Biogeographic barriers to gene flow are central to studies of plant phylogeography. There are many physical and geographic barriers in China, but few studies have used molecular ecological evidence to investigate the natural geographic isolation barrier of the Qinling Mountains-Huaihe River Line (QHL). Allium macrostemon is a precious Chinese perennial herb belonging to the Amaryllidaceae family. It is used as a food and medicine, with a variety of health and healing properties. Five SSR markers, three chloroplast DNA (cpDNA) markers (psbA-trnH, rps16 and trnL-F), one nuclear ribosomal DNA (nrDNA) marker (ITS), and simplified genome GBS sequencing were used to analyse the genetic diversity and structure of A. macrostemon. Combining SSR, cpDNA, nrDNA ITS data and GBS analysis results, we divided A. macrostemon populations into northern and southern groups, with the southern group further divided into southwestern and central-southeastern groups. Niche simulation results reveal that the distribution area of A. macrostemon will reach its maximum in the future. These data indicate that the regional separation of A. macrostemon has been maintained by the combined influence of a geographical barrier and Quaternary climate, and that the back-and-forth fluctuations of QHL and Quaternary climate have played an important role in this process. QHL acts as a north-south dividing line in phylogeography and population genetic structure, promoting physical geographic isolation. This study provides a theoretical basis for the conservation, development, and utilization of A. macrostemon resources. It further provides a reference for understanding the systematic geographical pattern of the large-scale spatial distribution of plants in China and enriches our understanding of Quaternary plant evolution in areas with complex terrain.

Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.

BACKGROUND: 6 - 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 - 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 - 4 photoproduct specific photolyase.
RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein.
CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan® probe method was employed for detection, with the amplified targets being the \"trnL (UAA)-intron\" or \"trnL-trnF intergenic spacer\" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called \'plastid nucleoid\', researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts.

BACKGROUND: The influence of native secondary succession associated with anthropogenic disturbance on the biodiversity of the forests in subtropical China remains uncertain. In particular, the evolutionary response of small understory shrubs, particularly pioneer species inhabiting continuously disturbed habitats, to topographic heterogeneity and climate change is poorly understood. This study aimed to address this knowledge gap by focusing on the Gaultheria crenulata group, a clade of small pioneer shrubs in subtropical China.
RESULTS: We examined the genetic structure and demographic history of all five species of the G. crenulata group with two maternally inherited chloroplast DNA (cpDNA) fragments and two biparentally inherited low-copy nuclear genes (LCG) over 89 natural populations. We found that the genetic differentiation of this group was influenced by the geomorphological boundary between different regions of China in association with Quaternary climatic events. Despite low overall genetic diversity, we observed an isolation-by-distance (IBD) pattern at a regional scale, rather than isolation-by-environment (IBE), which was attributed to ongoing human disturbance in the region.
CONCLUSIONS: Our findings suggest that the genetic structure of the G. crenulata group reflects the interplay of geological topography, historical climates, and anthropogenic disturbance during the Pliocene-Pleistocene-Holocene periods in subtropical China. The observed IBD pattern, particularly prominent in western China, highlights the role of limited dispersal and gene flow, possibly influenced by physical barriers or decreased connectivity over geographic distance. Furthermore, the east-to-west trend of gene flow, potentially facilitated by the East Asian monsoon system, underscores the complex interplay of biotic and abiotic factors shaping the genetic dynamics of pioneer species in subtropical China\'s secondary forests. These findings can be used to assess the impact of environmental changes on the adaptation and persistence of biodiversity in subtropical forest ecosystems.

BACKGROUND: Baolia H.W.Kung & G.L.Chu is a monotypic genus only known in Diebu County, Gansu Province, China. Its systematic position is contradictory, and its morphoanatomical characters deviate from all other Chenopodiaceae. Recent study has regarded Baolia as a sister group to Corispermoideae. We therefore sequenced and compared the chloroplast genomes of this species, and resolved its phylogenetic position based on both chloroplast genomes and marker sequences.
RESULTS: We sequenced 18 chloroplast genomes of 16 samples from two populations of Baolia bracteata and two Corispermum species. These genomes of Baolia ranged in size from 152,499 to 152,508 bp. Simple sequence repeats (SSRs) were primarily located in the LSC region of Baolia chloroplast genomes, and most of them consisted of single nucleotide A/T repeat sequences. Notably, there were differences in the types and numbers of SSRs between the two populations of B. bracteata. Our phylogenetic analysis, based on both complete chloroplast genomes from 33 species and a combination of three markers (ITS, rbcL, and matK) from 91 species, revealed that Baolia and Corispermoideae (Agriophyllum, Anthochlamys, and Corispermum) form a well-supported clade and sister to Acroglochin. According to our molecular dating results, a major divergence event between Acroglochin, Baolia, and Corispermeae occurred during the Middle Eocene, approximately 44.49 mya. Ancestral state reconstruction analysis showed that Baolia exhibited symplesiomorphies with those found in core Corispermoideae characteristics including pericarp and seed coat.
CONCLUSIONS: Comparing the chloroplast genomes of B. bracteata with those of eleven typical Chenopodioideae and Corispermoideae species, we observed a high overall similarity and a one notable noteworthy case of inversion of approximately 3,100 bp. of DNA segments only in two Atriplex and four Chenopodium species. We suggest that Corispermoideae should be considered in a broader sense, it includes Corispermeae (core Corispermoideae: Agriophyllum, Anthochlamys, and Corispermum), as well as two new monotypic tribes, Acroglochineae (Acroglochin) and Baolieae (Baolia).

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a \'legal high\' and has gained popularity as an alternative to opioids. However, kratom\'s increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

BACKGROUND: Kiwifruit (Actinidiaceae family) is an economically important fruit tree in China and New Zealand. It is a typical dioecious plant that has undergone frequent natural hybridization, along with chromosomal ploidy diversity within the genus Actinidia, resulting in higher genetic differences and horticultural diversity between interspecific and intraspecific traits. This diversity provides a rich genetic base for breeding. China is not only the original center of speciation for the Actinidia genus but also its distribution center, housing the most domesticated species: A. chinensis var. chinensis, A. chinensis var. deliciosa, A. arguta, and A. polygama. However, there have been relatively few studies on the application of DNA markers and the genetic basis of kiwifruit plants. By combining information from chloroplast-specific SNPs and nuclear SCoT (nSCoT) markers, we can uncover complementary aspects of genetic variation, population structure, and evolutionary relationships. In this study, one chloroplast DNA (cpDNA) marker pair was selected out of nine cpDNA candidate pairs. Twenty nSCoT markers were selected and used to assess the population structure and chloroplast-specific DNA haplotype diversity in 55 kiwifruit plants (Actinidia), including 20 samples of A. chinensis var. chinensis, 22 samples of A. chinensis var. deliciosa, 11 samples of A. arguta, and two samples of A. polygama, based on morphological observations collected from China.
RESULTS: The average genetic distance among the 55 samples was 0.26 with chloroplast-specific SNP markers and 0.57 with nSCoT markers. The Mantel test revealed a very small correlation (r = 0.21). The 55 samples were categorized into different sub-populations using Bayesian analysis, the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA), and the Principal Component Analysis (PCA) method, respectively. Based on the analysis of 205 variable sites, a total of 15 chloroplast-specific DNA haplotypes were observed, contributing to a higher level of polymorphism with an Hd of 0.78. Most of the chloroplast-specific DNA haplotype diversity was distributed among populations, but significant diversity was also observed within populations. H1 was shared by 24 samples, including 12 of A. chinensis var. chinensis and 12 of A. chinensis var. deliciosa, indicating that H1 is an ancient and dominant haplotype among the 55 chloroplast-specific sequences. H2 may not have evolved further.The remaining haplotypes were rare and unique, with some appearing to be exclusive to a particular variety and often detected in single individuals. For example, the H15 haplotype was found exclusively in A. polygama.
CONCLUSIONS: The population genetic variation explained by chloroplast-specific SNP markers has greater power than that explained by nSCoTs, with chloroplast-specific DNA haplotypes being the most efficient. Gene flow appears to be more evident between A. chinensis var. chinensis and A. chinensis var. deliciosa, as they share chloroplast-specific DNA haplotypes, In contrast, A.arguta and A. polygama possess their own characteristic haplotypes, derived from the haplotype of A. chinensis var. chinensis. Compared with A. chinensis, the A.arguta and A. polygama showed better grouping. It also seems crucial to screen out, for each type of molecular marker, especially haplotypes, the core markers of the Actinidia genus.