Gentiana section Cruciata is widely distributed across Eurasia at high altitudes, and some species in this section are used as traditional Chinese medicine. Accurate identification of these species is important for their utilization and conservation. Due to similar morphological and chemical characteristics, correct discrimination of these species still remains problematic. Here, we sequenced three complete chloroplast (cp) genomes (G. dahurica, G. siphonantha and G. officinalis). We further compared them with the previously published plastomes from sect. Cruciata and developed highly polymorphic molecular markers for species authentication. The eight cp genomes shared the highly conserved structure and contained 112 unique genes arranged in the same order, including 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. We analyzed the repeats and nucleotide substitutions in these plastomes and detected several highly variable regions. We found that four genes (accD, clpP, matK and ycf1) were subject to positive selection, and sixteen InDel-variable loci with high discriminatory powers were selected as candidate barcodes. Our phylogenetic analyses based on plastomes further confirmed the monophyly of sect. Cruciata and primarily elucidated the phylogeny of Gentianales. This study indicated that cp genomes can provide more integrated information for better elucidating the phylogenetic pattern and improving discriminatory power during species authentication.

The extreme conditions (e.g., cold, low oxygen, and strong ultraviolet radiation) of the high mountains provide an ideal natural laboratory for studies on speciation and the adaptive evolution of organisms. Up to now, few genome/transcriptome-based studies have been carried out on how plants adapt to conditions at extremely high altitudes. Notopterygium incisum and Notopterygium franchetii (Notopterygium, Apiaceae) are two endangered high-alpine herbal plants endemic to China. To explore the molecular genetic mechanisms of adaptation to high altitudes, we performed high-throughput RNA sequencing (RNA-seq) to characterize the transcriptomes of the two species. In total, more than 130 million sequence reads, 81,446 and 63,153 unigenes with total lengths of 86,924,837 and 62,615,693 bp, were generated for the two herbal species, respectively. OrthoMCL analysis identified 6375 single-copy orthologous genes between N. incisum and N. franchetii. In total, 381 positively-selected candidate genes were identified for both plants by using estimations of the non-synonymous to synonymous substitution rate. At least 18 of these genes potentially participate in RNA splicing, DNA repair, glutathione metabolism and the plant–pathogen interaction pathway, which were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains. Meanwhile, we detected various transcription factors that regulated the material and energy metabolism in N. incisum and N. franchetii, which probably play vital roles in the tolerance to stress in surroundings. In addition, 60 primer pairs based on orthologous microsatellite-containing sequences between the both Notopterygium species were determined. Finally, 17 polymorphic microsatellite markers (SSR) were successfully characterized for the two endangered species. Based on these candidate orthologous and SSR markers, we detected that the adaptive evolution and species divergence of N. incisum and N. franchetii were significantly associated with the extremely heterogeneous environments and climatic oscillations in high-altitude areas. This work provides important insights into the molecular mechanisms of adaptation to high-altitudes in alpine herbal plants.

The sex determination in gymnosperms is still poorly characterized due to the lack of genomic/transcriptome resources and useful molecular genetic markers. To enhance our understanding of the molecular mechanisms of the determination of sexual recognition of reproductive structures in conifers, the transcriptome of male and female conelets were characterized in a Chinese endemic conifer species, Pinus bungeana Zucc. ex Endl. The 39.62 Gb high-throughput sequencing reads were obtained from two kinds of sexual conelets. After de novo assembly of the obtained reads, 85,305 unigenes were identified, 53,944 (63.23%) of which were annotated with public databases. A total of 12,073 differentially expressed genes were detected between the two types of sexes in P. bungeana, and 5766 (47.76%) of them were up-regulated in females. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched analysis suggested that some of the genes were significantly associated with the sex determination process of P. bungeana, such as those involved in tryptophan metabolism, zeatin biosynthesis, and cysteine and methionine metabolism, and the phenylpropanoid biosynthesis pathways. Meanwhile, some important plant hormone pathways (e.g., the gibberellin (GA) pathway, carotenoid biosynthesis, and brassinosteroid biosynthesis (BR) pathway) that affected sexual determination were also induced in P. bungeana. In addition, 8791 expressed sequence tag-simple sequence repeats (EST-SSRs) from 7859 unigenes were detected in P. bungeana. The most abundant repeat types were dinucleotides (1926), followed by trinucleotides (1711). The dominant classes of the sequence repeat were A/T (4942) in mononucleotides and AT/AT (1283) in dinucleotides. Among these EST-SSRs, 84 pairs of primers were randomly selected for the characterization of potential molecular genetic markers. Finally, 19 polymorphic EST-SSR primers were characterized. We found low to moderate levels of genetic diversity (N_A = 1.754; H_O = 0.206; H_E = 0.205) across natural populations of P. bungeana. The cluster analysis revealed two distinct genetic groups for the six populations that were sampled in this endemic species, which might be caused by the fragmentation of habitats and long-term geographic isolation among different populations. Taken together, this work provides important insights into the molecular mechanisms of sexual identity in the reproductive organs of P. bungeana. The molecular genetic resources that were identified in this study will also facilitate further studies in functional genomics and population genetics in the Pinus species.