Conserved chloroplast genome sequences of the genus Clerodendrum Linn. (Lamiaceae) as a super-barcode.

BACKGROUND: The plants of the genus Clerodendrum L. have great potential for development as an ornamental and important herbal resource. There is no significant morphological difference among many species of the genus Clerodendrum, which will lead to confusion among the herbs of this genus and ultimately affect the quality of the herbs. The chloroplast genome will contribute to the development of new markers used for the identification and classification of species. METHODS AND RESULTS: Here, we obtained the complete chloroplast genome sequences of Clerodendrum chinense (Osbeck) Mabberley and Clerodendrum thomsoniae Balf.f. using the next generation DNA sequencing technology. The chloroplast genomes of the two species all encode a total of 112 unique genes, including 80 protein-coding, 28 tRNA, and four rRNA genes. A total of 44-42 simple sequence repeats, 19-16 tandem repeats and 44-44 scattered repetitive sequences were identified. Phylogenetic analyses showed that the nine Clerodendrum species were classified into two clades and together formed a monophyletic group. Selective pressure analyses of 77 protein-coding genes showed that there was no gene under positive selection in the Clerodendrum branch. Analyses of sequence divergence found two intergenic regions: trnH-GUG-psbA, nhdD-psaC, exhibiting a high degree of variations. Meanwhile, there was no hypervariable region identified in protein coding genes. However, the sequence identities of these two intergenic spacers (IGSs) are greater than 99% among some species, which will result in the two IGSs not being used to distinguish Clerodendrum species. Analysis of the structure at the LSC (Large single copy) /IR (Inverted repeat) and SSC (Small single copy)/IR boundary regions showed dynamic changes. The above results showed that the complete chloroplast genomes can be used as a super-barcode to identify these Clerodendrum species. The study lay the foundation for the understanding of the evolutionary process of the genus Clerodendrum.

Comparative assessment of ISSR, RAPD, and SCoT markers for genetic diversity in Clerodendrum species of North East India.

Clerodendrum belonging to the family of Lamiaceae is used in indigenous systems of medicine to treat various life-threatening diseases. The genus has complex morphological variations which lead to limits in its precise taxonomic classifications. Genetic diversity study could enhance taxonomic authentication and evolutionary relationship among the species of Clerodendrum. In this study, nine species of Clerodendrum collected from different regions of North East India were screened using ISSR, RAPD, and SCoT molecular markers. The markers of ISSR, RAPD, and SCoT generated a total of 79, 126, and 145 amplicons with an average of 6.58, 7.86, and 8.53 amplicon per primer. The polymorphism information contents (PIC) for ISSR, RAPD, and SCoT ranged from 0.28 to 0.37, 0.39 to 0.69, and 0.30 to 0.62 with resolving power (Rp) varying from 5.26 to 11.11, 4.04 to 9.67, and 4.54 to 8.65, respectively. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based clustering methods grouped 94 genotypes into 6 clusters for ISSR and 3 clusters each for RAPD and SCoT markers. Similarly, population structure-based analysis divided 94 genotypes into 6 populations for ISSR and RAPD and 4 populations for SCoT markers. AMOVA analysis revealed that SCoT markers generated maximum genetic variations within and among genotypes, contrary to ISSR and RAPD markers. Results in this study, suggest that the competence of three markers was relatively the same in genotypes fingerprinting, but SCoT was more efficient in the detection of polymorphism for Clerodendrum species. Further, these results could be integrated in the exploration of diverse Clerodendrum species and germplasm utilization.

DNA barcodes for delineating Clerodendrum species of North East India.

The diversified genus of Clerodendrum with its complex evolutionary history leads to taxonomic mystification. Unlike traditional taxonomic methods, DNA barcoding could be a promising tool for the identification and conservation of Clerodendrum species. This study was attempted to develop an efficient barcode locus in Clerodendrum species of North East India. We evaluated four barcode candidates (ITS2, matK, rbcL, ycf1) and its combinations in different Clerodendrum species. The reliability of barcodes to distinguish the species were calculated using genetic pairwise distances, intra- and inter-specific diversity, barcode gap, and phylogenetic tree-based methods. The results exemplify that matK posse's maximum number of variables and parsimony-informative sites (103/100), intra- (0.021 ± 0.001) and inter- (0.086 ± 0.005) specific divergences and species resolution rate (89.1%) followed by ITS2, ycf1, and rbcL. Among the combinatorial locus, ITS2 + matK showed the best species discrimination with distinctive barcode gaps. Therefore, we tentatively suggest that the combination of ITS2 + matK as core barcode for Clerodendrum and converted into quick response (QR) code. Hence, this finding indicates that DNA barcoding could provide consistent resources for species discrimination and resolve taxonomic controversies of the genus as well as set a preliminary assessment toward its biodiversity.

SSR marker development in Clerodendrum trichotomum using transcriptome sequencing.

Clerodendrum trichotomum, a member of the Lamiaceae (Verbenaceae) family, is an ornamental plant widely distributed in South Asia. Previous studies have focused primarily on its growth characteristics, stress resistance, and pharmacological applications; however, molecular investigations remain limited. Considering germplasm conservation and the extensive applications of this plant, it is necessary to explore transcriptome resources and SSR makers for C. trichotomum. In the present study, RNA sequencing was used to determine the transcriptome of C. trichotomum. Subsequently, unigene annotations and classifications were obtained, and SSRs were mined with MIcroSAtellite. Finally, primer pairs designed with Oligo 6.0 were selected for polymorphism validation. In total, 127,325,666 high-quality reads were obtained, and 58,345 non-redundant unigenes were generated, of which 36,900 (63.24%) were annotated. Among the annotated unigenes, 35,980 (97.51%) had significant similarity to 607 species in Nr databases. In addition, a total of 6,444 SSRs were identified in 5,530 unigenes, and 200 random primer pairs were designed for polymorphism validation. Furthermore, after primary polymorphism identification, 30 polymorphic primer pairs were selected for the further polymorphism screening, and 200 alleles were identified, 197 of which showed polymorphism. In this work, a large number of unigenes were generated, and numerous SSRs were detected. These findings should be beneficial for further investigations into germplasm conservation and various applications of C. trichotomum. These results should also provide a solid foundation for future molecular biology studies in C. trichotomum.

RNA-Seq analysis of Clerodendrum inerme (L.) roots in response to salt stress.

BACKGROUND: Clerodendrum inerme (L.) Gaertn, a halophyte, usually grows on coastal beaches as an important mangrove plant. The salt-tolerant mechanisms and related genes of this species that respond to short-term salinity stress are unknown for us. The de novo transcriptome of C. inerme roots was analyzed using next-generation sequencing technology to identify genes involved in salt tolerance and to better understand the response mechanisms of C. inerme to salt stress. RESULTS: Illumina RNA-sequencing was performed on root samples treated with 400 mM NaCl for 0 h, 6 h, 24 h, and 72 h to investigate changes in C. inerme in response to salt stress. The de novo assembly identified 98,968 unigenes. Among these unigenes, 46,085 unigenes were annotated in the NCBI non-redundant protein sequences (NR) database, 34,756 sequences in the Swiss-Prot database and 43,113 unigenes in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database. 52 Gene Ontology (GO) terms and 31 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were matched to those unigenes. Most differentially expressed genes (DEGs) related to the GO terms "single-organism process", "membrane" and "catalytic activity" were significantly enriched while numerous DEGs related to the plant hormone signal transduction pathway were also significantly enriched. The detection of relative expression levels of 9 candidate DEGs by qRT-PCR were basically consistent with fold changes in RNA sequencing analysis, demonstrating that transcriptome data can accurately reflect the response of C. inerme roots to salt stress. CONCLUSIONS: This work revealed that the response of C. inerme roots to saline condition included significant alteration in response of the genes related to plant hormone signaling. Besides, our findings provide numerous salt-tolerant genes for further research to improve the salt tolerance of functional plants and will enhance research on salt-tolerant mechanisms of halophytes.

Survey of traditional Dai medicine reveals species confusion and potential safety concerns: a case study on Radix Clerodendri Japonicum.

The adulteration of herbal products is a threat to consumer safety. In the present study, we surveyed the species composition of commercial Radix Clerodendri Japonicum products using DNA barcoding as a supervisory method. A reference database for plant-material DNA-barcode was successfully constructed with 48 voucher samples from 12 Clerodendrum species. The database was used to identify 27 Radix Clerodendri Japonicum decoction piece samples purchased from drug stores and hospitals. The DNA sequencing results revealed that only 1 decoction piece (3.70%) was authentic C. japonicum, as recorded in the Dai Pharmacopeia, whereas the other samples were all adulterants, indicating a potential safety issue. The results indicate that decoction pieces that are available in the market have complex origins and that DNA barcoding is a suitable tool for regulation of Dai medicines.

Development of nuclear SSRs for the insular shrub Clerodendrum izuinsulare (Verbenaceae) and the widespread C. trichotomum.

PREMISE OF THE STUDY: Nuclear microsatellite primers were developed in the insular shrub Clerodendrum izuinsulare and the closely related widespread C. trichotomum to provide molecular tools to undertake a comparative study of the reproductive systems and genetic structures of the two Clerodendrum species. METHODS AND RESULTS: Using an improved protocol for isolating codominant compound microsatellite markers, 19 primer sets were developed in C. izuinsulare and C. trichotomum. The primers amplified dinucleotide repeats with one to nine alleles per locus in C. izuinsulare and one to 15 alleles per locus in C. trichotomum. Tests of cross-amplification showed that four to 16 loci could be amplified using these markers in five other species of Clerodendrum that were studied. CONCLUSIONS: The microsatellite markers described here will be useful for comparative study of the reproductive systems and genetic structures of the narrow endemic C. izuinsulare and the widespread C. trichotomum.

Phylogenetic relationships between Clerodendrum (Lamiaceae) and other Ajugoid genera inferred from nuclear and chloroplast DNA sequence data.

Over the last two centuries the circumscription of the large, pan-tropical genus Clerodendrum (Lamiaceae) has changed frequently, as different authorities have added or removed taxa on the basis of various morphological characters. With the development of molecular methods for systematic research the process of circumscribing taxa has become increasingly analytical. When morphology signals the possibility that taxa are closely related, molecular methods can be used to test the hypothesis objectively. Aegiphila, Amasonia, Huxleya, and Kalaharia are similar morphologically to Clerodendrum. In this paper we use nuclear ribosomal ITS and chloroplast ndhF sequence data to clarify the positions of these four genera relative to Clerodendrum. We show that the Australian monotypic genus Huxleya evolved from within Clerodendrum. Accordingly, we sink Huxleya into Clerodendrum and make a new combination, Clerodendrum linifolium.