Analysis of complete chloroplast genome sequences and insight into the phylogenetic relationships of Ferula L.

BACKGROUND: Ferula L. is one of the largest and most taxonomically complicated genera as well as being an important medicinal plant resource in the family Apiaceae. To investigate the plastome features and phylogenetic relationships of Ferula and its neighboring genera Soranthus Ledeb., Schumannia Kuntze., and Talassia Korovin, we sequenced 14 complete plastomes of 12 species.  RESULTS: The size of the 14 complete chloroplast genomes ranged from 165,607 to 167,013 base pairs (bp) encoding 132 distinct genes (87 protein-coding, 37 tRNA, and 8 rRNA genes), and showed a typical quadripartite structure with a pair of inverted repeats (IR) regions. Based on comparative analysis, we found that the 14 plastomes were similar in codon usage, repeat sequence, simple sequence repeats (SSRs), and IR borders, and had significant collinearity. Based on our phylogenetic analyses, Soranthus, Schumannia, and Talassia should be considered synonymous with Ferula. Six highly divergent regions (rps16/trnQ-UUG, trnS-UGA/psbZ, psbH/petB, ycf1/ndhF, rpl32, and ycf1) were also detected, which may represent potential molecular markers, and combined with selective pressure analysis, the weak positive selection gene ccsA may be a discriminating DNA barcode for Ferula species. CONCLUSION: Plastids contain abundant informative sites for resolving phylogenetic relationships. Combined with previous studies, we suggest that there is still much room for improvement in the classification of Ferula. Overall, our study provides new insights into the plastome evolution, phylogeny, and taxonomy of this genus.

High-throughput sequencing reveals the diversity and community structure of rhizosphere fungi of Ferula Sinkiangensis at different soil depths.

Ferula sinkiangesis is a valuable medicinal plant that has become endangered. Improving the soil habitat of Ferula sinkiangesis can alleviate plant damage. Fungi play an important role in the soil, but current information on the fungal community structure in the habitat of Ferula sinkiangesis and the relationship between soil fungi and abiotic factors remains unclear. In this study, we analyzed the relative abundance of fungal species in the rhizosphere of Ferula sinkiangesis. Spearman correlation analysis showed that the abiotic factor total potassium (TK) significantly explained the alpha diversity of the fungal community. At altitude, available phosphorus (AP), nitrate nitrogen (NN) and TK were significantly associated with the fungal species. In addition, a two-way ANOVA showed that soil depth had no significant effects on the alpha diversity of rhizosphere and non-rhizosphere fungi. Interestingly, linear discriminant effect size (LEfSe) analysis indicated that different biomarkers were present at varying soil depths. These findings may be related to the growth and medicinal properties of Ferula Sinkiangensis.

Tissue-Specific Transcriptome Analysis Reveals Candidate Genes for Terpenoid and Phenylpropanoid Metabolism in the Medicinal Plant Ferula assafoetida.

Ferula assafoetida is a medicinal plant of the Apiaceae family that has traditionally been used for its therapeutic value. Particularly, terpenoid and phenylpropanoid metabolites, major components of the root-derived oleo-gum-resin, exhibit anti-inflammatory and cytotoxic activities, thus offering a resource for potential therapeutic lead compounds. However, genes and enzymes for terpenoid and coumarin-type phenylpropanoid metabolism have thus far remained uncharacterized in F. assafoetida Comparative de novo transcriptome analysis of roots, leaves, stems, and flowers was combined with computational annotation to identify candidate genes with probable roles in terpenoid and coumarin biosynthesis. Gene network analysis showed a high abundance of predicted terpenoid- and phenylpropanoid-metabolic pathway genes in flowers. These findings offer a deeper insight into natural product biosynthesis in F. assafoetida and provide genomic resources for exploiting the medicinal potential of this rare plant.

Genetic differentiation between bitter and sweet asafetida plants using ISSR markers.

Bitter (Ferula pseudalliacea) and sweet (Ferula assa-foetida) asafetida (Apiaceae family) are well-known economic and medicinal herbs owing to their gum. This study investigates genetic differentiation of F. pseudalliacea and F. assa-foetida using ISSR markers, to determine the effective primer and to assess the possibility of separating sweet and bitter plant populations from each other. Results showed that among 22 primers, eight markers reproduced obvious DNA patterns and revealed 234 scorable DNA bands. ISSR-16 and ISSR-55 primers had better performance than other primers according to the number of bands, PIC and Marker Index. Bitter population showed polymorphic loci (224), percentage of polymorphic loci (95.73%) and observed number of alleles (1.96 ± 0.2), while sweet populations showed the amount of these parameters as 218, 93.16% and 1.93 ± 0.25, respectively. Estimated Gst of sweet population was 0.09 and Gst of bitter population was 0.06. Comparing gene flow in bitter and sweet populations showed a lower level of gene flow between sweet populations (Nm = 4.93) compared to bitter ones (Nm = 7.89). Within group genetic similarity of sweet asafetida population was higher than between group variation of bitter and sweet populations. The highest similarity was observed between bitter populations (0.95). The highest genetic dissimilarity was also estimated between bitter and sweet populations (0.08). Cluster analysis grouped four studied populations into 13 clusters using Jaccard's similarity coefficient and UPGMA method. Principal coordinate analysis showed that 61.02% of total variance was explained using three components and it could completely separate populations as well as cluster analysis. These grouping correspond nearly with geographical distribution. Analysis of molecular variance showed that genetic variation within populations (87%) was more than among populations (13%). The results indicated that ISSR marker is suitable to investigate genetic diversity of asafetida populations and could separate populations of the same genera with similar germplasm.

Genetic diversity among and within Ferula asafoetida H. Karst. populations using molecular and phytochemical markers.

Ferula asafoetida is an herbaceous, perennial and monocarpic species of the Apiaceae family with medicinal properties. In this study, the genetic diversity of F. asafetida populations was assessed using SCoT and SRAP molecular markers. Nine SCoT and nine SRAP primer combinations amplified 211 and 194 bands, showing 96.07% and 92.87% of polymorphism, respectively. The dendrograms generated from Jaccard's similarity matrix and the UPGMA method revealed that genetic relationships among the F. asafoetida genotypes were not related to their geographical regions. The analysis also revealed a relatively acceptable differentiation in allele frequencies, illustrating the high genetic diversity and gene flows among the populations. The high percent of diversity among the populations indicated a rich resource of germplasm for breeding programs. Moreover, STRUCTURE analysis showed the high admixture of the studied accessions. According to AMOVA analysis, higher genetic variation occurred within the populations. Total phenolic content was also assessed using the folin-ciocalteu method, observing a relatively extent variety (0.163-0.938 mg TA/g DW) among the populations. In addition, HPLC analysis defined eleven compounds in which p-coumaric and 1,3- caffeoylquinic acids were the most abundant phenolic acids. Finally, Feragheh population possessed the highest TPC (total phenolic content) and gene diversity. Karaj population also exhibited the highest amount of flavonoids and phenolic acids such as kaempferol and p-coumaric acids.

Mining Ferula gummosa transcriptome to identify miRNAs involved in the regulation and biosynthesis of terpenes.

Ferula gummosa is a well-known medicinal and industrial plant for its oleo-gum-resin named galbanum. So far, there is no information about the role of miRNAs on the production of terpenes as the major secondary metabolite of galbanum. In the present study, RNA-seq data on the root and flower of the plant were used to predict miRNAs and their targets using computational approaches. Additionally, biological network analyses were used to unravel the direct or indirect regulatory effects of miRNAs on the targets involved in terpene biosynthesis. For the first time, 220 miRNAs from 94 families have been reported in F. gummosa. miR5658, miR1533, miR5021, miR414, and miR1436 are the top five miRNAs with high abundance. Gene ontology (GO) analysis of the identified targets showed that in the biological process category, the miRNA-regulated genes were highly involved in transcription. According to the KEGG and PlantCyc results, six miRNAs from five miRNA families including miR2919, miR5251, miR838, miR5021, and miR5658 were found to be related to the pathway of terpene biosynthesis. Moreover, network analysis showed that three terpene-regulating TFs namely SPL7, SPL11, and ATHB13 are putatively regulated by three miRNAs including miR1533, miR5021, and miR5658 respectively. Differential gene expression results showed that the expression levels of these miRNAs are negatively correlated to the expression levels of both TFs and their co-expressed terpene biosynthesis genes.

Transcriptome and metabolome analysis of Ferula gummosa Boiss. to reveal major biosynthetic pathways of galbanum compounds.

Ferula gummosa Boiss. is an industrial and pharmaceutical plant that has been highly recognized for its valuable oleo-gum-resin, namely galbanum. Despite the fabulous value of galbanum, very little information on the genetic and biochemical mechanisms of its production existed. In the present study, the oleo-gum-resin and four organs (root, flower, stem, and leaf) of F. gummosa were assessed in terms of metabolic compositions and the expression of genes involved in their biosynthetic pathways. Results showed that the most accumulation of resin and essential oils were occurred in the roots (13.99 mg/g) and flowers (6.01 mg/g), respectively. While the most dominant compound of the resin was ß-amyrin from triterpenes, the most abundant compounds of the essential oils were α-pinene and ß-pinene from monoterpenes and α-eudesmol and germacrene-D from sesquiterpenes. Transcriptome analysis was performed by RNA sequencing (RNA-seq) for the plant roots and flowers. Differential gene expression analysis showed that 1172 unigenes were differential between two organs that 934 (79.6%) of them were up-regulated in the flowers and 238 (20.4%) unigenes were up-regulated in the roots (FDR ≤0.001). The most important up-regulated unigenes in the roots were involved in the biosynthesis of the major components of galbanum, including myrcene, germacrene-D, α-terpineol, and ß-amyrin. The results obtained by RNA-Seq were confirmed by qPCR. These analyses showed that different organs of F. gummosa are involved in the production of oleo-gum-resin, but the roots are more active than other organs in terms of the biosynthesis of triterpenes and some mono- and sesquiterpenes. This study provides rich molecular and biochemical resources for further studies on molecular genetics and functional genomics of oleo-gum-resin production in F. gummosa.

The genetic diversity and spatial genetic structure of the Corso-Sardinian endemic Ferula arrigonii Bocchieri (Apiaceae).

Corsica and Sardinia represent major hotspots of plant diversity in the Mediterranean area and are priority regions for conservation due to their high number of endemic plant species. However, information supporting human decision-making on the conservation of these species is still scarce, especially at the genetic level. In this work, the first assessment is reported of the species-wide spatial genetic structure and diversity of Ferula arrigonii Bocchieri, a Corso-Sardinian endemic located in a few coastal sites and on small islands. Nine populations covering the entire natural range of the species were investigated by means of AFLP (amplified fragment length polymorphism) markers. Results indicate that this species is characterised by high levels of genetic polymorphism (92% polymorphic fragments) and of genetic diversity (H(w) = 0.317) and by relatively low differentiation among populations (F(st) = 0.057). PCoA, Bayesian analysis and neighbour-joining clustering were also employed to investigate the genetic structure of this species. Three genetically distinct groups were detected, although with considerable overlap between populations.

The phylogeographical history of the Iberian steppe plant Ferula loscosii (Apiaceae): a test of the abundant-centre hypothesis.

The geology and climate of the western Mediterranean area were strongly modified during the Late Tertiary and the Quaternary. These geological and climatic events are thought to have induced changes in the population histories of plants in the Iberian Peninsula. However, fine-scale genetic spatial architecture across western Mediterranean steppe plant refugia has rarely been investigated. A population genetic analysis of amplified fragment length polymorphism variation was conducted on present-day, relict populations of Ferula loscosii (Apiaceae). This species exhibits high individual/population numbers in the middle Ebro river valley and, according to the hypothesis of an abundant-centre distribution, these northern populations might represent a long-standing/ancestral distribution centre. However, our results suggest that the decimated southern and central Iberian populations are more variable and structured than the northeastern ones, representing the likely vestiges of an ancestral distribution centre of the species. Phylogeographical analysis suggests that F. loscosii likely originated in southern Spain and then migrated towards the central and northeastern ranges, further supporting a Late Miocene southern-bound Mediterranean migratory way for its oriental steppe ancestors. In addition, different glacial-induced conditions affected the southern and northern steppe Iberian refugia during the Quaternary. The contrasting genetic homogeneity of the Ebro valley range populations compared to the southern Iberian ones possibly reflects more severe bottlenecks and subsequent genetic drift experienced by populations of the northern Iberia refugium during the Pleistocene, followed by successful postglacial expansion from only a few founder plants.

Conservation genetics of the endangered Iberian steppe plant Ferula loscosii (Apiaceae).

Ferula loscosii (Lange) Willk (Apiaceae) is a threatened endemic species native to the Iberian Peninsula. The plant has a narrow and disjunct distribution in three regions, NE, C and SE Spain. Genetic variability within and among 11 populations from its natural distribution was assessed using allozymes. Intermediate levels of genetic diversity were detected in F. loscosii (P(99%) = 36.83; H(E) = 0.125; H(T) = 0.152). However, the highest genetic diversity (58%) corresponded to the threatened populations from SE and C Spain (H(T) = 0.169) rather than the more abundant and larger populations from NE Spain (Ebro valley) (H(T) = 0.122). Low to moderate levels of genetic structure were found among regional ranges (G(ST) = 0.134), and several statistical spatial correlation analyses corroborated substantial genetic differentiation among the three main regional ranges. However, no significant genetic differentiation was found among the NE Spain populations, except for a northernmost population that is geographically isolated. Outcrossing mating and other biological traits of the species could account for the maintenance of the present values of genetic diversity within populations. The existence of an ancestral late Tertiary wider distribution of the species in SE and C Spain, followed by the maintenance of different Quaternary refugia in these warmer areas, together with a more recent and rapid post-glacial expansion towards NE Spain, are arguments that could explain the low genetic variability and structure found in the Ebro valley and the higher levels of diversity in the southern Iberian populations.

Ferula gummosa Boiss. Embryogenic culture and karyological changes.

Ferula gummosa Boiss. a highly valuable medicinal plant which naturally propagates in very limited areas of the Middle East with specific environmental conditions. The production of Ferula gummosa somatic embryos and the karyological analysis of somatic seedlings were the purpose of this study. High frequency indirect embryogenesis was induced in callus derived from zygotic embryonic axes. Embryogenesis was obtained when callus tissues were placed onto an agar induction Murashige and Skoog medium with 1-naphthalene acetic acid and after the transfer of the cultures in a thermoperiod regime of 16 h, 19 degrees C/8 h, 7 degrees C under photoperiod of 16 h light/8h dark. Embryogenic callus tissues were maintained by subculture on induction medium. Globular proliferation was achieved with suspension culture in the Murashige and Skoog medium added with 1-naphthalene acetic acid or 2,4-dichlorophenoxyacetic acid for two weeks. Maturation of embryos and development of plantlets arose on the induction agar medium, but was better after transfer into the hormone free Murashige and Skoog medium. However, the level of abnormal embryos was high. Direct embryogenesis was obtained from somatic seedlings. The best results were obtained from hypocotyl explants. Embryo induction was achieved by two week culture of the explants in 2,4-dichlorophenoxyacetic acid liquid medium; somatic embryo growth and maturation was recovered on the hormone free medium. High level of abnormalities was recorded in the culture. Karyological analysis showed a high incidence level of cytochimerism in somatic seedlings with chromosome stickiness, polypoidy and aneuploidy in metaphase cells of the same root tip. The frequency of these karyological changes varied with the type of somatic embryos with regard to morphological abnormalities. Normal and abnormal rooted somatic seedlings were able to grow until production of the first leaf and then entered dormancy in the same manner as zygotic plantlets.

[Karyotypes analysis of Ferula fukanensis].

OBJECTIVE: Study on the karyotypes analysis of Ferula fukanensis. METHOD: The young roots were treated with 0.000 2 mol x L(1) 8-Hydroxyquinoline for 3 h, carnoy's for 3 h, 1 mol x L(-1) HCl in 5 min,carbol fuchsin coloration for 2 min and the treated roots were utilizied to make the plate for observation. RESULT: The fukanensis is diploid. The chromosome number of somatic cells was 2n = 22. The karyotype formula is 2n = 2x = 20 = 16m + 4sm. The 4th and 10th are submetacentric, and the others are metacentric. CONCLUSION: The karyotype of F. fukanensis belongs to "2A" type of stebbins', and it is a primitive species.