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The genus Gentiana is the largest in the Gentianaceae family with ca. 400 species. However, with most species growing on the Qinghai-Tibet plateau, the processes of adaptive evolution and speciation within the genus is not clear. Also, the genomic analyses could provide important information. So far, the complete chloroplast (cp) genome data of the genus are still deficient. As the second and third sequenced members within Gentianaceae, we report the construction of complete cp sequences of Gentiana robusta King ex Hook. f. and Gentiana crassicaulis Duthie ex Burk., and describe a comparative study of three Gentiana cp genomes, including the cp genome of Gentiana straminea Maxim. published previously. These cp genomes are highly conserved in gene size, gene content, and gene order and the rps16 pseudogene with exon2 missing was found common. Three repeat types and five SSR types were investigated, and the number and distribution are similar among the three genomes. Sixteen genome divergent hotspot regions were identified across these cp genomes that could provide potential molecular markers for further phylogenetic studies in Gentiana. The IR/SC boundary organizations in Gentianales cp genomes were compared and three different types of boundaries were observed. Six data partitions of cp genomes in Gentianales were used for phylogenetic analyses and different data partitions were largely congruent with each other. The ML phylogenetic tree was constructed based on the fragments in cp genomes commonly available in 33 species from Lamiids, including 12 species in Gentianales, 1 in Boraginaceae, 10 in Solanales, and 10 in Lamiales. The result strongly supports the position of Boraginaceae (Ehretia acuminata) as the sister of Solanales, with the bootstrap values of 97 %. This study provides a platform for further research into the molecular phylogenetics of species in the order Gentianales (family Gentianaceae) notably in respect of speciation and species identification.;
Scrophularia dentata is an important Tibetan medicinal plant and traditionally used for the treatment of exanthema and fever in Traditional Tibetan Medicine (TTM). However, there is little sequence and genomic information available for S. dentata. In this paper, we report the complete chloroplast genome sequence of S. dentata and it is the first sequenced member of the Sect. Tomiophyllum within Scrophularia (Scrophulariaceae). The gene order and organization of the chloroplast genome of S. dentata are similar to other Lamiales chloroplast genomes. The plastome is 152,553 bp in length and includes a pair of inverted repeats (IRs) of 25,523 bp that separate a large single copy (LSC) region of 84,058 bp and a small single copy (SSC) region of 17,449 bp. It has 38.0% GC content and includes 114 unique genes, of which 80 are protein-coding, 30 are transfer RNA, and 4 are ribosomal RNA. Also, it contains 21 forward repeats, 19 palindrome repeats and 41 simple sequence repeats (SSRs). The repeats and SSRs within S. dentata were compared with those of S. takesimensis and present certain discrepancies. The chloroplast genome of S. dentata was compared with other five publicly available Lamiales genomes from different families. All the coding regions and non-coding regions (introns and intergenic spacers) within the six chloroplast genomes have been extracted and analysed. Furthermore, the genome divergent hotspot regions were identified. Our studies could provide basic data for the alpine medicinal species conservation and molecular phylogenetic researches of Scrophulariaceae and Lamiales.
The alpine plant Gentiana robusta is an endemic species to the Sino-Himalayan subregion. Also, it is one of the original plants used as traditional Tibetan medicine Jie-Ji. We sequence the nuclear ribosomal internal transcribed spacer (ITS) regions, matK, rbcL, rpoC1, trnL (UAA), psbA-trnH, atpB-rbcL, trnS( GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF( GAA) fragments of cp DNA in both G. robusta and such relative species as G. straminea, G. crassicaulis and G. waltonii. With Halenia elliptica as the outgroup, molecular systematic analysis reveals that G. robusta is a natural hybrid. G. straminea is the mother of hybrids, but the father is not very clear. In addition, the molecular markers for distinguishing G. robusta from the parental species or closely related species are identified, respectively. Our studies may provide valuable reference for the species identifications of medicinal plants with complex genetic backgrounds.
Objective: To identify the common Tibetan herb Chuan-Bu.; Method: Local herbalists were visited to observe which plants were being used as Chuan-Bu. Samples of the indigenous plants were collected at the same time. Leaf materials were collected from field surveys. Total genomic DNA was extracted from silica gel-dried leaf samples. The PCR products were purified and directly sequenced.; Result: As the origin of Chuan-Bu in Tibet autonomous region was authenticated, two species were determined, i. e. Euphorbia stracheyiand E. wallichii. Also, based on our earlier research, the origin of Chuan-Bu in Gansu province, is from E. kansuensis. The sequences of ITS1 for E. stracheyi and E. wallichii were 261 bp in size, and 221 bp in ITS2, respectively. The size of the 5.8S coding region was 164 bp for all species examined in the genus. Especially, there was a heterozygous locus in ITS1 (C/G; position 72) for E. stracheyi. The nucleotide divergence between sequences of the 6 species in pairwise comparisons was calculated and the result showed that the variable site could be detected in each pairwise comparison of sequences. Also, there were 8 point mutations in the 5.8S coding region.; Conclusion: nrDNA ITS sequences can be used as the molecular markers to identify the Tibetan herb Chuan-Bu and such Traditional Chinese Medicines from the same genus Euphorbia as E. lathyris, E. humifusa and E. pekinensis.;