Lactobacillus casei-fermented Amomum xanthioides ameliorates metabolic dysfunction in high-fat diet-induced obese mice.

Amomum xanthioides (AX) has been used as an edible herbal medicine to treat digestive system disorders in Asia. Additionally, Lactobacillus casei is a well-known probiotic commonly used in fermentation processes as a starter. The current study aimed to investigate the potential of Lactobacillus casei-fermented Amomum xanthioides (LAX) in alleviating metabolic disorders induced by high-fat diet (HFD) in a mouse model. LAX significantly reduced the body and fat weight, outperforming AX, yet without suppressing appetite. LAX also markedly ameliorated excessive lipid accumulation and reduced inflammatory cytokine (IL-6) levels in serum superior to AX in association with UCP1 activation and adiponectin elevation. Furthermore, LAX noticeably improved the levels of fasting blood glucose, serum insulin, and HOMA-IR through positive regulation of glucose transporters (GLUT2, GLUT4), and insulin receptor gene expression. In conclusion, the fermentation of AX demonstrates a pronounced mitigation of overnutrition-induced metabolic dysfunction, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and obesity, compared to non-fermented AX. Consequently, we proposed that the fermentation of AX holds promise as a potential candidate for effectively ameliorating metabolic disorders.

Phenolics and terpenoids with good anti-inflammatory activity from the fruits of Amomum villosum and the anti-inflammatory mechanism of active diterpene.

The fruits of Amomum villosum are often considered a medicinal and food homologous material and have been found to have therapeutic effects in chronic enteritis, gastroenteritis, and duodenal ulcer. The aim of this study is to discover the anti-inflammatory active ingredients from dried ripe fruits of A. villosum and to elucidate the molecular mechanisms. We verified that the inhibitory activity of the ethyl acetate extract was superior to Dexamethasone (Dex), so we ultimately chose to study the ethyl acetate extract from the fruits of A. villosum. A total of 33 compounds were isolated from its ethyl acetate extract, including nine known diterpenoids (compounds 1-9), twelve known sesquiterpenoids (compounds 10-21), ten known phenolics (compounds 22, 23, 25-29, 31-33) and two new phenolics (24 and 30). On the basis of chemical evidences and spectral data analysis (UV, ECD, Optical rotation data, 1D and 2D-NMR, HR-ESI-MS, NMR chemical shift calculations), the structures of new compounds were elucidated. Among these compounds, isocoronarin D (5) was found to have good anti-inflammatory activity. Further research has found that isocoronarin D can down-regulate the protein levels of COX2 and NOS2, activate Nrf2/Keap1 and suppress NF-κB signaling pathway in LPS-induced RAW264.7 cells. In addition, isocoronarin D inhibited inflammasome assembly during inflammasome activation by hampering the binding of NLRP3 and ASC. Further evidence revealed that isocoronarin D suppressed the assembly of the NLRP3 inflammasome via blocking the formation of ASC specks. From these results, isocoronarin D may be the important bioactive compound of A. villosum and exhibits anti-inflammatory effects by regulating the NF-κB/Nrf2/NLRP3 axis in macrophages.

Antioxidant aromatic compounds from Amomum villosum and target prediction of active ingredients.

The dried fruit of Amomum villosum is an important spice and medicinal plant that has received great attention in recent years due to its high content of bioactive components and its potential for food additives and drug development. However, the stems and leaves of A. villosum are usually disposed of as waste. Based on the study of the fruits of A. villosum, we also systematically studied its stems and leaves. Fourteen aromatic compounds (1-14) were isolated and identified from A. villosum, including five new compounds (1-5) and nine known compounds (6-14). Among them, compounds 2-5, 8-10, 12-13 were obtained from the fruits of A. villosum, and compounds 1, 6-7,11, 14 were isolated from the stems and leaves of A. villosum. Based on chemical evidence and spectral data analysis (UV, ECD, Optical rotation data, 1D and 2D-NMR, and HR-ESI-MS), the structures of new compounds were elucidated. Furthermore, all compounds were tested for their effects on the survival rate of BV-2 cells in the presence of hydrogen peroxide. Among them, compound 5 showed antioxidant effects. Through network pharmacology screening and the cell thermal shift assay (CETSA), the Phosphoglycerate Mutase 5 (PGAM5) protein was identified as the antioxidant target of compound 5. Molecular docking results showed that compound 5 maintains binding to PGAM5 by forming hydrogen bond interactions with Lys93 and Agr214. In summary, A. villosum had potential medicinal and food values due to the diverse bioactive components.

Assessing heavy metal contamination in Amomum villosum Lour. fruits from plantations in Southern China: Soil-fungi-plant interactions.

Amomum villosum Lour. fruit is a common healthy food widely cultivated in southern China. Heavy metal contamination of farmland soils has becomes a serious environmental concern in China. Heavy metals in soil can be introduced into the food chain and pose health risks to humans. However, microbial communities may play beneficial roles in plants grown in metal-polluted soils. This study aimed to assess the potential health risks of heavy metals in soils and A. villosum fruits from different production areas and to explore the soil-microbe-plant regulation pattern for heavy metals in A. villosum fruits. Soil and A. villosum fruit samples were collected from nine planting fields in four provinces of southern China. The results showed that soils from seven areas were polluted with heavy metals to different degrees. Cr and Mn were the most serious contaminating elements. However, the accumulation of heavy metals in A. villosum fruit was negligible with no expected human health risks. Partial least squares path analysis of structural equation modeling showed that the accumulation of heavy metals in A. villosum fruits was influenced by multiple factors. More importantly, the PLS-SEM revealed that the heavy metal content in A. villosum fruits was indirectly affected by soil heavy metals through the regulation of the microbial community. Furthermore, some fungal phyla (e.g., Ascomycota and Chytridiomycota) and genera (e.g., Mucor) were related to the heavy metal content in the soil and in A. villosum fruits. The results of this study verified that soil fungal community play an important role in the accumulation of heavy metals in A. villosum fruits. Using fungi provides a potential biological strategy for reducing the health risk posed by heavy metals in food.

Integrating chemical similarity and bioequivalence: an overall evaluation of the quality consistency of traditional decoction and dispensing granule decoction of Amomum villosum.

OBJECTIVE: This study aims to investigate the quality consistency between traditional decoction (TD) of Amomum villosum and its dispensing granule decoction (DGD). Fifteen batches of TD and nine batches of dispensing granules (manufactured by A, B, and C) were prepared and evaluated for their consistency. METHODS: Firstly, The chemical similarity of TD and DGD was examined using GC and HPLC, coupled with hierarchical cluster analysis (HCA), criteria importance though intercrieria correlation(CRITIC) weighting method, and principal component analysis (PCA). Secondly, the gastrointestinal motility experiments in mice, along with the CRITIC weighting method, were employed to assess the bioequivalence of TD and DGD of Amomum villosum. Finally, the entropy weight technique-gray relative analysis(GRA) method was used to compare the quality of Amomum villosum decoctions. RESULTS: ①The CRITIC weighting method indicated significantly higher scores for TD than DGD (p < 0.01). HCA and PCA results demonstrated a clear distinction between TD and DGD. ②Gastrointestinal motility test results revealed no significant difference between TD and DGD in other indicators (p > 0.05).③Gray relative analysis results showed that the relative correlation of TD was more significant than that of DGD. CONCLUSION: The chemical composition of DGD and TD differed. The biological activity of DGD-A/B was consistent with that of TD, while the difference between DGD-C and TD was significant. A comprehensive evaluation showed that TD exhibited better quality than DGD. DGD manufacturers should optimize the preparation process to enhance product quality.

Black cardamom (Amomum subulatum) extract improves growth potential, antioxidant status, immune parameters and response to crowding stress in Catla catla.

Natural herbs are excellent alternatives to synthetic compounds to enhance the growth performance and health status of fish. This study was conducted to evaluate the efficacy of black cardamom (Amomum subulatum) extract (BCE) as an herbal supplement in improving the growth, antioxidant status, haematology, and serum biochemistry of Catla catla. The acclimatized fish (N = 900; average initial weight = 14.44 ± 0.33 g) were allocated into five groups (60 fish/group in triplicate) in hapas (4 × 2 × 2.5 ft) and provided with feed containing 0 (control), 0.5, 1, 2 and 4 g/kg BCE for 90 days before being subjected to 8 days of crowding stress. After 90 days, significantly higher feed utilization and growth were observed in all BCE-fed treatments compared to the control; however, the maximum values of these parameters were noted in the 2 g/kg BCE-fed treatment. Moreover, the BCE-fed groups exhibited a significant increase in antioxidant indices (glutathione peroxidase, superoxide dismutase and catalase), with a significant reduction in malondialdehyde levels, indicating a higher antioxidant capacity compared to the control. Significant improvements in haematological parameters, such as an increase in haematocrit, haemoglobin and red blood cells and a decrease in white blood cells, were observed in BCE-fed treatments compared to the control. Furthermore, BCE-fed groups showed a significant decrease in serum glucose, cortisol and triglycerides, while total protein, globulin and albumin levels were significantly higher compared with the control. During the feeding trial of 90 days, no mortality was observed; however, the lowest cumulative mortality was noted in the 2 g/kg BCE group after crowding stress of 8 days. In conclusion, the use of BCE in feed can enhance the growth performance, antioxidant status, haematology, and serum biochemistry of C. catla and improve the resistance against crowding stress. The optimum levels of BCE for C. catla were estimated based on weight gain % (1.78 g/kg; R2 = 0.97), feed conversion ratio (1.65 g/kg; R2 = 0.98), MDA content (1.66 g/kg; R2 = 0.93) and serum lysozyme activity (1.72 g/kg; R2 = 1) using broken-line regression analysis.

[Multi-index evaluation of different parts of Amomum villosum].

To investigate the quality differences between the seeds and husks of Amomum villosum and explore the rationality of using the seeds without husks, this study determined the content of protocatechuic acid, vanillic acid, epicatechin, quercitrin, volatile oil, water extract, and ethanol extract. The 2,2-diphenyl-1-picrylhydrazyl(DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), and hydroxyl radical scavenging activities were determined to evaluate the antioxidant activities of seeds and husks. The quality differences between the seeds and husks were assessed through orthogonal partial least squares-discriminant analysis(OPLS-DA) and analytic hierarchy process(AHP) combined with the entropy weight method(EWM). Significant differences(P<0.05) were observed in all 10 indicators between the seeds and husks. The levels of epicatechin, quercetin, and volatile oil were higher in the seeds, whereas those of protocatechuic acid, vanillic acid, water extract, and ethanol extract were higher in the husks. The seeds showed stronger scavenging ability against DPPH and ABTS radicals, while the husks showed a stronger scavenging effect on hydroxyl radicals. OPLS-DA significantly discriminated between the seeds and husks. Furthermore, volatile oil, water extract, DPPH radical scavenging rate, quercitrin, ABTS radical scavenging rate, hydroxyl radical scavenging rate, and vanillic acid were selected as the main differential indicators by variable importance in projection(VIP). Comprehensive scores calculated by AHP combined with EWM indicated that the seeds were superior to husks in terms of overall quality. However, there are still some dominant components and a certain antioxidant effect in the husks. Therefore, it is suggested to using Amomi Fructus with a certain amount of husks or utilizing the husks for other purposes.

Transcriptome and proteome analyses reveal the potential mechanism of seed dormancy release in Amomum tsaoko during warm stratification.

BACKGROUND: In Amomum tsaoko breeding, the low germination rate is the major limitation for their large-scale reproduction. We found that warm stratification was an effective treatment to break the seed dormancy of A. tsaoko prior to sowing and could be an important component of improving breeding programs. The mechanism of seed dormancy release during warm stratification remains unclear. Therefore, we studied the differences between transcripts and proteomes at 0, 30, 60, and 90 days of warm stratification, to identify some regulatory genes and functional proteins that may cause seed dormancy release in A. tsaoko and reveal their regulatory mechanism. RESULTS: RNA-seq was performed for the seed dormancy release process, and the number of differentially expressed genes (DEGs) was 3196 in three dormancy release periods. Using TMT-labelling quantitative proteome analysis, a total of 1414 proteins were defined as differentially expressed proteins (DEPs). Functional enrichment analyses revealed that the DEGs and DEPs were mainly involved in signal transduction pathways (MAPK signaling, hormone) and metabolism processes (cell wall, storage and energy reserves), suggesting that these differentially expressed genes and proteins are somehow involved in response to seed dormancy release process, including MAPK, PYR/PYL, PP2C, GID1, GH3, ARF, AUX/IAA, TPS, SPS, and SS. In addition, transcription factors ARF, bHLH, bZIP, MYB, SBP, and WRKY showed differential expression during the warm stratification stage, which may relate to dormancy release. Noteworthy, XTH, EXP, HSP and ASPG proteins may be involved in a complex network to regulate cell division and differentiation, chilling response and the seed germination status in A. tsaoko seed during warm stratification. CONCLUSION: Our transcriptomic and proteomic analysis highlighted specific genes and proteins that warrant further study in fully grasping the precise molecular mechanisms that control the seed dormancy and germination of A. tsaoko. A hypothetical model of the genetic regulatory network provides a theoretical basis for overcoming the physiological dormancy in A. tsaoko in the future.

Phylogenomics and genome size evolution in Amomum s. s. (Zingiberaceae): Comparison of traditional and modern sequencing methods.

BACKGROUND AND AIMS: A targeted enrichment NGS approach was used to construct the phylogeny of Amomum Roxb. (Zingiberaceae). Phylogenies based on hundreds of nuclear genes, the whole plastome and the rDNA cistron were compared with an ITS-based phylogeny. Trends in genome size (GS) evolution were examined, chromosomes were counted and the geographical distribution of phylogenetic lineages was evaluated. METHODS: In total, 92 accessions of 54 species were analysed. ITS was obtained for 79 accessions, 37 accessions were processed with Hyb-Seq and sequences from 449 nuclear genes, the whole cpDNA, and the rDNA cistron were analysed using concatenation, coalescence and supertree approaches. The evolution of absolute GS was analysed in a phylogenetic and geographical context. The chromosome numbers of 12 accessions were counted. KEY RESULTS: Four groups were recognised in all datasets though their mutual relationships differ among datasets. While group A (A. subulatum and A. petaloideum) is basal to the remaining groups in the nuclear gene phylogeny, in the cpDNA topology it is sister to group B (A. repoeense and related species) and, in the ITS topology, it is sister to group D (the Elettariopsis lineage). The former Elettariopsis makes a monophyletic group. There is an increasing trend in GS during evolution. The largest GS values were found in group D in two tetraploid taxa, A. cinnamomeum and A. aff. biphyllum (both 2n = 96 chromosomes). The rest varied in GS (2C = 3.54-8.78 pg) with a constant chromosome number 2n = 48. There is a weak connection between phylogeny, GS and geography in Amomum. CONCLUSIONS: Amomum consists of four groups, and the former Elettariopsis is monophyletic. Species in this group have the largest GS. Two polyploids were found and GS greatly varied in the rest of Amomum.

Antibacterial activity and synergic effects of the essential oils of Amomum verum Blackw and Zanthoxylum limonella (Dennst.) Alston.

The antibacterial activity of Amomum verum Blackw, Zanthoxylum limonella (Dennst.) Alston, Zanthoxylum bungeanum, and Zingiber montanum (J. Koenig) Link ex A. Dietr essential oils were investigated against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. The essential oils of A. verum Blackw, Z. limonella (Dennst.) Alston, Z. bungeanum, and Z. montanum (J. Koenig) Link ex A. Dietr displayed strong antibacterial activity with a minimum inhibitory concentration and minimumbactericidal concentration ranging from 0.31 to 1.25 µg/mL and 0.62-5.00 µg/mL, respectively. The chemical composition of A. verum Blackw, Z. limonella (Dennst.) Alston, Z. bungeanum, and Z. montanum (J. Koenig) Link ex A. Dietr essential oils were analysed using gas chromatography-mass spectrometry. 1,8-Cineole and limonene were detected in high amounts in the A. verum Blackw and Z. limonella (Dennst.) Alston essential oils, respectively. The major compound in Z. bungeanum and Z. montanum (J. Koenig) Link ex A. Dietr essential oil was 2,4-dimethylether-phloroacetophenone and terpinene-4-ol, respectively. The antibacterial activities and synergistic effects between these essential oils were further analysed. The combination of A. verum Blackw and Z. limonella (Dennst.) Alston essential oils showed a synergistic effect against all bacterial strains, while the other essential oil combinations showed additive, antagonistic effects, and no interaction. The synergistic effect of the combination between A. verum Blackw and Z. limonella (Dennst.) Alston essential oils could be resulted from 1,8-cineole and limonene which was evaluated to possess strong antibacterial activity.

Chemical constituents from the stems and leaves of Amomum villosum Lour. and their anti-inflammatory and antioxidant activities.

Amomum villosum Lour. is a medicinal and edible plant, whose medicinal parts are dried and mature fruits, and its stems and leaves are always treated as waste. HPLC-MS/MS analysis showed that the chemical components contained in the stems/leaves of A. villosum and those in fruits are quite different. To discover potential active ingredients from the stems/leaves of A. villosum, phytochemical evaluation of the stems/leaves of A. villosum was conducted to isolate and identify-four undescribed compounds (1, 2a, 2b, and 3) along with 41 known ones (4a, 4b, 5a, 5b, and 6-42). All isolated compounds were assessed for their anti-inflammatory and antioxidant activities. Among them, compounds 5b, 33, 34, and 38 exhibited anti-inflammatory activity, and compounds 1, 4a, 4b, 6, 7, 15, 33, 35, 37, and 41 showed antioxidant effects. Among them, the new compound 1 showed a significant antioxidant effect via activation of NRF2/HO-1 pathways. Therefore, the leaves and stems of A. villosum may be served as a potential medicine or dietary supplement for preventing and treating diseases resulting from inflammation and oxidative stress.

Three New Labdane-Type Diterpenoids from the Fruits of Amomum villosum and Their Anti-Inflammatory Activities.

Three new labdane-type diterpenoids, calcaratarin E, villosumtriol, and 12-epi-villosumtriol (1-3) were isolated from the fruits of Amomum villosum, along with seven known diterpenoids (4-10). Through comprehensive analysis of chemical evidence and spectral data including UV, 1D and 2D NMR, HR-ESI-MS, IR, and X-ray crystallography, the structures of these novel compounds were successfully determined. Additionally, the inhibitory effects of compounds 2-10 on NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells were evaluated. Notably, compound 6 exhibited the most significant inhibitory effect with an IC50 value of 1.74±0.69 µM.

Flavonoids in Amomum tsaoko Crevost et Lemarie Ameliorate Loperamide-Induced Constipation in Mice by Regulating Gut Microbiota and Related Metabolites.

Amomum tsaoko (AT) is a dietary botanical with laxative properties; however, the active ingredients and mechanisms are still unclear. The active fraction of AT aqueous extract (ATAE) for promoting defecation in slow transit constipation mice is the ethanol-soluble part (ATES). The total flavonoids of ATES (ATTF) were the main active component. ATTF significantly increased the abundance of Lactobacillus and Bacillus and reduced the dominant commensals, such as Lachnospiraceae, thereby changing the gut microbiota structure and composition. Meanwhile, ATTF changed the gut metabolites mainly enriched in pathways such as the serotonergic synapse. In addition, ATTF increased the serum serotonin (5-HT) content and mRNA expression of 5-hydroxytryptamine receptor 2A (5-HT2A), Phospholipase A2 (PLA2), and Cyclooxygenase-2 (COX2), which are involved in the serotonergic synaptic pathway. ATTF increased Transient receptor potential A1 (TRPA1), which promotes the release of 5-HT, and Myosin light chain 3(MLC3), which promotes smooth muscle motility. Notably, we established a network between gut microbiota, gut metabolites, and host parameters. The dominant gut microbiota Lactobacillus and Bacillus, prostaglandin J2 (PGJ2) and laxative phenotypes showed the most significant associations. The above results suggest that ATTF may relieve constipation by regulating the gut microbiota and serotonergic synaptic pathway and has great potential for laxative drug development in the future.

Chemical Constituents from the Fruits of Amomum kravanh and Their Role in Activating Alcohol Dehydrogenase.

Alcoholism is a worldwide health problem, and diseases caused by alcoholism are killing people every year. Amomum kravanh is a traditional Chinese medicine used to relieve hangovers. However, whether its bioactive components improve alcohol metabolism is not clear. In this study, ten new (amomumols A-J, 1-10) and thirty-five known (11-45) compounds were isolated from the fruits of Amomum kravanh by an activity-guided separation. Ten novel compounds were identified as four sesquiterpenoids (1-4), three monoterpene derivatives (5-7), two neolignans (8, 9), and a novel norsesquiterpenoid (10) with a new C14 nor-bisabolane skeleton. Their structures were determined by the comprehensive analysis of high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculation. The effects of all isolated compounds on the activity of alcohol dehydrogenase were evaluated in vitro, and it was found that eight compounds (11, 12, 15, 18, 26, and 36-38) exhibited significant activation effects on the alcohol dehydrogenase at 50 µM.

Tsaokoic Acid: A New Bicyclic Nonene from the Fruits of Amomum tsao-ko with Acetylcholinesterase Inhibitory Activity.

A new bicyclic nonene, tsaokoic acid (1), was isolated from the fruits of Amomum tsao-ko, together with three known compounds (2-4). The structure of 1 was elucidated by analyzing spectroscopic data including 1D and 2D NMR spectra and compounds 2-4 were identified as tsaokoin, vanillin, and tsaokoarylone, respectively, by comparing their NMR spectra with previously reported data. Compounds 1-4 showed possible inhibitory activity against acetylcholinesterase (AChE) in silico molecular docking simulations. They were submitted to in vitro assay system and exhibited moderate inhibitory activity with IC50 values of 32.78, 41.70, 39.25, and 31.13 µM, respectively.

Chemical Composition of Essential Oils and Supercritical Carbon Dioxide Extracts from Amomum kravanh, Citrus hystrix and Piper nigrum 'Kampot'.

The fruits of Amomum kravanh, Citrus hystrix and Piper nigrum 'Kampot' are traditionally used as spices in Cambodian cuisine. In this study, the chemical composition of essential oils (EOs) and supercritical CO2 extracts from all three species was determined using GC-MS, with two columns of different polarity (HP-5/DB-HeavyWAX). Differences between the chemical profile of the EOs and CO2 extracts were observed for all species. The greatest difference was detected in A. kravanh EO containing mainly eucalyptol (78.8/72.6%), while the CO2 extract was rich in fatty acids (13/55.92%) and long-chain alkanes (25.55/9.54%). Furthermore, the results for the CO2 extract of this species differed, where tricosane (14.74%) and oleic acid (29.26%) were the main compounds identified when utilizing the HP-5 or DB-HeavyWAX columns, respectively. Moreover, the EO and CO2 extract from P. nigrum 'Kampot' fruits and the CO2 extract from C. hystrix fruit peel, containing respective amounts 34.84/39.55% (for EO) and 54.21/55.86% (for CO2 extract) of ß-caryophyllene and 30.2/28.9% of ß-pinene, were isolated and analyzed for the first time. Generally, these findings suggest that supercritical CO2 could potentially be used for the extraction of all three spices. Nevertheless, further research determining the most efficient extraction parameters is required before its commercial application.

PLGA nanoparticle with Amomum longiligulare polysaccharide 1 increased the immunogenicity of infectious bursal disease virus VP2 protein.

1. The purpose of this study was to create ALP1-VP2-PLGA nanoparticle (AVPN) and to study the immunogenicity of AVPN. AVPN was prepared and observed by scanning and transmission electron microscopies.2. Chickens were divided into five groups and vaccinated with normal saline, VP2 protein, ALP1 and VP2 protein, AVPN or PLGA, respectively. After 28 days, the immune organ indexes were calculated; specific antibody levels in blood were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, the spleen and bursa of Fabricius were determined by HE staining, immunological cytokine mRNA levels in bursa of Fabricius were detected by qPCR andchicken body weight was determined.3. The results indicated that AVPN was a spherical nanoparticle with a diameter of about 85 nm. It increased bursal indexes and IBDV-specific antibody levels and promoted the expression of IL-2 mRNA in blood and TNF-α and IgG mRNA in bursa of Fabricius. This promoted growth.4. This study suggested that AVPN can increase immunogenicity of VP2 protein, and it could possibly be used as an IBDV subunit vaccine.

Comparative chloroplast genome analyses of Amomum: insights into evolutionary history and species identification.

BACKGROUND: Species in genus Amomum always have important medicinal and economic values. Classification of Amomum using morphological characters has long been a challenge because they exhibit high similarity. The main goals of this study were to mine genetic markers from cp genomes for Amomum species identification and discover their evolutionary history through comparative analysis. RESULTS: Three species Amomum villosum, Amomum maximum and Amomum longipetiolatum were sequenced and annotated for the complete chloroplast (cp) genomes, and the cp genomes of A. longipetiolatum and A. maximum were the first reported. Three cp genomes exhibited typical quadripartite structures with 163,269-163,591 bp in length. Each genome encodes 130 functional genes including 79 protein-coding, 26 tRNAs and 3 rRNAs genes. 113-152 SSRs and 99 long repeats were identified in the three cp genomes. By designing specific primers, we amplified the highly variable loci and the mined genetic marker ccsA exhibited a relatively high species identification resolution in Amomum. The nonsynonymous and synonymous substitution ratios (Ka/Ks) in Amomum and Alpinia showed that most genes were subjected to a purifying selection. Phylogenetic analysis revealed the evolutionary relationships of Amomum and Alpinia species and proved that Amomum is paraphyletic. In addition, the sequenced sample of A. villosum was found to be a hybrid, becoming the first report of natural hybridization of this genus. Meanwhile, the high-throughput sequencing-based ITS2 analysis was proved to be an efficient tool for interspecific hybrid identification and with the help of the chloroplast genome, the hybrid parents can be also be determined. CONCLUSION: The comparative analysis and mined genetic markers of cp genomes were conducive to species identification and evolutionary relationships of Amomum.

De novo transcriptome assembly, gene annotation, and EST-SSR marker development of an important medicinal and edible crop, Amomum tsaoko (Zingiberaceae).

BACKGROUND: Amomum tsaoko is a medicinal and food dual-use crop that belongs to the Zingiberaceae family. However, the lack of transcriptomic and genomic information has limited the understanding of the genetic basis of this species. Here, we performed transcriptome sequencing of samples from different A. tsaoko tissues, and identified and characterized the expressed sequence tag-simple sequence repeat (EST-SSR) markers. RESULTS: A total of 58,278,226 high-quality clean reads were obtained and de novo assembled to generate 146,911 unigenes with an N50 length of 2002 bp. A total of 128,174 unigenes were successfully annotated by searching seven protein databases, and 496 unigenes were identified as annotated as putative terpenoid biosynthesis-related genes. Furthermore, a total of 55,590 EST-SSR loci were detected, and 42,333 primer pairs were successfully designed. We randomly selected 80 primer pairs to validate their polymorphism in A. tsaoko; 18 of these primer pairs produced distinct, clear, and reproducible polymorphisms. A total of 98 bands and 96 polymorphic bands were amplified by 18 pairs of EST-SSR primers for the 72 A. tsaoko accessions. The Shannon's information index (I) ranged from 0.477 (AM208) to 1.701 (AM242) with an average of 1.183, and the polymorphism information content (PIC) ranged from 0.223 (AM208) to 0.779 (AM247) with an average of 0.580, indicating that these markers had a high level of polymorphism. Analysis of molecular variance (AMOVA) indicated relatively low genetic differentiation among the six A. tsaoko populations. Cross-species amplification showed that 14 of the 18 EST-SSR primer pairs have transferability between 11 Zingiberaceae species. CONCLUSIONS: Our study is the first to provide transcriptome data of this important medicinal and edible crop, and these newly developed EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity, and molecular marker-assisted selection in A. tsaoko.

Silky dogwood (Swida amomum) seed dispersal by freshwater turtles.

PREMISE: Effective seed dispersal is essential to the success of plant species. Swida amomum (silky dogwood) has a seed-dispersal syndrome characteristic of autumn-ripening shrubs with fleshy fruits; attached fruits are ingested and defecated by birds, while fallen fruits are consumed by ground-foraging birds and mammals. METHODS: We documented that fallen fruits of this shrub were consumed by two aquatic turtle species (eastern painted turtle [Chrysemys picta] and red-eared slider [Trachemys scripta]) and that their seeds were defecated. We compared germination success (percentage of seeds germinated) of defecated seeds, seeds collected from a pond surface, and seeds removed from shrubs. RESULTS: While four seed species were identified in fecal samples, seeds of S. amomum were the most frequent (93%) among samples and the most numerous (106 seeds) in any sample. Average proportion of fecal seeds germinated (85.99%) exceeded that of seeds from the pond surface (82.76%) and from shrubs (60.24%), albeit the difference in germination success was insignificant. When analyzed using fecal samples from painted turtles only, the difference in germination success between fecal seeds and those collected from pond or shrub became significant. CONCLUSIONS: Our findings represent the first report of S. amomum seeds being dispersed by turtle gut passage and suggest aquatic turtles could be an important part of a secondary seed dispersal process influencing woody plant community composition in temperate wetland ecosystems.