackii, and performed a series of analyses to investigate gene structure, GC content, sequence alignment, and nucleic acid diversity, with the objectives of identifying positive selection genes and understanding evolutionary relationships. The results indicated that the Euonymus cp genome was 156,860–157,611bp in length and exhibited a typical circular tetrad structure. Similar to the majority of angiosperm chloroplast genomes, the results yielded a large single-copy region (LSC) (85,826–86,299bp) and a small single-copy region (SSC) (18,319–18,536bp), separated by a pair of sequences (IRA and IRB; 26,341–26,700bp) with the same encoding but in opposite directions. The chloroplast genome was annotated to 130–131 genes, including 85–86 protein coding genes, 37 tRNA genes, and eight rRNA genes, with GC contents of 37.26–37.31%. The GC content was variable among regions and was highest in the inverted repeat (IR) region. The IR boundary of Euonymus happened expanding resulting that the rps19 entered into IR region and doubled completely. Such fluctuations at the border positions might be helpful in determining evolutionary relationships among Euonymus. The simple-sequence repeats (SSRs) of Euonymus species were composed primarily of single nucleotides (A)n and (T)n, and were mostly 10–12bp in length, with an obvious A/T bias. We identified several loci with suitable polymorphism with the potential use as molecular markers for inferring the phylogeny within the genus Euonymus. Signatures of positive selection were seen in rpoB protein encoding genes. Based on data from the whole chloroplast genome, common single copy genes, and the LSC, SSC, and IR regions, we constructed an evolutionary tree of Euonymus and related species, the results of which were consistent with traditional taxonomic classifications. It showed that E. fortunei sister to the Euonymus japonicus, whereby E. maackii appeared as sister to Euonymus hamiltonianus. Our study provides important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Euonymus species.

CFM1, a member of the CRM-domain protein family, functions in chloroplast group II intron splicing in Setaria viridis.

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.

Molecular markers from the chloroplast genome of rose provide a complementary tool for variety discrimination and profiling

The rose is one of the most important ornamental woody plants because of its extensive use and high economic value. Herein, we sequenced a complete chloroplast genome of the miniature rose variety Rosa ‘Margo Koster’ and performed comparative analyses with sequences previously published for other species in the Rosaceae family. The chloroplast genome of Rosa ‘Margo Koster’, with a size of 157,395 bp, has a circular quadripartite structure typical of angiosperm chloroplast genomes and contains a total of 81 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Conjunction regions in the chloroplast genome of Rosa ‘Margo Koster’ were verified and manually corrected by Sanger sequencing. Comparative genome analysis showed that the IR contraction and expansion events resulted in rps19 and ycf1 pseudogenes. The phylogenetic analysis within the Rosa genus showed that Rosa ‘Margo Koster’ is closer to Rosa odorata than to other Rosa species. Additionally, we identified and screened highly divergent sequences and cpSSRs and compared their power to discriminate rose varieties by Sanger sequencing and capillary electrophoresis. The results showed that 15 cpSSRs are polymorphic, but their discriminating power is only moderate among a set of rose varieties. However, more than 150 single nucleotide variations (SNVs) were discovered in the flanking region of cpSSRs, and the results indicated that these SNVs have a higher divergence and stronger power for profiling rose varieties. These findings suggest that nucleotide mutations in the chloroplast genome may be an effective and powerful tool for rose variety discrimination and DNA profiling. These molecular markers in the chloroplast genome sequence of Rosa spp. will facilitate population and phylogenetic studies and other related studies of this species.

Study of spontaneous mutations in the transmission of poplar chloroplast genomes from mother to offspring

BackgroundChloroplasts have their own genomes, independent from nuclear genomes, that play vital roles in growth, which is a major targeted trait for genetic improvement in Populus. Angiosperm chloroplast genomes are maternally inherited, but the chloroplast’ variation pattern of poplar at the single-base level during the transmission from mother to offspring remains unknown.ResultsHere, we constructed high-quality and almost complete chloroplast genomes for three poplar clones, ‘NL895’ and its parents, ‘I69’ and ‘I45’, from the short-read datasets using multi-pass sequencing (15–16 times per clone) and ultra-high coverage (at least 8500× per clone), with the four-step strategy of Simulation–Assembly–Merging–Correction. Each of the three resulting chloroplast assemblies contained contigs covering > 99% of Populus trichocarpa chloroplast DNA as a reference. A total of 401 variant loci were identified by a hybrid strategy of genome comparison-based and mapping-based single nucleotide polymorphism calling. The genotypes of 94 variant loci were different among the three poplar clones. However, only 1 of the 94 loci was a missense mutation, which was located in the exon region of rpoC1 encoding the β’ subunit of plastid-encoded RNA polymerase. The genotype of the loci in NL895 and its female parent (I69) was different from that of its male parent (I45).ConclusionsThis research provides resources for further chloroplast genomic studies of a F1 full-sibling family derived from a cross between I69 and I45, and will improve the application of chloroplast genomic information in modern Populus breeding programs.

Comparative Chloroplast Genomics of Dipsacales Species: Insights Into Sequence Variation, Adaptive Evolution, and Phylogenetic Relationships.

In general, the chloroplast genomes of angiosperms are considered to be highly conserved and affected little by adaptive evolution. In this study, we tested this hypothesis based on sequence differentiation and adaptive variation in the plastid genomes in the order Dipsacales. We sequenced the plastid genomes of one Adoxaceae species and six Caprifoliaceae species, and together with seven previously released Dipsacales chloroplasts, we determined the sequence variations, evolutionary divergence of the plastid genomes, and phylogeny of Dipsacales species. The chloroplast genomes of Adoxaceae species ranged in size from 157,074 bp (Sinadoxa corydalifolia) to 158,305 bp (Sambucus williamsii), and the plastid genomes of Caprifoliaceae varied from 154,732 bp (Lonicera fragrantissima var. lancifolia) to 156,874 bp (Weigela florida). The differences in the number of genes in Caprifoliaceae and Adoxaceae species were largely due to the expansion and contraction of inverted repeat regions. In addition, we found that the number of dispersed repeats (Adoxaceae = 37; Caprifoliaceae = 384) was much higher than that of tandem repeats (Adoxaceae = 34; Caprifoliaceae = 291) in Dipsacales species. Interestingly, we determined 19 genes with positive selection sites, including three genes encoding ATP protein subunits (atpA, atpB, and atpI), four genes for ribosome protein small subunits (rps3, rps7, rps14, and rps15), four genes for photosystem protein subunits (psaA, psaJ, psbC, and pabK), two genes for ribosome protein large subunits (rpl22 and rpl32), and the clpP, infA, matK, rbcL, ycf1, and ycf2 genes. These gene regions may have played key roles in the adaptation of Dipsacales to diverse environments. In addition, phylogenetic analysis based on the plastid genomes strongly supported the division of 14 Dipsacales species into two previously recognized sections. The diversification of Adoxaceae and Caprifoliaceae was dated to the late Cretaceous and Tertiary periods. The availability of these chloroplast genomes provides useful genetic information for studying taxonomy, phylogeny, and species evolution in Dipsacales.

Awards of JSE Outstanding Papers (2016)

Awards of JSE Outstanding Papers (2016)

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

AbstractChloroplast genome information helps improve the phylogenetic resolution and can act as organelle‐scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long‐range polymerase chain reaction and using next‐generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel‐dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel‐dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.

The Complete Chloroplast Genome Sequence of Ampelopsis: Gene Organization, Comparative Analysis, and Phylogenetic Relationships to Other Angiosperms.

Ampelopsis brevipedunculata is an economically important plant that belongs to the Vitaceae family of angiosperms. The phylogenetic placement of Vitaceae is still unresolved. Recent phylogenetic studies suggested that it should be placed in various alternative families including Caryophyllaceae, asteraceae, Saxifragaceae, Dilleniaceae, or with the rest of the rosid families. However, these analyses provided weak supportive results because they were based on only one of several genes. Accordingly, complete chloroplast genome sequences are required to resolve the phylogenetic relationships among angiosperms. Recent phylogenetic analyses based on the complete chloroplast genome sequence suggested strong support for the position of Vitaceae as the earliest diverging lineage of rosids and placed it as a sister to the remaining rosids. These studies also revealed relationships among several major lineages of angiosperms; however, they highlighted the significance of taxon sampling for obtaining accurate phylogenies. In the present study, we sequenced the complete chloroplast genome of A. brevipedunculata and used these data to assess the relationships among 32 angiosperms, including 18 taxa of rosids. The Ampelopsis chloroplast genome is 161,090 bp in length, and includes a pair of inverted repeats of 26,394 bp that are separated by small and large single copy regions of 19,036 bp and 89,266 bp, respectively. The gene content and order of Ampelopsis is identical to many other unrearranged angiosperm chloroplast genomes, including Vitis and tobacco. A phylogenetic tree constructed based on 70 protein-coding genes of 33 angiosperms showed that both Saxifragales and Vitaceae diverged from the rosid clade and formed two clades with 100% bootstrap value. The position of the Vitaceae is sister to Saxifragales, and both are the basal and earliest diverging lineages. Moreover, Saxifragales forms a sister clade to Vitaceae of rosids. Overall, the results of this study will contribute to better support of the evolution, molecular biology and genetic improvement of the plant Ampelopsis.

An improved method for chloroplast genome sequencing in non-model forest tree species

Chloroplast genomes can provide a large amount of information and resources for use in studies on plant evolution and molecular ecology. However, a rapid and efficient method for obtaining chloroplast genome sequences is still lacking. In this study, we report a modified method for the isolation of intact chloroplasts, which needs less than 0.5 g leaf material. Coupled with rolling circle amplification (RCA), next-generation sequencing, and a pipeline combining de novo assembly and reference-guided assembly (RGA), we successfully obtained a complete chloroplast genome for the non-model forest tree species, evergreen oak Quercus spinosa, with as many as 36 % of the sequence reads mapped to the chloroplast genome. The Q. spinosa cpDNA is 160,825 bp in length and codes for 134 genes (89 protein coding, 8 ribosomal RNAs (rRNAs), and 36 distinct transfer RNAs (tRNAs)). The genome organization and arrangement are similar to those found among most angiosperm chloroplast genomes. Our inexpensive and efficient protocol can be applied to the reconstruction of chloroplast genomes for plant evolutionary studies, especially in non-model tree species.

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.

Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae)

The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699bp consists of two inverted repeat (IR) regions of 26,360bp each, a large single copy of 83,372bp, and a small single copy of 17,607bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a potential medicinal plant, will contribute to research on the genetic applications of this genus.

The chloroplast genome sequence of Syzygium cumini (L.) and its relationship with other angiosperms

Only a few studies to date have conducted comparative genomics in the Myrtaceae family. Here, we report the complete sequence and bioinformatics analysis of the chloroplast genome of Syzygium cumini (L.), one of the family members. The size of S. cumini cp genome was within the range of reported angiosperm chloroplast genomes. Comparison of S. cumini cpDNA sequence with previously reported partial sequences of S. cumini revealed several SNPs that resulted in non-synonymous mutations in maturase K and NADH-plastoquinone oxidoreductase subunit-5. These polymorphic characters might serve as intra-specific markers to address whether lineage sorting from polymorphic ancestry has occurred. Comparison of the S. cumini chloroplast genome with related dicots revealed an expansion in the intergenic spacer located between IRA/large single copy (LSC) border and the first gene of LSC region, driven by sequence of 54 bp. This type of variation in the intergenic regions can be utilized in the development of species-specific vectors for chloroplast genetic engineering. Several of the longer (30–40 bp) repeats were found to be conserved in other dicot species, suggesting that they might be widespread in angiosperm chloroplast genomes.

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

The chloroplast genome of Pyrus was found to be 159,922 bp in length which included a pair of inverted repeats (IRs) of 26,392 bp, separated by a small single-copy region of 19,237 bp and a large single-copy region (LSC) of 87,901 bp. A total of 130 predicted genes (113 unique genes and 17 genes, which were duplicated in the IR) including 79 protein-coding genes, four ribosomal RNA genes and 30 tRNA genes were identified based on similarity to homologs from the chloroplast genome of Nicotiana tabacum. Genome organization was very similar to the inferred ancestral angiosperm chloroplast genome. Comparisons between Pyrus, Malus, and Prunus in Rosaceae revealed 220 indels (≥10 bp). Excluding ycf1 and ycf2, which contained deletions in the coding region, all of these were detected in the spacer or intron regions. Three insertions and 13 deletions were detected in Pyrus compared to the same loci in Malus and Prunus. After comparing 89 noncoding chloroplast DNA regions in Pyrus and Malus, highly variable regions such as ndhC-trnV and trnR-atpA were identified. In Pyrus and Malus, the IR/LSC borders were 62 bp shorter than those of Prunus. In addition, there were length mutations at the IRa/LSC junction and in trnH. A total of 67 simple sequence repeats (more than 10 repeated motifs) were identified in the Pyrus chloroplast genome. The indels and simple sequence repeats will be useful evolutionary tools at both intra- and interspecific levels. Phylogenetic analysis demonstrated a close relationship between Pyrus and Prunus in the Rosaceae.

Complete sequence and organisation of the Jatropha curcas (Euphorbiaceae) chloroplast genome

Jatropha curcas is an important non-edible oil seed tree species and is considered a promising source of biodiesel. The complete nucleotide sequence of J. curcas chloroplast genome (cpDNA) was determined by pyrosequencing and gaps filled by Sanger sequencing. The cpDNA is a circular molecule of 163,856 bp in length and codes for 110 distinct genes (78 protein coding, four rRNA and 28 distinct tRNA). Genome organisation and arrangement are similar to the reported angiosperm chloroplast genome. However, in Jatropha, the infA and the rps16 genes are non-functional. The inverted repeat (IR) boundary is within the rpl2 gene, and the 13 nucleotides at the ends of the two duplicate genes are different. Repeat analysis suggests the presence of 72 repeat regions (>30 bp) apart from the IR; of these, 48 were direct and 24 were palindromic repeats. Phylogenetic analysis of 81 protein coding chloroplast genes from 65 taxa by maximum parsimony, maximum likelihood and minimum evolution analyses at 100 bootstraps provide strong support for the placement of inaperturate crotonoids of which Jatropha is a member as sister to articulated crotonoids of which Manihot is a member.

Different status of the gene for ribosomal protein S16 in the chloroplast genome during evolution of the genus Arabidopsis and closely related species

The ribosomal protein S16 (RPS16), the product of the rps16, is generally encoded in the chloroplast genomes of flowering plants. However, it has been reported that chloroplast-encoded RPS16 in mono- and dicotyledonous plants has been substituted by the product of nuclear-encoded rps16, which was transferred from the mitochondria to the nucleus before the early divergence of angiosperms. Current databases show that the chloroplast-encoded rps16 has become a pseudogene in four species of the Brassicaceae (Aethionema grandiflorum, Arabis hirsuta, Draba nemorosa, and Lobularia maritima). Further analysis of Arabidopsis thaliana and its close relatives has shown that pseudogenization has also occurred via the loss of its splicing capacity (Arabidopsis thaliana and Olimarabidopsis pumila). In contrast, the spliced product of chloroplast-encoded rps16 is observed in close relatives of Arabidopsis thaliana (Arabidopsis arenosa, Arabidopsis lyrata, and Crucihimalaya lasiocarpa). In this study, we identified the different functional status of rps16 in several chloroplast genomes in the genus Arabidopsis and its close relatives. Our results strongly suggest that nuclear- and chloroplast-encoded rps16 genes coexisted for at least 126 million years. We raise the possibility of the widespread pseudogenization of rps16 in the angiosperm chloroplast genomes via the loss of its splicing capacity, even when the rps16 encoded in the chloroplast genome is transcriptionally active.

Extensive Rearrangements in the Chloroplast Genome of Trachelium caeruleum Are Associated with Repeats and tRNA Genes

Chloroplast genome organization, gene order, and content are highly conserved among land plants. We sequenced the chloroplast genome of Trachelium caeruleum L. (Campanulaceae), a member of an angiosperm family known for highly rearranged genomes. The total genome size is 162,321 bp, with an inverted repeat (IR) of 27,273 bp, large single-copy (LSC) region of 100,114 bp, and small single-copy (SSC) region of 7,661 bp. The genome encodes 112 different genes, with 17 duplicated in the IR, a tRNA gene (trnI-cau) duplicated once in the LSC region, and a protein-coding gene (psbJ) with two duplicate copies, for a total of 132 putatively intact genes. ndhK may be a pseudogene with internal stop codons, and clpP, ycf1, and ycf2 are so highly diverged that they also may be pseudogenes. ycf15, rpl23, infA, and accD are truncated and likely nonfunctional. The most conspicuous feature of the Trachelium genome is the presence of 18 internally unrearranged blocks of genes inverted or relocated within the genome relative to the ancestral gene order of angiosperm chloroplast genomes. Recombination between repeats or tRNA genes has been suggested as a mechanism of chloroplast genome rearrangements. The Trachelium chloroplast genome shares with Pelargonium and Jasminum both a higher number of repeats and larger repeated sequences in comparison to eight other angiosperm chloroplast genomes, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so some combination of repeats and tRNA genes may have mediated these rearrangements.