Chloroplast Genomes Evolution and Phylogenetic Relationships of Caragana species.

Caragana sensu lato (s.l.) includes approximately 100 species that are mainly distributed in arid and semi-arid regions. Caragana species are ecologically valuable for their roles in windbreaking and sand fixation. However, the taxonomy and phylogenetic relationships of the genus Caragana are still unclear. In this study, we sequenced and assembled the chloroplast genomes of representative species of Caragana and reconstructed robust phylogenetic relationships at the section level. The Caragana chloroplast genome has lost the inverted repeat region and wascategorized in the inverted repeat loss clade (IRLC). The chloroplast genomes of the eight species ranged from 128,458 bp to 135,401 bp and contained 110 unique genes. All the Caragana chloroplast genomes have a highly conserved structure and gene order. The number of long repeats and simple sequence repeats (SSRs) showed significant variation among the eight species, indicating heterogeneous evolution in Caragana. Selective pressure analysis of the genes revealed that most of the protein-coding genes evolved under purifying selection. The phylogenetic analyses indicated that each section forms a clade, except the section Spinosae, which was divided into two clades. This study elucidated the evolution of the chloroplast genome within the widely distributed genus Caragana. The detailed information obtained from this study can serve as a valuable resource for understanding the molecular dynamics and phylogenetic relationships within Caragana.

Historical climate change and vicariance events contributed to the intercontinental disjunct distribution pattern of ash species (Fraxinus, Oleaceae).

The Northern Hemisphere temperate forests exhibit a disjunct distributional pattern in Europe, North America, and East Asia. Here, to reveal the promoter of intercontinental disjunct distribution, Fraxinus was used as a model organism to integrate abundant fossil evidence with high-resolution phylogenies in a phytogeographic analysis. We constructed a robust phylogenetic tree using genomic data, reconstructed the geographic ancestral areas, and evaluated the effect of incorporating fossil information on the reconstructed biogeographic history. The phylogenetic relationships of Fraxinus were highly resolved and divided into seven clades. Fraxinus originated in western North America during Eocene, and six intercontinental dispersal events and five intercontinental vicariance events were occured. Results suggest that climate change and vicariance contributed to the intercontinental disjunct distribution pattern of Fraxinus. Moreover, results highlight the necessity of integrating phylogenetic relationship and fossil to improve the reliability of inferred biogeographic events and our understanding of the processes underlying disjunct distributions.

Comparative analysis of jujube and sour jujube gave insight into their difference in genetic diversity and suitable habitat.

Jujube (Ziziphus jujuba var. jujuba Mill.) and sour jujube (Z. jujuba var. spinosa (Bunge) Hu ex H.F.Chow.) are economically, nutritionally, and ecologically significant members of the Rhamnaceae family. Despite their importance, insufficient research on their genetics and habitats has impeded effective conservation and utilization. To address this knowledge gap, we conducted plastome sequencing, integrated distribution data from China, and assessed genetic diversity and suitable habitat. The plastomes of both species exhibited high conservation and low genetic diversity. A new-found 23 bp species-specific Indel in the petL-petG enabled us to develop a rapid Indel-based identification marker for species discrimination. Phylogenetic analysis and dating illuminated their genetic relationship, showing speciation occurred 6.9 million years ago, in a period of dramatic global temperature fluctuations. Substantial variations in suitable climatic conditions were observed, with the mean temperature of the coldest quarter as the primary factor influencing distributions (-3.16°C-12.73°C for jujube and -5.79°C to 4.11°C for sour jujube, suitability exceeding 0.6). Consequently, distinct conservation strategies are warranted due to differences in suitable habitats, with jujube having a broader distribution and sour jujube concentrated in Northern China. In conclusion, disparate habitats and climatic factors necessitate tailored conservation approaches. Comparing genetic diversity and developing rapid species-specific primers will further enhance the sustainable utilization of these valuable species.

From genomics to metabolomics: Deciphering sanguinarine biosynthesis in Dicranostigma leptopodum.

Dicranostigma leptopodum (Maxim) Fedde (DLF) is a renowned medicinal plant in China, known to be rich in alkaloids. However, the unavailability of a reference genome has impeded investigation into its plant metabolism and genetic breeding potential. Here we present a high-quality chromosomal-level genome assembly for DLF, derived using a combination of Nanopore long-read sequencing, Illumina short-read sequencing and Hi-C technologies. Our assembly genome spans a size of 621.81 Mb with an impressive contig N50 of 93.04 Mb. We show that the species-specific whole-genome duplication (WGD) of DLF and Papaver somniferum corresponded to two rounds of WGDs of Papaver setigerum. Furthermore, we integrated comprehensive homology searching, gene family analyses and construction of a gene-to-metabolite network. These efforts led to the discovery of co-expressed transcription factors, including NAC and bZIP, alongside sanguinarine (SAN) pathway genes CYP719 (CFS and SPS). Notably, we identified P6H as a promising gene for enhancing SAN production. By providing the first reference genome for Dicranostigma, our study confirms the genomic underpinning of SAN biosynthesis and establishes a foundation for advancing functional genomic research on Papaveraceae species. Our findings underscore the pivotal role of high-quality genome assemblies in elucidating genetic variations underlying the evolutionary origin of secondary metabolites.

Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae).

Hypericum perforatum (St. John's Wort) is a medicinal plant from the Hypericaceae family. Here, we sequenced the whole chloroplast genome of H. perforatum and compared the genome variation among five Hypericum species to discover dynamic changes and elucidate the mechanisms that lead to genome rearrangements in the Hypericum chloroplast genomes. The H. perforatum chloroplast genome is 139,725 bp, exhibiting a circular quadripartite structure with two copies of inverted repeats (IRs) separating a large single-copy region and a small single-copy region. The H. perforatum chloroplast genome encodes 106 unique genes, including 73 protein-coding genes, 29 tRNAs, and 4 rRNAs. Hypericum chloroplast genomes exhibit genome rearrangement and significant variations among species. The genome size variation among the five Hypericum species was remarkably associated with the expansion or contraction of IR regions and gene losses. Three genes-trnK-UUU, infA, and rps16-were lost, and three genes-rps7, rpl23, and rpl32-were pseudogenized in Hypericum. All the Hypericum chloroplast genomes lost the two introns in clpP, the intron in rps12, and the second intron in ycf3. Hypericum chloroplast genomes contain many long repeat sequences, suggesting a role in facilitating rearrangements. Most genes, according to molecular evolution assessments, are under purifying selection.

[Species identification of Ligustrum lucidum].

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Molecular evolution and phylogenomic analysis of complete chloroplast genomes of Cotinus (Anacardiaceae).

Cotinus is an oligo-specific ornamentally valuable genus with a disjunct distribution in the Northern Hemisphere. Traditionally, the taxonomy of Cotinus was mainly based on leaf morphological characteristics. However, the limited availability of genomic information greatly hindered the study of molecular evolution and phylogeny of this genus. This study sequenced the chloroplast (cp) genomes of all currently recognized taxa of Cotinus, including three species and four varieties. A comparative analysis was performed to investigate their cp genome characteristics and evolution. Furthermore, we inferred the phylogenetic relationships of Cotinus based on whole cp genomes, protein-coding genes, and nuclear ITS data. All cp genomes exhibited a typical quadripartite structure with genome sizes ranging from 158,865 to 160,155 bp. A total of 113-114 genes were identified in the genomes. Seven non-coding and four coding regions were identified as the most divergent hotspots for potential molecular barcodes and phylogenetic markers. Selection pressure analysis showed that there had been positive selection on genes matK and rps8 in the Cotinus cp genomes. Phylogenetic results confirmed that Cotinus is a monophyletic group but the widely distributed species Cotinus coggygria is not monophyletic. The divergence-time analysis suggested that Cotinus underwent an evolutionary divergence from the middle Eocene and rapid adaptive radiation from the middle Miocene. This study revealed new insights into the cp genome evolution and phylogeny of Cotinus and related taxa.

Pueraria montana Population Structure and Genetic Diversity Based on Chloroplast Genome Data.

Despite having a generally conserved structure, chloroplast genome data have been helpful for plant population genetics and evolution research. To mine Pueraria montana chloroplast genome variation architecture and phylogeny, we investigated the chloroplast variation architecture of 104 P. montana accessions from across China. P. montana's chloroplast genome showed high diversity levels, with 1674 variations, including 1118 single nucleotide polymorphisms and 556 indels. The intergenic spacers, psbZ-trnS and ccsA-ndhD, are the two mutation hotspot regions in the P. montana chloroplast genome. Phylogenetic analysis based on the chloroplast genome dataset supported four P. montana clades. P. montana variations were conserved among and within clades, which showed high gene flow levels. Most P. montana clades were estimated to have diverged at 3.82-5.17 million years ago. Moreover, the East Asian summer monsoon and South Asian summer monsoon may have accelerated population divergence. Our results show that chloroplast genome sequences were highly variable and can be used as molecular markers to assess genetic variation and relationships in P. montana.

Genetic diversity and population divergence of Leonurus japonicus and its distribution dynamic changes from the last interglacial to the present in China.

BACKGROUND: Leonurus japonicus, a significant medicinal plant known for its therapeutic effects on gynecological and cardiovascular diseases, has genetic diversity that forms the basis for germplasm preservation and utilization in medicine. Despite its economic value, limited research has focused on its genetic diversity and divergence. RESULTS: The avg. nucleotide diversity of 59 accessions from China were 0.00029 and hotspot regions in petN-psbM and rpl32-trnL(UAG) spacers, which can be used for genotype discrimination. These accessions divided into four clades with significant divergence. The four subclades, which split at approximately 7.36 Ma, were likely influenced by the Hengduan Mountains uplift and global temperature drop. The initial divergence gave rise to Clade D, with a crown age estimated at 4.27 Ma, followed by Clade C, with a crown age estimated at 3.39 Ma. The four clades were not showed a clear spatial distribution. Suitable climatic conditions for the species were identified, including warmest quarter precipitation 433.20 mm ~ 1,524.07 mm, driest month precipitation > 12.06 mm, and coldest month min temp > -4.34 °C. The high suitability distribution showed contraction in LIG to LGM, followed by expansion from LGM to present. The Hengduan Mountains acted as a glacial refuge for the species during climate changes. CONCLUSIONS: Our findings reflected a clear phylogenetic relationships and divergence within species L. japonicus and the identified hotspot regions could facilitate the genotype discrimination. The divergence time estimation and suitable area simulation revealed evolution dynamics of this species and may propose conservation suggestions and exploitation approaches in the future.

Maternal Donor and Genetic Variation of Lagerstroemia indica Cultivars.

Lagerstroemia indica L. is a well-known ornamental plant with large pyramidal racemes, long flower duration, and diverse colors and cultivars. It has been cultivated for nearly 1600 years and is essential for investigating the germplasm and assessing genetic variation to support international cultivar identification and breeding programs. In this study, 20 common Lagerstroemia indica cultivars from different varietal groups and flower morphologies, as well as multiple wild relative species, were analyzed to investigate the maternal donor of Lagerstroemia indica cultivars and to discover the genetic variation and relationships among cultivars based on plastome and nuclear ribosomal DNA (nrDNA) sequences. A total of 47 single nucleotide polymorphisms (SNPs) and 24 insertion/deletions (indels) were identified in the 20 L. indica cultivars' plastome and 25 SNPs were identified in the nrDNA. Phylogenetic analysis based on the plastome sequences showed that all the cultivars formed a clade with the species of L. indica, indicating that L. indica was the maternal donor of the cultivars. Population structure and PCA analyses supported two clades of cultivars, which exhibited significant genetic differences according to the plastome dataset. The results of the nrDNA supported that all 20 cultivars were divided into three clades and most of the cultivars had at least two genetic backgrounds and higher gene flow. Our results suggest that the plastome and nrDNA sequences can be used as molecular markers for assessing the genetic variation and relationships of L. indica cultivars.

Plant Disease Resistance-Related Pathways Recruit Beneficial Bacteria by Remodeling Root Exudates upon Bacillus cereus AR156 Treatment.

The environmentally friendly biological control strategy that relies on beneficial bacterial inoculants to improve plant disease resistance is a promising strategy. Previously, it has been demonstrated that biocontrol bacteria treatments can change the plant rhizosphere microbiota but whether plant signaling pathways, especially those related to disease resistance, mediate the changes in rhizosphere microbiota has not been explored. Here, we investigated the complex interplay among biocontrol strains, plant disease resistance-related pathways, root exudates, rhizosphere microorganisms, and pathogens to further clarify the biocontrol mechanism of biocontrol bacteria by using plant signaling pathway mutants. Bacillus cereus AR156, which was previously isolated from forest soil by our laboratory, can significantly control tomato bacterial wilt disease in greenhouse and field experiments. Moreover, compared with the control treatment, the B. cereus AR156 treatment had a significant effect on the soil microbiome and recruited 35 genera of bacteria to enrich the rhizosphere of tomato. Among them, the relative rhizosphere abundance of nine genera, including Ammoniphilus, Bacillus, Bosea, Candidimonas, Flexivirga, Brevundimonas, Bordetella, Dyella, and Candidatus_Berkiella, was regulated by plant disease resistance-related signaling pathways and B. cereus AR156. Linear correlation analysis showed that the relative abundances of six genera in the rhizosphere were significantly negatively correlated with pathogen colonization in roots. These rhizosphere bacteria were affected by plant root exudates that are regulated by signaling pathways. IMPORTANCE Our data suggest that B. cereus AR156 can promote the enrichment of beneficial microorganisms in the plant rhizosphere by regulating salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling pathways in plants, thereby playing a role in controlling bacterial wilt disease. Meanwhile, Spearman correlation analysis showed that the relative abundances of these beneficial bacteria were correlated with the secretion of root exudates. Our study reveals a new mechanism for SA and JA/ET signals to participate in the adjustment of plant resistance whereby the signaling pathways adjust the rhizosphere microecology by changing the root exudates and thus change plant resistance. On the other hand, biocontrol strains can utilize this mechanism to recruit beneficial bacteria by activating disease resistance-related signaling pathways to confine the infection and spread of pathogens. Finally, our data also provide a new idea for the in-depth study of biocontrol mechanisms.

Plastome evolution and phylogeny of the tribe Ruteae (Rutaceae).

Rutaceae is a large family, and the genus-level classification in the subfamilies or tribes of this family is not unified based on different taxonomic treatments. Until now, phylogenetic relationships of some genera in traditional tribe Ruteae have not been clearly resolved. In this study, seven new complete plastomes of this tribe were sequenced, and a comparative analysis was performed to investigate their plastome characteristics and evolution. In addition, we inferred the phylogenetic relationships of Ruteae based on complete plastome and nuclear ITS data. All plastomes exhibited a typical quadripartite structure and were relatively conserved in their structure and gene arrangement. Their genome sizes ranged from 154,656 bp to 160,677 bp, and the size variation was found to be associated with differences in IR expansion and gene loss. A total of 112 to 114 genes were identified in the genomes, including 78 to 79 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 2 pseudogenes. Sequence divergence analysis indicated that non-coding regions exhibited a higher percentage of variable characters, and nine non-coding and six coding regions were identified as divergent hotspots. Phylogenetic results based on different datasets showed that this tribe was divided into three reciprocally exclusive groups. The phylogenetic analyses between plastome and nuclear ITS data were partly incongruent with each other. This study provides new insights into plastome evolution of Ruteae as well as Rutaceae. The availability of these plastomes provides useful genomic resources for molecular DNA barcodes and phylogenetically informative markers and deepens our understanding of the phylogeny in Ruteae.

Insights into the phylogeny and chloroplast genome evolution of Eriocaulon (Eriocaulaceae).

BACKGROUND: Eriocaulon is a wetland plant genus with important ecological value, and one of the famous taxonomically challenging groups among angiosperms, mainly due to the high intraspecific diversity and low interspecific variation in the morphological characters of species within this genus. In this study, 22 samples representing 15 Eriocaulon species from China, were sequenced and combined with published samples of Eriocaulon to test the phylogenetic resolution using the complete chloroplast genome. Furthermore, comparative analyses of the chloroplast genomes were performed to investigate the chloroplast genome evolution of Eriocaulon. RESULTS: The 22 Eriocaulon chloroplast genomes and the nine published samples were proved highly similar in genome size, gene content, and order. The Eriocaulon chloroplast genomes exhibited typical quadripartite structures with lengths from 150,222 bp to 151,584 bp. Comparative analyses revealed that four mutation hotspot regions (psbK-trnS, trnE-trnT, ndhF-rpl32, and ycf1) could serve as effective molecular markers for further phylogenetic analyses and species identification of Eriocaulon species. Phylogenetic results supported Eriocaulon as a monophyletic group. The identified relationships supported the taxonomic treatment of section Heterochiton and Leucantherae, and the section Heterochiton was the first divergent group. Phylogenetic tree supported Eriocaulon was divided into five clades. The divergence times indicated that all the sections diverged in the later Miocene and most of the extant Eriocaulon species diverged in the Quaternary. The phylogeny and divergence times supported rapid radiation occurred in the evolution history of Eriocaulon. CONCLUSION: Our study mostly supported the taxonomic treatment at the section level for Eriocaulon species in China and demonstrated the power of phylogenetic resolution using whole chloroplast genome sequences. Comparative analyses of the Eriocaulon chloroplast genome developed molecular markers that can help us better identify and understand the evolutionary history of Eriocaulon species in the future.

What determines plant species diversity along the Modern Silk Road in the east?

As primary producers, plants provide food, oxygen, and other resources for global ecosystems, and should therefore be given priority in biodiversity protection. Most biodiversity research focuses on biodiversity hotspots, while biodiversity coldspots, such as deserts, are largely ignored. We propose that the factors shaping plant species diversity differ between biodiversity hot spots and cold spots, especially for desert ecosystems. To test this hypothesis, we investigated plant species diversity along the Modern Silk Road in the Northwest China desert, an area characterized by low precipitation, scarce vegetation, a limited number of species, and variable human activities. Surface soil was sampled from 144 plots, environmental DNA (eDNA) was extracted from soil samples, and seed plant species were identified using DNA metabarcoding technology. A total of 671 seed plant species were detected, which was more diverse than indicated by plot survey data. Plant species diversity gradually decreased from east to west along the Silk Road. In this area, temperature determines plant species diversity more than precipitation. Additionally, human activity has altered plant species diversity by introducing crops and invasive plants and eliminating environmentally adapted indigenous plants. Our results demonstrate the potential of eDNA metabarcoding technology for plant species diversity surveying. Desert plants have adapted to dry environments by relying on underground water or utilizing occasional rainfall as ephemerals, which are often not visible during surface surveys because of their short aboveground life cycle but can be detected with eDNA metabarcoding technology. Groundwater maintenance and human activity control are recommended for plant species diversity conservation and desertification control.

Dynamic evolution of the plastome in the Elm family (Ulmaceae).

MAIN CONCLUSION: This study compared the plastomes of Ulmaceae allowing analyses of the dynamic evolution, including genome structure, codon usage bias, repeat sequences, molecular mutation rates, and phylogenetic inferences. Ulmaceae is a small family in the order Rosales. This family consists of seven genera, including Ulmus, Zelkova, Planera, Hemiptelea, Phyllostylon, Ampelocera, and Holoptelea. Ulmaceae is an interesting lineage from plant biogeographic, systematic, evolutionary, and paleobotanic perspectives. It is also a good model to investigate the evolution of the plastomes in woody plants. In this study, we sequenced and assembled the complete plastomes of the six Ulmaceae genera to compare genomic structures and reveal the molecular evolutionary patterns. The size of the quadripartite plastomes ranged from 158,290 bp to 161,886 bp. The genomes contained 131 genes, including 87 coding genes, 36 tRNA, and 8 rRNA. The gene number, gene content, and genomic structure were highly consistent among the Ulmaceae genera. Nine variable regions including ndhA intron, ndhF-rpl32, ycf1, psbK-trnS, rps16-trnQ, trnT-trnL, trnT-psbD, trnS-trnG, and rpl32-trnL, were identified in Ulmaceae plastomes according to the nucleotide diversity values. Condon usage was biased among the genes and showed consistent trends in the seven genera. Molecular evolution analyses revealed that most of the genes and all gene groups were under widespread purifying selection. Twelve genes (ccsA, matK, psbH, psbK, rbcL, rpl22, rpl32, rpoA, rps12, rps15, rps16, and ycf2) were under positive selection. Phylogenetic analyses supported that Ulmaceae should be divided into two main clades, such as the temperate clade, including Ulmus, Zelkova, Planera, and Hemiptelea and the tropical clade, including Phyllostylon, Ampelocera and Holoptelea. This study reports the structure and evolutionary characteristics of the Elm family. These new genomic data will benefit assessments of genomic evolution and provide information to elucidate the phylogenetic relationships among Ulmaceae species.

Evolutionary history of genus Coptis and its dynamic changes in the potential suitable distribution area.

The genus Coptis belongs to the Ranunculaceae family, containing 15 recognized species highly diverse in morphology. It is a conspicuous taxon with special evolutionary position, distribution pattern and medicinal value, which makes it to be of great research and conservation significance. In order to better understand the evolutionary dynamics of Coptis and promote more practical conservation measures, we performed plastome sequencing and used the sequencing data in combination with worldwide occurrence data of Coptis to estimate genetic diversity and divergence times, rebuild biogeographic history and predict its potential suitable distribution area. The average nucleotide diversity of Coptis was 0.0067 and the hotspot regions with the highest hypermutation levels were located in the ycf1 gene. Coptis is most likely to have originated in North America and Japanese archipelago and has a typical Eastern Asian and North American disjunct distribution pattern, while the species diversity center is located in Mid-West China and Japan. The crown age of the genus is estimated at around 8.49 Mya. The most suitable climatic conditions for Coptis were as follows: precipitation of driest quarter > 25.5 mm, annual precipitation > 844.9 mm and annual mean temperature -3.1 to 19 °C. The global and China suitable area shows an upward trend in the future when emission of greenhouse gases is well controlled, but the area, especially in China, decreases significantly without greenhouse gas policy interventions. The results of this study provide a comprehensive insight into the Coptis evolutionary dynamics and will facilitate future conservation efforts.

Mitogen-Activated Protein Kinases Associated Sites of Tobacco Repression of Shoot Growth Regulates Its Localization in Plant Cells.

Plant defense and growth rely on multiple transcriptional factors (TFs). Repression of shoot growth (RSG) is a TF belonging to a bZIP family in tobacco, known to be involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG previously. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG-interacting protein kinase. Moreover, the mutation of the predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74, and Thr-135) significantly altered the intracellular localization of the NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acid mutants (T30A and S74A) were observed after being treated with plant defense elicitor peptide flg22 within 5 min, and the two mutated RSG swiftly re-localized in tobacco cytoplasm within 30 min. In addition, triple-point mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and is predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no re-localization effect under the treatments of flg22, B. cereus AR156, or GA3, and over-expression of this mutant in tobacco resulted in lower expression levels of downstream gene GA20ox1. Our results suggest that MAPK-associated phosphorylation sites of RSG regulate its localization in tobacco, and that constant unphosphorylation of RSG in Thr-30, Ser-74, and Thr-135 keeps RSG predominantly localized in cytoplasm.

Phylogenomics and Genetic Diversity of Arnebiae Radix and Its Allies (Arnebia, Boraginaceae) in China.

Arnebiae Radix is a traditional medicine with pleiotropic properties that has been used for several 100 years. There are five species of Arnebia in China, and the two species Arnebia euchroma and Arnebia guttata are the source plants of Arnebiae Radix according to the Chinese Pharmacopoeia. Molecular markers that permit species identification and facilitate studies of the genetic diversity and divergence of the wild populations of these two source plants have not yet been developed. Here, we sequenced the chloroplast genomes of 56 samples of five Arnebia species using genome skimming methods. The Arnebia chloroplast genomes exhibited quadripartite structures with lengths from 149,539 and 152,040 bp. Three variable markers (rps16-trnQ, ndhF-rpl32, and ycf1b) were identified, and these markers exhibited more variable sites than universal chloroplast markers. The phylogenetic relationships among the five Arnebia species were completely resolved using the whole chloroplast genome sequences. Arnebia arose during the Oligocene and diversified in the middle Miocene; this coincided with two geological events during the late Oligocene and early Miocene: warming and the progressive uplift of Tianshan and the Himalayas. Our analyses revealed that A. euchroma and A. guttata have high levels of genetic diversity and comprise two and three subclades, respectively. The two clades of A. euchroma exhibited significant genetic differences and diverged at 10.18 Ma in the middle Miocene. Three clades of A. guttata diverged in the Pleistocene. The results provided new insight into evolutionary history of Arnebia species and promoted the conservation and exploitation of A. euchroma and A. guttata.

[Complete chloroplast genome of Ligustrum lucidum and highly variable marker identification for Ligustrum].

Ligustri Lucidi Fructus, the sun-dried mature fruit of Ligustrum lucidum, is cool, plain, sweet, and bitter, which can be used as both food and medicine, with the effects of improving vision, blacking hair, and tonifying liver and kidney. It takes effect slowly. However, little is known about the genetic information of the medicinal plant and it is still a challenge to distinguish Ligustrum species. In this study, the complete chloroplast genome of L. lucidum was obtained by genome skimming and then compared with that of five other Ligustrum species, which had been reported. This study aims to evaluate the interspecific variation of chloroplast genome within the genus and develop molecular markers for species identification of the genus. The result showed that the chloroplast genome of L. lucidum was 162 162 bp with a circular quadripartite structure of two single-copy regions separated by a pair of inverted repeats. The Ligustrum chloroplast genomes were conserved with small interspecific difference. Comparative analysis of six Ligustrum chloroplast genomes revealed three variable regions(rbcL-accD, ycf1a, and ycf1b), and ycf1a and ycf1b can be used as the species-specific DNA barcode for Ligustrum. Phylogeny analysis provided the best resolution of Ligustrum and supported that L. lucidum was sister to L. gracile. This study clarified the genetic diversity of L. lucidum from provenance, which can serve as a reference for further analysis of pharmacological differences and breeding of excellent varieties with stable drug effects.