Development of Species-Specific InDel Markers in Citrus

Citrus taxonomy is complex owing to the existence of a wide range of species: Poncirus is used mainly for rootstock; Fortunella produces small fruit and edible pericarp; and Citrus comprises the most widespread fruit crop species worldwide. Rapidly increasing genome resources from different citrus species facilitate the development of convenient and genome-wide molecular markers that can be applied to both inter- and intra-species analyses. In this study, by comparing the genome sequences of four citrus species, a set of 1958 InDels were identified and 453 candidate InDels were converted into PCR-based markers. Among these candidate InDels, 268 (65%) exhibited length polymorphisms from 30 bp to 200 bp when applied to seven species from the genera Poncirus, Fortunella and Citrus. Seven InDel markers exhibited high intraspecific polymorphisms in a natural pummelo population. The results showed that the InDel markers are effective for both inter- and intra-specific variation and identification analyses. These InDel markers are expected to be applied to germplasm identification, phylogenetic analysis, genetic diversity evaluation and marker-assisted breeding in citrus.     

Phylogenetic Relationships of Citrus and Its Relatives Based on matK Gene Sequences

The genus Citrus includes mandarin, orange, lemon, grapefruit and lime, which have high economic and nutritional value. The family Rutaceae can be divided into 7 subfamilies, including Aurantioideae. The genus Citrus belongs to the subfamily Aurantioideae. In this study, we sequenced the chloroplast matK genes of 135 accessions from 22 genera of Aurantioideae and analyzed them phylogenetically. Our study includes many accessions that have not been examined in other studies. The subfamily Aurantioideae has been classified into 2 tribes, Clauseneae and Citreae, and our current molecular analysis clearly discriminate Citreae from Clauseneae by using only 1 chloroplast DNA sequence. Our study confirms previous observations on the molecular phylogeny of Aurantioideae in many aspects. However, we have provided novel information on these genetic relationships. For example, inconsistent with the previous observation, and consistent with our preliminary study using the chloroplast rbcL genes, our analysis showed that Feroniella oblata is not nested in Citrus species and is closely related with Feronia limonia. Furthermore, we have shown that Murraya paniculata is similar to Merrillia caloxylon and is dissimilar to Murraya koenigii. We found that “true citrus fruit trees” could be divided into 2 subclusters. One subcluster included Citrus, Fortunella, and Poncirus, while the other cluster included Microcitrus and Eremocitrus. Compared to previous studies, our current study is the most extensive phylogenetic study of Citrus species since it includes 93 accessions. The results indicate that Citrus species can be classified into 3 clusters: a citron cluster, a pummelo cluster, and a mandarin cluster. Although most mandarin accessions belonged to the mandarin cluster, we found some exceptions. We also obtained the information on the genetic background of various species of acid citrus grown in Japan. Because the genus Citrus contains many important accessions, we have comprehensively discussed the classification of this genus.     

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.     

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.     

Identification and physiological evaluation of the components from Citrus fruits as potential drugs for anti-corpulence and anticancer

On the basis of monitoring the prevention of accumulation of lipid droplets in mouse 3T3-L1 preadipocyte cells and inhibition of the proliferation of human colon cancer HT-29 cells, effective anti-corpulence and anticancer compounds were isolated from the peel of Citrus fruits. These bioactive components were identified as polymethoxyflavones and coumarin derivatives by spectroscopic analyses. 5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone had the greatest anti-corpulence effects and 3,5,6,7,8,3′,5′-heptamethoxyflavone had the greatest anticancer effects. Furthermore, distributions of those bioactive components in the peel of 10 species of Citrus fruits were demonstrated by HPLC analyses.     

Phylogenetic relationships of <Emphasis Type="Italic">Citrus</Emphasis> and its relatives based on <Emphasis Type="Italic">rbcL</Emphasis> gene sequences

We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed.     

Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs)

Twenty-four simple sequence repeat (SSR) markers were used to detect molecular polymorphisms among 370 mostly sexually derived Citrus accessions from the collection of citrus germplasm maintained at the University of California, Riverside. A total of 275 alleles were detected with an average of 11.5 alleles per locus and an average polymorphism information content of 0.625. Genetic diversity statistics were calculated for each individual SSR marker, the entire population, and for specified Citrus groups. Phylogenetic relationships among all citrus accessions and putative non-hybrid Citrus accessions were determined by constructing neighbor-joining trees. There was strong support for monophyly at the species level when hybrid taxa were removed from the data set. Both of these trees indicate that Fortunella clusters within the genus Citrus but Poncirus is a sister genus to Citrus. Additionally, Citrus accessions were probabilistically assigned to populations or multiple populations if their genotype indicated an admixture by a model-based clustering approach. This approach identified five populations in this data set. These separate analyses (distance and model based) both support the hypothesis that there are only a few naturally occurring species of Citrus and most other types of Citrus arose through various hybridization events between these naturally occurring forms.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999     

利用DNA片段测序方法探究枳属和富民枳的分类地位

富民枳(Poncirus polyandra)属于芸香科(Rutaceae)枳属(Poncirus Raf.).自发表以来,分类地位一直备受争议,其中在Flora of China中认为富民枳为柑橘杂交种(Poncirus polyandra),把枳属归并于柑橘属(Citrus).该研究选取枳属的富民枳、枳(Ponci...     

Phytochemical,phylogenetic, and anti-inflammatory evaluation of 43 Urtica accessions (stinging nettle) based on UPLC–Q-TOF-MS metabolomic profiles

Several species of the genus Urtica (especially Urtica dioica, Urticaceae), are used medicinally to treat a variety of ailments. To better understand the chemical diversity of the genus and to compare different accessions and different taxa of Urtica, 63 leaf samples representing a broad geographical, taxonomical and morphological diversity were evaluated under controlled conditions. A molecular phylogeny for all taxa investigated was prepared to compare phytochemical similarity with phylogenetic relatedness. Metabolites were analyzed via UPLC–PDA–MS and multivariate data analyses. In total, 43 metabolites were identified, with phenolic compounds and hydroxy fatty acids as the dominant substance groups. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) provides a first structured chemotaxonomy of the genus. The molecular data present a highly resolved phylogeny with well-supported clades and subclades. U. dioica is retrieved as both para- and polyphyletic. European members of the U. dioica group and the North American subspecies share a rather similar metabolite profile and were largely retrieved as one, nearly exclusive cluster by metabolite data. This latter cluster also includes – remotely related – Urtica urens, which is pharmaceutically used in the same way as U. dioica. However, most highly supported phylogenetic clades were not retrieved in the metabolite cluster analyses. Overall, metabolite profiles indicate considerable phytochemical diversity in the genus, which largely falls into a group characterized by high contents of hydroxy fatty acids (e.g., most Andean-American taxa) and another group characterized by high contents of phenolic acids (especially the U. dioica-clade). Anti-inflammatory in vitro COX1 enzyme inhibition assays suggest that bioactivity may be predicted by gross metabolic profiling in Urtica.     

Characterization of genes associated with polyembryony and in vitro somatic embryogenesis in Citrus

Apomixis is a common reproduction system in the genus Citrus. To investigate the molecular mechanism of apomictic embryogenesis in Citrus, genes expressed specifically in an apomictic genotype were isolated by PCR-selected suppression subtractive hybridization with total RNAs obtained from the ovule at anthesis. Several genes showing conspicuously different expressions between polyembryonic (apomictic) and monoembryonic (nonapomictic) genotypes were selected, and their expression profiles during ovule development were analyzed in detail. This analysis identified two apomictic and three nonapomictic genotype-specific genes. Among the latter, msg-2 was highly expressed in the late stage of somatic embryogenesis. Specific expression during ovule development in monoembryonic cultivars and in the late stage of somatic embryogenesis indicated that msg-2 is not expressed in the initiation stage of polyembryogenesis and somatic embryogenesis, suggesting its role in suppressing initial cell formation of somatic embryos. The full-length complementary DNA of msg-2 contained small open reading frames in its sequence but showed no homology to functionally known genes in the public databases. As sequences similar to msg-2 were frequently found among Citrus expressed sequence tags, msg-2 may be associated with polyembryogenesis and somatic embryogenesis in a Citrus-specific manner.     

A Six Nuclear Gene Phylogeny of Citrus (Rutaceae) Taking into Account Hybridization and Lineage Sorting

Background

Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species.

Methodology and Principal Findings

(1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test.

Conclusions

We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We conclude that identifying hybridization as a frequent cause of incongruence among gene trees is critical to correctly infer the phylogeny among species of Citrus.     

Composition of Peel Oil from Citrus Unshu

Constituents of aroma of peel oil (both oleoresin and cold-pressed oil) from Citrus Unshu were isolated by column chromatography and gas chromatography (GLC). and identified mostly by infrared (IR), partially by mass (MS) and nuclear magnetic resonance (NMR) spectroscopy.

One hundred and seven compounds were found in the oil and among them, four sesquiterpene hydrocarbons, five acetates, five carbonyls and five alcohols were identified as a main constituents which contributed to difference in flavor from other citrus peel oils. Characteristics of aroma of C. Unshu was discussed on the basis of quantitative analysis.     

Antioxidant activities of the volatile oils and methanol extracts from olive stems

This study was designed to examine the chemical composition and antioxidant activities of the volatile oils and methanol extracts of Olea europaea L. (cvs) chemlali and neb jmel stems. GC and GC–MS analyses of the volatile oils resulted in the identification of 38 and 35 compounds, representing 91.1 and 87.4 % of the volatile oils. Phenylethyl alcohol was found in the volatile oil of each cultivar, which was also the major volatile component of cv. chemlali and cv. neb jmel stems. Besides benzyl alcohol, methyl salicylate and 3-ethenylpyridine were the main volatile compounds of cv. chemlali, while nonanal, 3-ethenylpyridine and benzyl alcohol of cv. neb jmel stems were also the main constituents. Significant differences were also found in total tannin contents among two cultivars, representing 8.10 mg CEQ/g DW in cv. chemlali and 20.47 mg CEQ/g DW in cv. neb jmel. The highest contents of total phenols and o-diphenols were observed in stems extracts of cv. neb jmel (78.26, and 9.56 mg/100 g, respectively). The HPLC profiles for methanol extracts from stems of cv. chemlali and cv. neb jmel showed that oleuropein, vanillic acid and gallic acid were the predominant free phenolic compounds. Antioxidant activities of the volatile oils and the methanolic extract from stems parts were evaluated by DPPH and ABTS⁺ radical-scavenging activity assays. In all tests, methanolic extracts obtained from stems parts showed better antioxidant activity than volatile oils. Principal components analysis of the phenolics content and antioxidant activities showed discrimination between methanol extracts of the two cultivars.     

Genome-Wide Comparative Analysis Reveals Similar Types of NBS Genes in Hybrid Citrus sinensis Genome and Original Citrus clementine Genome and Provides New Insights into Non-TIR NBS Genes

In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.     

Elektronenmikroskopische Beobachtungen an den Drüsenzellen vonPoncirus trifoliata; zugleich ein Beitrag zur Wirkung ätherischer Öle auf Pflanzenzellen und eine Methode zur Unterscheidung flüchtiger von nichtflüchtigen lipophilen Komponenten

Zusammenfassung Die Feinstruktur der Zellen aus den Drüsenkomplexen der Fruchtschale vonPoncirus trifoliata wurde in allen Altersstufen festgehalten. Die Piastiden zeigen stark geschlängelte, in alle Richtungen verlaufende Tubuli und enthalten lipophile Substanzen. Diese können nicht mit den Plastoglobuli verwechselt werden, die in den Piastiden des Flavedos reifenderPoncirus-Früchte in großer Zahl auftreten. Mit Hilfe von Gefriertrocknung und durch Einbringen verschiedener ätherischer Öle in Testpflanzen wird bewiesen, daß die osmiophilen Substanzen in der Plastide größtenteils ätherische Öle sind. Gefriertrocknung erlaubt es, flüchtige von nichtflüchtigen Komponenten in e-optischen Präparationen bei ausreichend guter Strukturerhaltung zu unterscheiden. Ätherische Öle, die in Gewebe eingebracht werden, zerstören rasch die Membransysteme von Pflanzen, die Monoterpene nicht in größerer Menge synthetisieren. In ähnlicher Weise schädigt Ölsäure die Zellen. Die in fixierte Zellen eingebrachten Öle sind anders verteilt als die genuinen Öle der intakten Drüsenzelle vonPoncirus. Glutaraldehyd-OsO₄ gestattet ätherische Ölein situ zu fixieren. Diese werden dann während der nachfolgenden Entwässerung weniger extrahiert als nach alleinigem Einwirken von OsO₄. Tritiummarkiertes Mevalonat dürfte nur sehr wenig eingebaut werden. Die in die Terpene inkorporierte Mevalonsäure wird durch die Acetonentwässerung extrahiert, die nicht eingebaute geht infolge der guten Wasserlöslichkeit während der Einbettung ebenfalls verloren. Es wird vermutet, daß in den Drüsenzellen derPoncirus-Frucht hauptsächlich die Plastiden Monoterpene erzeugen, wobei angelieferte Vorstufen der Terpensynthese in oder an den Tubuli der Piastiden zu Monoterpenen konvertiert werden. Die ätherischen Öle verbleiben zu einem großen Prozentsatz solange in der Plastide, bis diese degeneriert.

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The ultrastructure of gland cells inPoncirus trifoliata; at the same time a contribution to the effect of essential oils on plant cells and a method how to distinguish between volatile and nonvolatile lipophilic components under the electron microscope

Summary The ultrastructure of gland cells from the peel ofPoncirus trifoliata fruits of different age was investigated. The plastids have undulating tubuli which are oriented in all directions and contain lipophilic substances. These substances may not be mixed up with plastoglobuli which are found in great number in plastids of the epicarp of maturingPoncirus fruits. The osmiophilic spots and rings in the plastid are for the most part composed of essential oils as indicated by freeze-drying and application of different essential oils. Freeze-drying makes it possible to distinguish between volatile and non-volatil components under the electron microscope. D-limonene, which is the major component of the oil fromPoncirus andCitrus as well asCitrus oil, when incorporated into test plants give rings similar to those occuring in plastids ofPoncirus. When essential oils are brought into tissues of plants which do not synthesize appreciable amounts of monoterpenes, destruction of the membrane systeme occurs. Oleic acid damages the cell in a similar way. The oil applied to fixed tissue are distributed in a different pattern as the genuine oils of intact gland cells ofPoncirus. Essential oils which are fixedin situ with glutaraldehyde-osmic acid are extracted to a smaller amount than those fixed with osmic acid only. Tritium labelled mevalonate is incorporated only to a small amount. Mevalonic acid which is incorporated into terpenes is extracted during specimen dehydration which acetone. The mevalonic acid which is not incorporated is lost in the course of the embedding process due to its water solubility. It is surmised that mainly the plastids of gland cells ofPoncirus fruits produce monoterpenes. During its synthesis precursors of terpenes are converted into monoterpenes within or at the tubuli of the plastid. The essential oils rest within the plastid until its degeneration.

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Herrn Dir. Dr. W.Bernhard und seinen Mitarbeitern danke ich für die freundliche Aufnahme im Institut de Recherches Scientifiques sur le cancer, Villejuif, Seine, France, und für die Einführung in die Autoradiographie im EM. Der Deutschen Forschungsgemeinschaft bin ich für großzügige finanzielle Unterstützung zu Dank verpflichtet.     

Essential Oil Composition of Three Globularia Species

The chemical composition of the essential oils obtained by hydrodistillation from the aerial parts of Globularia cordifolia L., G. meridionalis (Podp.) O.Schwarz , and G. punctata Lapeyr . was characterized by GC‐FID and GC/MS analyses. Among the 33 identified compounds, the most abundant present in all investigated samples were oct‐1‐en‐3‐ol (2.9–47.0%), 6‐(1,5‐dimethylhex‐4‐enyl)‐3‐methylcyclohex‐2‐enone (8.2–40.9%), and fukinanolid (7.4–31.6%). Multivariate statistical analyses (PCA and HCA) of the hitherto studied Globularia volatile compounds confirmed to some extent the assumed phylogenetic relationships of the Globularia species studied, including the close relationship between the morphologically similar species G. cordifolia and G. meridionalis, but also evidenced several discrepancies in the current classification of Globularia species.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.