Chloroplast Genomic Resources Research Articles

Comparative chloroplast genomics, phylogenetic relationships and molecular markers development of Aglaonema commutatum and seven green cultivars of Aglaonema

Aglaonema commutatum is a famous species in the Aglaonema genus, which has important ornamental and economic value. However, its chloroplast genome information and phylogenetic relationships among popular green cultivars of Aglaonema in southern China have not been reported. Herein, chloroplast genomes of one variety of A. commutatum and seven green cultivars of Aglaonema, namely, A. commutatum ‘San Remo’, ‘Kai Sa’, ‘Pattaya Beauty’, ‘Sapphire’, ‘Silver Queen’, ‘Snow White’, ‘White Gem’, and ‘White Horse Prince’, were sequenced and assembled for comparative analysis and phylogeny. These eight genomes possessed a typical quadripartite structure that consisted of a LSC region (90,799–91,486 bp), an SSC region (20,508–21,137 bp) and a pair of IR regions (26,661–26,750 bp). Each genome contained 112 different genes, comprising 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes. The gene orders, GC contents, codon usage frequency, and IR/SC boundaries were highly conserved among these eight genomes. Long repeats, SSRs, SNPs and indels were analyzed among these eight genomes. Comparative analysis of 15 Aglaonema chloroplast genomes identified 7 highly variable regions, including trnH-GUG-exon1-psbA, trnS-GCU-trnG-UCC-exon1, trnY-GUA-trnE-UUC, psbC-trnS-UGA, trnF-GAA-ndhJ, ccsA-ndhD, and rps15-ycf1-D2. Reconstruction of the phylogenetic trees based on chloroplast genomes, strongly supported that Aglaonema was a sister to Anchomanes, and that the Aglaonema genus was classified into two sister clades including clade I and clade II, which corresponded to two sections, Aglaonema and Chamaecaulon, respectively. One variety and five cultivars, including A. commutatum ‘San Remo’, ‘Kai Sa’, ‘Pattaya Beauty’, ‘Silver Queen’, ‘Snow White’, and ‘White Horse Prince’, were classified into clade I; and the rest of the two cultivars, including ‘Sapphire’ and ‘White Gem’, were classified into clade II. Positive selection was observed in 34 protein-coding genes at the level of the amino acid sites among 77 chloroplast genomes of the Araceae family. Based on the highly variable regions and SSRs, 4 DNA markers were developed to differentiate the clade I and clade II in Aglaonema. In conclusion, this study provided chloroplast genomic resources for Aglaonema, which were useful for its classification and phylogeny.

Assembly and analysis of the chloroplast genome of Amorphophallus kachinensis Engler & Gehrmann (Araceae) from Southwestern China: implications for conservation and utilization

Konjac glucomannan consists of D-mannose and D-glucose units and is a hydrocolloid obtained from the corm of Amorphophallus species. Due to its bioactive properties, biodegradability, and hydrophilic ability, glucomannan is widely used in the fields of food, medicine, and industry. Amorphophallus species have been cultivated as cash crops in many Asian countries. Amorphophallus kachinensis Engler & Gehrmann 1911 is naturally distributed in southwestern China, Laos, and northern Thailand. To help the genetic assessment and conservation of this species, the first chloroplast genome of A. kachinensis was sequenced on the Illumina sequencing platform. We assembled the chloroplast genome using the software GetOrganelle and annotated the genome by Geseq and Cpgavas 2. The assembled chloroplast genome was 173,330 bp long, and the average GC content was 35% (GenBank accession number: PP072244). The chloroplast genome of A. kachinensis contained one large single copy, one small single copy, and two inverted repeats, with lengths of 92,030 bp, 15,118 bp, 33,091 bp, and 33,091 bp, respectively. We successfully annotated 132 genes across the genome, which was consistent with other Amorphophallus species. The phylogenetic tree indicates a sub-divergence in the Amorphophallus genus with two main genetic groups detected among eight species. The two genetic groups should be treated as distinct evolutionarily significant units when making conservation strategies. Our study enriched the chloroplast genome resources of the Amorphophallus genus and could help future phylogeographic studies, protection, and utilization of wild resources.

Complete Chloroplast Genomes of Four Oaks from the Section Cyclobalanopsis Improve the Phylogenetic Analysis and Understanding of Evolutionary Processes in the Genus Quercus.

Quercus is a valuable genus ecologically, economically, and culturally. They are keystone species in many ecosystems. Species delimitation and phylogenetic studies of this genus are difficult owing to frequent hybridization. With an increasing number of genetic resources, we will gain a deeper understanding of this genus. In the present study, we collected four Quercus section Cyclobalanopsis species (Q. poilanei, Q. helferiana, Q. camusiae, and Q. semiserrata) distributed in Southeast Asia and sequenced their complete genomes. Following analysis, we compared the results with those of other species in the genus Quercus. These four chloroplast genomes ranged from 160,784 bp (Q. poilanei) to 161,632 bp (Q. camusiae) in length, with an overall guanine and cytosine (GC) content of 36.9%. Their chloroplast genomic organization and order, as well as their GC content, were similar to those of other Quercus species. We identified seven regions with relatively high variability (rps16, ndhk, accD, ycf1, psbZ-trnG-GCC, rbcL-accD, and rpl32-trnL-UAG) which could potentially serve as plastid markers for further taxonomic and phylogenetic studies within Quercus. Our phylogenetic tree supported the idea that the genus Quercus forms two well-differentiated lineages (corresponding to the subgenera Quercus and Cerris). Of the three sections in the subgenus Cerris, the section Ilex was split into two clusters, each nested in the other two sections. Moreover, Q. camusiae and Q. semiserrata detected in this study diverged first in the section Cyclobalanopsis and mixed with Q. engleriana in the section Ilex. In particular, 11 protein coding genes (atpF, ndhA, ndhD, ndhF, ndhK, petB, petD, rbcL, rpl22, ycf1, and ycf3) were subjected to positive selection pressure. Overall, this study enriches the chloroplast genome resources of Quercus, which will facilitate further analyses of phylogenetic relationships in this ecologically important tree genus.

Phylogenomics and plastome evolution of Indigofera (Fabaceae).

Indigofera L. is the third largest genus in Fabaceae and includes economically important species that are used for indigo dye-producing, medicinal, ornamental, and soil and water conservation. The genus is taxonomically difficult due to the high level of overlap in morphological characters of interspecies, fewer reliability states for classification, and extensive adaptive evolution. Previous characteristic-based taxonomy and nuclear ITS-based phylogenies have contributed to our understanding of Indigofera taxonomy and evolution. However, the lack of chloroplast genomic resources limits our comprehensive understanding of the phylogenetic relationships and evolutionary processes of Indigofera. Here, we newly assembled 18 chloroplast genomes of Indigofera. We performed a series of analyses of genome structure, nucleotide diversity, phylogenetic analysis, species pairwise Ka/Ks ratios, and positive selection analysis by combining with allied species in Papilionoideae. The chloroplast genomes of Indigofera exhibited highly conserved structures and ranged in size from 157,918 to 160,040 bp, containing 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Thirteen highly variable regions were identified, of which trnK-rbcL, ndhF-trnL, and ycf1 were considered as candidate DNA barcodes for species identification of Indigofera. Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods based on complete chloroplast genome and protein-coding genes (PCGs) generated a well-resolved phylogeny of Indigofera and allied species. Indigofera monophyly was strongly supported, and four monophyletic lineages (i.e., the Pantropical, East Asian, Tethyan, and Palaeotropical clades) were resolved within the genus. The species pairwise Ka/Ks ratios showed values lower than 1, and 13 genes with significant posterior probabilities for codon sites were identified in the positive selection analysis using the branch-site model, eight of which were associated with photosynthesis. Positive selection of accD suggested that Indigofera species have experienced adaptive evolution to selection pressures imposed by their herbivores and pathogens. Our study provided insight into the structural variation of chloroplast genomes, phylogenetic relationships, and adaptive evolution in Indigofera. These results will facilitate future studies on species identification, interspecific and intraspecific delimitation, adaptive evolution, and the phylogenetic relationships of the genus Indigofera.

Seven newly sequenced chloroplast genomes from the order Watanabeales (Trebouxiophyceae, Chlorophyta): Phylogenetic and comparative analysis

The little-known order Watanabeales currently includes 10 genera with Chlorella-like species that reproduce by unequal-sized autospores and are predominantly solitary or terrestrial. The taxonomic scheme of Watanabeales has only been primarily inferred by short and less informative rDNA phylogenetic analysis. In the present study, seven newly sequenced genomes and one reported chloroplast genome representing the existing major branches of Watanabeales were harvested to phylogenetically reconstruct this order and to further understand its evolution. All chloroplast genomes of Watanabeales ranging from 133 to 274 kb were circular mapping and lacked a quadripartite structure. The chloroplast genome size, GC content, number of introns, and length of intergenic region proportion of the Watanabeales showed consistent trends, with Calidiella yingdensis D201 and Kalinella pachyderma 2601 having the lowest and highest values, respectively, echoing the positive correlation between organismal size and genome size. Phylogenetic analysis of Watanabeales based on 76 protein-coding genes coupled with the establishment of various complex analytical methods determined the unique robust taxonomic scheme which was incongruent with rDNA. Comparative genomic analyses revealed that the chloroplast genomes of Watanabeales accounted for numerous complex rearrangements and inversions which indicated high cryptic diversity. Substitution rate estimation indicated that the chloroplast genomes of Watanabeales were under purifying selection and similar evolutionary pressure and supported the view that genus Symbiochloris should be excluded from Watanabeales. Our results enrich the chloroplast genome resources of Watanabeales, clarify the phylogenetic status of species within this order, and provide more reference information for subsequent taxonomic and phylogenetic study.

Comparative chloroplast genome and transcriptome analysis on the ancient genus Isoetes from China

Isoetes is a famous living fossil that plays a significant role in the evolutionary studies of the plant kingdom. To explore the adaptive evolution of the ancient genus Isoetes from China, we focused on Isoetes yunguiensis (Q.F. Wang and W.C. Taylor), I. shangrilaensis (X. Li, Y.Q. Huang, X.K. Dai & X. Liu), I. taiwanensis (DeVol), I. sinensis (T.C. Palmer), I. hypsophila_GHC (Handel-Mazzetti), and I. hypsophila_HZS in this study. We sequenced, assembled, and annotated six individuals’ chloroplast genomes and transcriptomes, and performed a series of analyses to investigate their chloroplast genome structures, RNA editing events, and adaptive evolution. The six chloroplast genomes of Isoetes exhibited a typical quadripartite structure with conserved genome sequence and structure. Comparative analyses of Isoetes species demonstrated that the gene organization, genome size, and GC contents of the chloroplast genome are highly conserved across the genus. Besides, our positive selection analyses suggested that one positively selected gene was statistically supported in Isoetes chloroplast genomes using the likelihood ratio test (LRT) based on branch-site models. Moreover, we detected positive selection signals using transcriptome data, suggesting that nuclear-encoded genes involved in the adaption of Isoetes species to the extreme environment of the Qinghai-Tibetan Plateau (QTP). In addition, we identified 291–579 RNA editing sites in the chloroplast genomes of six Isoetes based on transcriptome data, well above the average of angiosperms. RNA editing in protein-coding transcripts results from amino acid changes to increase their hydrophobicity and conservation in Isoetes, which may help proteins form functional three-dimensional structure. Overall, the results of this study provide comprehensive transcriptome and chloroplast genome resources and contribute to a better understanding of adaptive evolutionary and molecular biology in Isoetes.

Comparative Genomic and Phylogenetic Analysis of Chloroplast Genomes of Hawthorn (Crataegus spp.) in Southwest China.

The hawthorns (Crataegus spp.) are widely distributed and famous for their edible and medicinal values. There are ∼18 species and seven varieties of hawthorn in China distributed throughout the country. We now report the chloroplast genome sequences from C. scabrifolia, C. chungtienensis and C. oresbia, from the southwest of China and compare them with the previously released six species in Crataegus and four species in Rosaceae. The chloroplast genome structure of Crataegus is typical and can be divided into four parts. The genome sizes are between 159,654 and 159,898bp. The three newly sequenced chloroplast genomes encode 132 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Comparative analysis of the chloroplast genomes revealed six divergent hotspot regions, including ndhA, rps16-trnQ-UUG, ndhF-rpl32, rps16-psbK, trnR-UCU-atpA and rpl32-trnL-UAG. According to the correlation and co-occurrence analysis of repeats with indels and SNPs, the relationship between them cannot be ignored. The phylogenetic tree constructed based on the complete chloroplast genome and intergenic region sequences indicated that C. scabrifolia has a different origin from C. chungtienensis and C. oresbia. We support the placement of C. hupehensis, C. cuneata, C. scabrifolia in C. subg. Crataegus and C. kansuensis, C. oresbia, C. kansuensis in C. subg. Sanguineae. In addition, based on the morphology, geographic distribution and phylogenetic relationships of C. chungtienensis and C. oresbia, we speculate that these two species may be the same species. In conclusion, this study has enriched the chloroplast genome resources of Crataegus and provided valuable information for the phylogeny and species identification of this genus.

Chloroplast Genomic Resources and Genetic Divergence of Endangered Species Bretschneidera sinensis (Bretschneideraceae)

Bretschneidera sinensis is an endangered woody species found in East and South China. Comprehensive intraspecies chloroplast genome studies have demonstrated novel genetic resources to assess the genetic variation and diversity of this species. Using genome skimming method, we assembled the whole chloroplast genome of 12 genotypes of B. sinensis from different geographical locations, covering most wild populations. The B. sinensis chloroplast genome size ranged from 158,959 to 159,045 base pairs (bp) and displayed a typical circular quadripartite structure. Comparative analyses of 12 B. sinensis chloroplast genome revealed 33 polymorphic simple sequence repeats (SSRs), 105 polymorphic single nucleotide polymorphisms (SNPs), and 55 indels. Phylogenetic analysis showed that the 12 genotypes were grouped into 2 branches, which is consistent with the geographical distribution (Eastern clade and Western clade). Divergence time estimates showed that the two clades were divergent from 0.6 Ma in the late Pleistocene. Ex situ conservation is essential for this species. In this study, we identified SNPs, indels, and microsatellites of B. sinensis by comparative analyses of chloroplast genomes and determined genetic variation between populations using these genomic markers. Chloroplast genomic resources are also important for further domestication, population genetic, and phylogenetic analysis, possibly in combination with molecular markers of mitochondrial and/or nuclear genomes.

Complete Chloroplast Genome of the Inverted Repeat-Lacking Species Vicia bungei and Development of Polymorphic Simple Sequence Repeat Markers.

BackgroundVicia bungei is an economically important forage crop in South Korea and China. Although detailed genetic and genomic data can improve population genetic studies, conservation efforts, and improved breeding of crops, few such data are available for Vicia species in general and none at all for V. bungei. Therefore, the main objectives of this study were to sequence, assemble, and annotate V. bungei chloroplast genome and to identify simple sequence repeats (SSRs) as polymorphic genetic markers.ResultsThe whole-genome sequence of V. bungei was generated using an Illumina MiSeq platform. De novo assembly of complete chloroplast genome sequences was performed for the low-coverage sequence using CLC Genome Assembler with a 200–600-bp overlap size. Vicia bungei chloroplast genome was 130,796-bp long. The genome lacked an inverted repeat unit and thus resembled those of species in the inverted repeat-lacking clade within Fabaceae. Genome annotation using Dual OrganellarGenoMe Annotator (DOGMA) identified 107 genes, comprising 75 protein-coding, 28 transfer RNA, and 4 ribosomal RNA genes. In total, 432 SSRs were detected in V. bungei chloroplast genome, including 64 mononucleotides, 14 dinucleotides, 5 trinucleotides, 4 tetranucleotides, 233 pentanucleotides, 90 hexanucleotides, and 14 complex repeated motifs. These were used to develop 232 novel chloroplast SSR markers, 39 of which were chosen at random to test amplification and genetic diversity in Vicia species (20 accessions from seven species). The unweighted pair group method with arithmetic mean cluster analysis identified seven clusters at the interspecies level and intraspecific differences within clusters.ConclusionThe complete chloroplast genome sequence of V. bungei was determined. This reference genome should facilitate chloroplast resequencing and future searches for additional genetic markers using population samples. The novel chloroplast genome resources and SSR markers will greatly contribute to the conservation of the genus Vicia and facilitate genetic and evolutionary studies of this genus and of other higher plants.

Complete Chloroplast Genome of Cnidium monnieri (Apiaceae) and Comparisons with Other Tribe Selineae Species

Cnidium monnieri is an economically important traditional Chinese medicinal plant. In this study, the complete chloroplast (cp) genome of C. monnieri was determined using the Illumina paired-end sequencing, the GetOrganelle de novo assembly strategy, as well as the GeSeq annotation method. Our results showed that the cp genome was 147,371 bp in length with 37.4% GC content and included a large single-copy region (94,361 bp) and a small single-copy region (17,552 bp) separated by a pair of inverted repeat regions (17,729 bp). A total of 129 genes were contained in the cp genome, including 85 protein-coding genes, 36 tRNA genes, and eight rRNA genes. We also investigated codon usage, RNA editing, repeat sequences, simple sequence repeats (SSRs), IR boundaries, and pairwise Ka/Ks ratios. Four hypervariable regions (trnD-trnY-trnE-trnT, ycf2, ndhF-rpl32-trnL, and ycf1) were identified as candidate molecular markers for species authentication. The phylogenetic analyses supported non-monophyly of Cnidium and C. monnieri located in tribe Selineae based on the cp genome sequences and internal transcribed spacer (ITS) sequences. The incongruence of the phylogenetic position of C. monnieri between ITS and cpDNA phylogenies suggested that C. monnieri might have experienced complex evolutions with hybrid and incomplete lineage sorting. All in all, the results presented herein will provide plentiful chloroplast genomic resources for studies of the taxonomy, phylogeny, and species authentication of C. monnieri. Our study is also conducive to elucidating the phylogenetic relationships and taxonomic position of Cnidium.

Identification of the Complete Chloroplast Genome of Malus zhaojiaoensis Jiang and Its Comparison and Evolutionary Analysis with Other Malus Species.

The genus Malus is rich in species and many of its plastid genomes have been released. However, limited resources and few markers are not conducive to the comparison of differences among species and resource identification and evaluation. In this study, the complete chloroplast genome of Malus zhaojiaoensis was studied by NGS sequencing, with a total length of 159998 bp. It consists of four regions, LSC (88,070 bp), IRB (26,359 bp), SSC (19,210 bp) and IRA (26,359 bp). M. zhaojiaoensis cp genome contained a total of 111 genes made up of three classes: 78 coding sequences, 29 tRNA genes, and four rRNA genes. In addition, a total of 91 SSRs and 43 INEs were found in the M. zhaojiaoensis cp genome, which was slightly different from M. baccata and M. hupehensis in number. The analysis of codon usage and RNA editing showed that high-frequency codons tended to end at A/U bases and RNA editing mainly occurred at the second codon. Comparative genome analysis suggested that the cp genomes of eight Malus species had higher overall similarity, but there were more variation hotspots (rps16_trnK-UUU, trnG-UCC_atpA, atpH_atpF, trnT-GGU_psbD, etc.) in the LSC region. By building evolutionary trees, it can be clearly observed that M. zhaojiaoensis formed a large group with eight species of Malus, but was relatively independent in differentiation. In conclusion, this study provides high-quality chloroplast genome resources of M. zhaojiaoensis and discusses the genetic variation characteristics of Malus genus. The findings of this study will provide a good reference for plastid genome assembly and interspecific comparison in the future.

Insights Into Comparative Analyses and Phylogenomic Implications of Acer (Sapindaceae) Inferred From Complete Chloroplast Genomes.

Acer L. (Sapindaceae) is one of the most diverse and widespread plant genera in the Northern Hemisphere. It comprises 124–156 recognized species, with approximately half being native to Asia. Owing to its numerous morphological features and hybridization, this genus is taxonomically and phylogenetically ranked as one of the most challenging plant taxa. Here, we report the complete chloroplast genome sequences of five Acer species and compare them with those of 43 published Acer species. The chloroplast genomes were 149,103–158,458 bp in length. We conducted a sliding window analysis to find three relatively highly variable regions (psbN-rps14, rpl32-trnL, and ycf1) with a high potential for developing practical genetic markers. A total of 76–103 SSR loci were identified in 48 Acer species. The positive selection analysis of Acer species chloroplast genes showed that two genes (psaI and psbK) were positively selected, implying that light level is a selection pressure for Acer species. Using Bayes empirical Bayes methods, we also identified that 20 cp gene sites have undergone positive selection, which might result from adaptation to specific ecological niches. In phylogenetic analysis, we have reconfirmed that Acer pictum subsp. mono and A. truncatum as sister species. Our results strongly support the sister relationships between sections Platanoidea and Macrantha and between sections Trifoliata and Pentaphylla. Moreover, series Glabra and Arguta are proposed to promote to the section level. The chloroplast genomic resources provided in this study assist taxonomic and phylogenomic resolution within Acer and the Sapindaceae family.

Six Newly Sequenced Chloroplast Genomes From Trentepohliales: The Inflated Genomes, Alternative Genetic Code and Dynamic Evolution.

Cephaleuros is often known as an algal pathogen with 19 taxonomically valid species, some of which are responsible for red rust and algal spot diseases in vascular plants. No chloroplast genomes have yet been reported in this genus, and the limited genetic information is an obstacle to understanding the evolution of this genus. In this study, we sequenced six new Trentepohliales chloroplast genomes, including four Cephaleuros and two Trentepohlia. The chloroplast genomes of Trentepohliales are large compared to most green algae, ranging from 216 to 408 kbp. They encode between 93 and 98 genes and have a GC content of 26–36%. All new chloroplast genomes were circular-mapping and lacked a quadripartite structure, in contrast to the previously sequenced Trentepohlia odorata, which does have an inverted repeat. The duplicated trnD-GTC, petD, and atpA genes in C. karstenii may be remnants of the IR region and shed light on its reduction. Chloroplast genes of Trentepohliales show elevated rates of evolution, strong rearrangement dynamics and several genes display an alternative genetic code with reassignment of the UGA/UAG codon presumably coding for arginine. Our results present the first whole chloroplast genome of the genus Cephaleuros and enrich the chloroplast genome resources of Trentepohliales.

Plastome Evolution in the Hyperdiverse Genus Euphorbia (Euphorbiaceae) Using Phylogenomic and Comparative Analyses: Large-Scale Expansion and Contraction of the Inverted Repeat Region.

With c. 2,000 species, Euphorbia is one of the largest angiosperm genera, yet a lack of chloroplast genome (plastome) resources impedes a better understanding of its evolution. In this study, we assembled and annotated 28 plastomes from Euphorbiaceae, of which 15 were newly sequenced. Phylogenomic and comparative analyses of 22 plastome sequences from all four recognized subgenera within Euphorbia revealed that plastome length in Euphorbia is labile, presenting a range of variation c. 42 kb. Large-scale expansions of the inverted repeat (IR) region were identified, and at the extreme opposite, the near-complete loss of the IR region (with only 355 bp left) was detected for the first time in Euphorbiaceae. Other structural variations, including gene inversion and duplication, and gene loss/pseudogenization, were also observed. We screened the most promising molecular markers from both intergenic and coding regions for phylogeny-based utilities, and estimated maximum likelihood and Bayesian phylogenies from four datasets including whole plastome sequences. The monophyly of Euphorbia is supported, and its four subgenera are recovered in a successive sister relationship. Our study constitutes the first comprehensive investigation on the plastome structural variation in Euphorbia and it provides resources for phylogenetic research in the genus, facilitating further studies on its taxonomy, evolution, and conservation.

Comparison among the chloroplast genomes of five species of Chamaerhodos (Rosaceae: Potentilleae): phylogenetic implications

Chamaerhodos is a small genus with ca 7–8 species with a disjunct distribution in Asia and western North America. Due to limited sampling of species and genes in previous studies, little is known about the phylogenetic relationships among the species. Moreover, chloroplast genomic resources for Chamaerhodos have been limited. Herein, we conducted a comparative analysis of the complete chloroplast (cp) genomes of five Chamaerhodos species. The five cp genomes had a typical quadripartite structure with high conservation of gene content and gene order. These five cp genomes encoded an identical set of 129 genes, including 84 protein‐coding genes, 37 tRNA genes and eight rRNA genes. Comparison of the boundaries between the IRs and single copy regions revealed only very slight boundary differences and the five cp genomes showed only little sequence divergence. Seven regions (trnR‐UCU‐atpA, trnS‐GCU‐trnG‐UCC, TrnG‐GCC‐trnfM‐CAU, trnL‐UAA intron, trnH‐GUG‐psbA, trnF‐GAA‐ndhJ, matK‐5′‐trnK‐UUU) were identified as excellent candidate markers. A total of 117, 108, 116, 117 and 118 perfect SSRs were detected in the cp genomes of Ch. altaica, Ch. canescens, Ch. erecta, Ch. sabulosa and Ch. trifida, respectively. A phylogenetic analysis support the monophyly of Chamaerhodos and place it as part of the Fragariinae clade sister to the clade of Dasiphora, Drymocallis and Potaninia. Within Chamaerhodos, Ch. canescens was inferred sister to a clade containing the other four species. The detailed characterization of the Chamaerhodos chloroplast genomes sets the foundation for further studies on the genus and its close relatives in the economically important Rosaceae.

Chloroplast genomes elucidate diversity, phylogeny, and taxonomy of Pulsatilla (Ranunculaceae)

Pulsatilla (Ranunculaceae) consists of about 40 species, and many of them have horticultural and/or medicinal value. However, it is difficult to recognize and identify wild Pulsatilla species. Universal molecular markers have been used to identify these species, but insufficient phylogenetic signal was available. Here, we compared the complete chloroplast genomes of seven Pulsatilla species. The chloroplast genomes of Pulsatilla were very similar and their length ranges from 161,501 to 162,669 bp. Eight highly variable regions and potential sources of molecular markers such as simple sequence repeats, large repeat sequences, and single nucleotide polymorphisms were identified, which are valuable for studies of infra- and inter-specific genetic diversity. The SNP number differentiating any two Pulsatilla chloroplast genomes ranged from 112 to 1214, and provided sufficient data for species delimitation. Phylogenetic trees based on different data sets were consistent with one another, with the IR, SSC regions and the barcode combination rbcL + matK + trnH-psbA produced slightly different results. Phylogenetic relationships within Pulsatilla were certainly resolved using the complete cp genome sequences. Overall, this study provides plentiful chloroplast genomic resources, which will be helpful to identify members of this taxonomically challenging group in further investigation.

Complete chloroplast genome sequence of Amomum villosum and comparative analysis with other Zingiberaceae plants

ObjectiveAmomum villosum (AV) is an herb whose dried fruit has been extensively used in modern medicine to treat digestive system diseases such as dysentery, vomiting and abdominal pain. This paper aims to supplement chloroplast (cp) genomic resources and to be used in phylogenetic studies and identification of AV related plants. MethodsHigh-throughput sequencing technology was used to determine the complete sequence of the AV cp genome, and the sequence was then compared with three related species. ResultsThe genome size of AV we obtained was 163,968 bp with an obvious tetrad structure. The AV cp genome was observed to contain 125 unique genes and 81 simple sequence repeat (SSRs) had been determined and the majority of which were adenine–thymine (AT)-rich. Comparative analysis of genome sequence of four ginger plants showed that the atpF, clpP and rpl32 genes are potential markers for identifying Amomum species. Phylogenetic analysis suggested that AV was closely related to A. kravanh and A. compactum. ConclusionThese results have brought useful genetic resources for further identification researches, DNA barcoding, resolving taxonomy and understanding the evolutionary mode of Zingiberaceae cp genome.

The Complete Chloroplast Genome Sequences of 14 Curcuma Species: Insights Into Genome Evolution and Phylogenetic Relationships Within Zingiberales.

Zingiberaceae is taxonomically complex family where species are perennial herb. However, lack of chloroplast genomic information severely hinders our understanding of Zingiberaceae species in the research of evolution and phylogenetic relationships. In this study, the complete chloroplast (cp) genomes of fourteen Curcuma species were assembled and characterized using next-generation sequencing. We compared the genome features, repeat sequences, sequence divergence, and constructed the phylogenetic relationships of the 25 Zingiberaceae species. In each Zingiberaceae species, the 25 complete chloroplast genomes ranging from 155,890 bp (Zingiber spectabile) to 164,101 bp (Lanxangia tsaoko) contained 111 genes consisting of 77 protein coding genes, 4 ribosomal RNAs and 30 transfer RNAs. These chloroplast genomes are similar to most angiosperm that consisted of a four-part circular DNA molecules. Moreover, the characteristics of the long repeats sequences and simple sequence repeats (SSRs) were found. Six divergent hotspots regions (matK-trnk, Rps16-trnQ, petN-psbM, rpl32, ndhA, and ycf1) were identified in the 25 Zingiberaceae chloroplast genomes, which could be potential molecular markers. In addition to Wurfbainia longiligularis, the ψycf1 was discovered among the 25 Zingiberaceae species. The shared protein coding genes from 52 Zingiberales plants and four other family species as out groups were used to construct phylogenetic trees distinguished by maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) and showed that Musaceae was the basal group in Zingiberales, and Curcuma had a close relationship with Stahlianthu. Besides this, Curcuma flaviflora was clustered together with Zingiber. Its distribution area (Southeast Asia) overlaps with the latter. Maybe hybridization occur in related groups within the same region. This may explain why Zingiberaceae species have a complex phylogeny, and more samples and genetic data were necessary to confirm their relationship. This study provide the reliable information and high-quality chloroplast genomes and genome resources for future Zingiberaceae research.

Complete chloroplast genome sequence of turnip (Brassica rapa. ssp. rapa): genome structure and phylogenetic analysis

Turnip (Brassica rapa. ssp. rapa) is considered worldwide to be one of the most important leaf and root vegetable crops in the Brassicaceae family. However, to date, few chloroplast (cp) genomic resources have been reported for this genus. Here, we determined the complete cp genome sequences of Brassica rapa ssp. rapa. A 153,621 bp quadripartite cycle without any gap was obtained with a large single-copy region (LSC) of 83,512 bp, a small single-copy region (SSC) of 17,683 bp, and two inverted repeat (IR), IRa and IRb of 26,213 bp. A total of 132 genes were identified, including 87 protein-coding genes (PCG), 37 transfer RNA (tRNA), and 8 ribosomal RNA (rRNA). The phylogenetic analysis of ten other crops selected showed that the turnip was most closely related to the Brassica rapa.

Comparative analyses of 32 complete plastomes of Tef (Eragrostis tef ) accessions from Ethiopia: phylogenetic relationships and mutational hotspots.

Eragrostis tef is an important cereal crop in Ethiopia with excellent storage properties, high–quality food, and the unique ability to thrive in extreme environmental conditions. However, the application of advanced molecular tools for breeding and conservation of these species is extremely limited. Therefore, developing chloroplast genome resources and high-resolution molecular markers are valuable to E. tef population and biogeographic studies. In the current study, we assembled and compared the complete plastomes of 32 E. tef accessions. The size of the plastomes ranged from 134,349 to 134,437 bp with similar GC content (∼38.3%). Genomes annotations revealed 112 individual genes, including 77 protein-coding, 31 tRNA, and 4 rRNA genes. Comparison of E. tef plastomes revealed a low degree of intraspecific sequence variations and no structural differentiations. Furthermore, we found 34 polymorphic sites (13 cpSSRs, 12 InDels, and 9 SNPs) that can be used as valuable DNA barcodes. Among them, the majority (88%) of the polymorphic sites were identified in the noncoding genomic regions. Nonsynonymous (ka) and synonymous (ks) substitution analysis showed that all PCGs were under purifying selection (ka/ks <1). The phylogenetic analyses of the whole plastomes and polymorphic region sequences were able to distinguish the accession from the southern population, indicating its potential to be used as a super-barcode. In conclusion, the newly generated plastomes and polymorphic markers developed here could be a useful genomic resource in molecular breeding, population genetics and the biogeographical study of E. tef.