Transcriptomic and physiological analysis of atractylodes chinensis in response to drought stress reveals the putative genes related to sesquiterpenoid biosynthesis.

BACKGROUND: Atractylodes chinensis (DC) Koidz., a dicotyledonous and hypogeal germination species, is an important medicinal plant because its rhizome is enriched in sesquiterpenes. The development and production of A. chinensis are negatively affected by drought stress, especially at the seedling stage. Understanding the molecular mechanism of A. chinensis drought stress response plays an important role in ensuring medicinal plant production and quality. In this study, A. chinensis seedlings were subjected to drought stress treatment for 0 (control), 3 (D3), and 9 days (D9). For the control, the sample was watered every two days and collected on the second morning after watering. The integration of physiological and transcriptomic analyses was carried out to investigate the effects of drought stress on A. chinensis seedlings and to reveal the molecular mechanism of its drought stress response. RESULTS: The malondialdehyde, proline, soluble sugar, and crude protein contents and antioxidative enzyme (superoxide dismutase, peroxidase, and catalase) activity were significantly increased under drought stress compared with the control. Transcriptomic analysis indicated a total of 215,665 unigenes with an average length of 759.09 bp and an N50 of 1140 bp. A total of 29,449 differentially expressed genes (DEGs) were detected between the control and D3, and 14,538 DEGs were detected between the control and D9. Under drought stress, terpenoid backbone biosynthesis had the highest number of unigenes in the metabolism of terpenoids and polyketides. To identify candidate genes involved in the sesquiterpenoid and triterpenoid biosynthetic pathways, we observed 22 unigene-encoding enzymes in the terpenoid backbone biosynthetic pathway and 15 unigene-encoding enzymes in the sesquiterpenoid and triterpenoid biosynthetic pathways under drought stress. CONCLUSION: Our study provides transcriptome profiles and candidate genes involved in sesquiterpenoid and triterpenoid biosynthesis in A. chinensis in response to drought stress. Our results improve our understanding of how drought stress might affect sesquiterpenoid and triterpenoid biosynthetic pathways in A. chinensis.

[Difference of effective component content and key enzyme gene expression of biosynthesis in Atractylodes chinensis with different leaf shapes].

In this study, four Atractylodes chinensis(A. chinensis) with different leaf shapes, such as the split leaf, long and narrow leaf, oval leaf, and large round leaf, were used as experimental materials to establish a method for simultaneously determining atractylodin, atractylenolide Ⅰ, ß-eudesmol, and atractylon in the rhizome of A. chinensis. The expression of key enzyme genes for biosynthesis of acetyl-CoA carboxylase(ACC), 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR), and farnesyl pyrophosphate synthase(FPPS) was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR). High performance liquid chromatography(HPLC) was used to compare the difference in the content of four active components in A. chinensis with different leaf shapes, and the correlation between the content of active components and the expression of key enzyme genes in biosynthesis was discussed. The results show that there was good linearity among atractylodin, atractylenolide Ⅰ, ß-eudesmol, and atractylon in the range of 3.30-33.00 µg·mL~(-1)(r =0.999 7), 12.04-120.40 µg·mL~(-1)(r =0.999 5), 29.16-291.60 µg·mL~(-1)(r =0.999 5), and 14.20-142.00 µg·mL~(-1)(r =0.999 5), respectively. The average recoveries were 99.77%(RSD=2.1%), 98.56%(RSD=1.2%), 103.0%(RSD=1.2%), and 100.6%(RSD=1.5%), respectively. The method was accurate and had good reproducibility, which could be used to simultaneously detect atractylodin, atractylenolide Ⅰ, ß-eudesmol, and atractylon. The results showed that there were significant differences in the content of four active components in A. chinensis with different leaf shapes. The content of atractylodin, atractylenolide Ⅰ, and ß-eudesmol in A. chinensis with split leaves was the highest, which were 1.341 9, 5.237 2, and 12.084 3 mg·g~(-1), respectively. The content of atractylon in A. chinensis with long and narrow leaves was the highest(5.470 1 mg·g~(-1)). The content of atractylodin, atractylenolide Ⅰ, ß-eudesmol, and atractylon in A. chinensis with oval leaves was the lowest. The total content of the four effective components in descending order was A. chinensis with split leaves > A. chinensis with long and narrow leaves > A. chinensis with large round leaves > A. chinensis with oval leaves. The gene expression levels of key enzymes ACC, HMGR, and FPPS in A. chinensis with split leaves were the highest(P < 0.05), and the gene expression levels of key enzymes ACC and HMGR in A. chinensis with oval leaves were the lowest(P < 0.05). The gene expression level of key enzyme FPPS in A. chinensis with large round leaves was the lowest. In A. chinensis with different leaf shapes, the key enzyme gene ACC was significantly positively correlated with the polyacetylene component, namely atractylodin(P < 0.01), and the key enzyme genes HMGR and FPPS were positively correlated with the sesquiterpene components, namely atractylenolide Ⅰ, ß-eudesmol, and atractylon. In summary, the quality of A. chinensis with split leaves is the best, and the biosynthesis of atractylodin is significantly correlated with the gene expression of key enzyme ACC, which provides a theoretical basis for screening and optimizing the germplasm resources of A. chinensis and improving the quality of medicinal materials.

Isolation, characterisation, and expression profiling of DXS and DXR genes in Atractylodes lancea.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

[Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province].

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Effects of PAMK on lncRNA, miRNA, and mRNA expression profiles of thymic epithelial cells.

Polysaccharides from Atractylodes macrocephala Koidz (PAMK) can promote the proliferation of thymocytes and improve the body's immunity. However, the effect of PAMK on thymic epithelial cells has not been reported. Studies have shown that miRNAs and lncRNAs are key factors in regulating cell proliferation. In this study, we found that PAMK could promote the proliferation of mouse medullary thymic epithelial cell line 1 (MTEC1) cells through CCK-8 and EdU experiments. To further explore its mechanism, we detected the effect of PAMK on the expression profiles of lncRNAs, miRNAs, and mRNAs in MTEC1 cells. The results showed that PAMK significantly affected the expression of 225 lncRNAs, 29 miRNAs, and 800 mRNAs. Functional analysis showed that these differentially expressed genes were significantly enriched in cell cycle, cell division, NF-kappaB signaling, apoptotic process, and MAPK signaling pathway. Finally, we used Cytoscape to visualize lncRNA-miRNA-mRNA(14 lncRNAs, 17 miRNAs, 171 mRNAs) networks based on ceRNA theory. These results suggest that lncRNAs and miRNAs may be involved in the effect of PAMK on the proliferation of MTEC1 cells, providing a new research direction for exploring the molecular mechanism of PAMK promoting the proliferation of thymic epithelial cells.

An overlooked dispersal route of Cardueae (Asteraceae) from the Mediterranean to East Asia revealed by phylogenomic and biogeographical analyses of Atractylodes.

BACKGROUND AND AIMS: The East Asian-Tethyan disjunction pattern and its mechanisms of formation have long been of interest to researchers. Here, we studied the biogeographical history of Asteraceae tribe Cardueae, with a particular focus on the temperate East Asian genus Atractylodes DC., to understand the role of tectonic and climatic events in driving the diversification and disjunctions of the genus. METHODS: A total of 76 samples of Atractylodes from 36 locations were collected for RAD-sequencing. Three single nucleotide polymorphism (SNP) datasets based on different filtering strategies were used for phylogenetic analyses. Molecular dating and ancestral distribution reconstruction were performed using both chloroplast DNA sequences (127 Cardueae samples) and SNP (36 Atractylodes samples) datasets. KEY RESULTS: Six species of Atractylodes were well resolved as individually monophyletic, although some introgression was identified among accessions of A. chinensis, A. lancea and A. koreana. Dispersal of the subtribe Carlininae from the Mediterranean to East Asia occurred after divergence between Atractylodes and Carlina L. + Atractylis L. + Thevenotia DC. at ~31.57 Ma, resulting in an East Asian-Tethyan disjunction. Diversification of Atractylodes in East Asia mainly occurred from the Late Miocene to the Early Pleistocene. CONCLUSIONS: Aridification of Asia and the closure of the Turgai Strait in the Late Oligocene promoted the dispersal of Cardueae from the Mediterranean to East China. Subsequent uplift of the Qinghai-Tibet Plateau as well as changes in Asian monsoon systems resulted in an East Asian-Tethyan disjunction between Atractylodes and Carlina + Atractylis + Thevenotia. In addition, Late Miocene to Quaternary climates and sea level fluctuations played major roles in the diversification of Atractylodes. Through this study of different taxonomic levels using genomic data, we have revealed an overlooked dispersal route between the Mediterranean and far East Asia (Japan/Korea) via Central Asia and East China.

Alternaria koreana sp. nov., a new pathogen isolated from leaf spot of ovate-leaf Atractylodes in South Korea.

BACKGROUND: A new species within the genus Alternaria was isolated from the leaf spot of Atractylodes ovata in the Mungyeong and Hwabuk-myeon districts of the Gyeongbuk province of Korea. The leaves showed disease symptoms such as circular or irregular leaf spots and brown to dark brown with gray spots at the center. The leaves also showed that concentric rings were surrounded with yellow halos. METHODS AND RESULTS: Phylogenetic analysis was conducted using the sequence dataset of the internal transcribed spacer region and part of the glyceraldehyde-3-phosphate dehydrogenase. The RNA polymerase II second largest subunit, endopolygalacturonase, Alternaria major allergen gene, anonymous gene region, and translation elongation factor 1-alpha genes were used as well. Results showed that present fungal isolates were distinct from other species of the sect. Alternaria. Morphologically, the present isolates also differed from other members of the sect. Alternaria in their production of solitary conidia or conidial chains (two units) and conidial body features. Similarly, it exhibited moderate pathogenicity in the host plant. CONCLUSIONS: This study described and illustrated A. koreana as a new species and the causal agent of the leaf-spot disease on A. ovata in Korea.

Differences in gene expression and endophytic bacterial diversity in Atractylodes macrocephala Koidz. rhizomes from different growth years.

Atractylodes macrocephala Koidz. (AMK) is widely used in traditional Chinese medicine owing to its pharmacological activity. Here, we aimed to characterize the differentially expressed genes (DEGs) of one- and three-year growth (OYG and TYG) rhizomes of AMK, combined with endophytic bacterial diversity analysis using high-throughput RNA sequencing. A total of 114 572 unigenes were annotated using six public databases. In all, 3570 DEGs revealed a clear difference, of which 936 and 2634 genes were upregulated and downregulated, respectively. The results of KEGG pathway analysis indicated that DEGs corresponding to terpenoid synthesis gene were downregulated in TYG rhizomes. In addition, 414 424 sequences corresponding to the 16S rRNA gene were divided into 1267 operational taxonomic units (OTUs). Moreover, the diversity of endophytic bacteria changed with species in the OYG (773) and TYG (1201) rhizomes at the OTU level, and Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla. A comparison of species differences among different growth years revealed that some species were significantly different, such as Actinomycetes, Variovorax, and Cloacibacterium. Interestingly, the decrease in the function-related metabolism of terpenoids and polyketides was correlated with the low expression of terpene synthesis genes in TYG rhizomes, as assessed using PICRUSt2. These data provide a scientific basis for elucidating the mechanisms underlying metabolite accumulation and endophytic bacterial diversity in relation to the growth years in AMK.

[Comparison of transcriptome of Atractylodes lancea rhizome and exploration of genes for sesquiterpenoid biosynthesis].

This study compared the transcriptome of Atractylodes lancea rhizome at different development stages and explored genes encoding the key enzymes of the sesquiterpenoid biosynthesis pathway. Specifically, Illumina NovaSeq 6000 was employed for sequencing the cDNA libraries of A. lancea rhizome samples at the growth stage(SZ), flowering stage(KH), and harvesting stage(CS), respectively. Finally, a total of 388 201 748 clean reads were obtained, and 16 925, 8 616, and 13 702 differentially expressed genes(DEGs) were identified between SZ and KH, KH and CS, and SZ and CS, separately. Among them, 53 genes were involved in the sesquiterpenoid biosynthesis pathways: 9 encoding 6 enzymes of the mevalonic acid(MVA) pathway, 15 encoding 7 enzymes of the 2-C-methyl-D-erythritol-4-phosphate(MEP) pathway, and 29 of sesquiterpenoid and triterpenoid biosynthesis pathway. Weighted gene co-expression network analysis(WGCNA) yielded 12 genes related to sesquiterpenoid biosynthesis for the SZ, 1 gene for the KH, and 1 gene for CS, and several candidate genes for sesquiterpenoid biosynthesis were discovered based on the co-expression network. This study laid a solid foundation for further research on the sesquiterpenoid biosynthesis pathway, analysis of the regulation mechanism, and mechanism for the accumulation of sesquiterpenoids in A. lancea.

Chloroplast genome variation and phylogenetic relationships of Atractylodes species.

BACKGROUND: Atractylodes DC is the basic original plant of the widely used herbal medicines "Baizhu" and "Cangzhu" and an endemic genus in East Asia. Species within the genus have minor morphological differences, and the universal DNA barcodes cannot clearly distinguish the systemic relationship or identify the species of the genus. In order to solve these question, we sequenced the chloroplast genomes of all species of Atractylodes using high-throughput sequencing. RESULTS: The results indicate that the chloroplast genome of Atractylodes has a typical quadripartite structure and ranges from 152,294 bp (A. carlinoides) to 153,261 bp (A. macrocephala) in size. The genome of all species contains 113 genes, including 79 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Four hotspots, rpl22-rps19-rpl2, psbM-trnD, trnR-trnT(GGU), and trnT(UGU)-trnL, and a total of 42-47 simple sequence repeats (SSR) were identified as the most promising potentially variable makers for species delimitation and population genetic studies. Phylogenetic analyses of the whole chloroplast genomes indicate that Atractylodes is a clade within the tribe Cynareae; Atractylodes species form a monophyly that clearly reflects the relationship within the genus. CONCLUSIONS: Our study included investigations of the sequences and structural genomic variations, phylogenetics and mutation dynamics of Atractylodes chloroplast genomes and will facilitate future studies in population genetics, taxonomy and species identification.

Molecular cloning and functional characterization of two squalene synthase genes in Atractylodes lancea.

MAIN CONCLUSION: Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.

[Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea].

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid ß-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid ß-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid ß-oxidation in A. lancea.

Evaluation of heritability of ß-eudesmol/hinesol content ratio in Atractylodes lancea De Candolle.

BACKGROUND: Atractylodes lancea De Candolle is a medicinal plant distributed in East Asia. Its rhizome has been used as an important crude drug in traditional Chinese and Japanese medicines for the treatment of numerous diseases and disorders. In recent years, the demand for mass production of the crude drug with a stable quality has increased. Its major active compounds are sesquiterpenoids, such as ß-eudesmol and hinesol that have closely related chemical structures with each other. As the criteria for evaluating the quality of A. lancea, the ß-eudesmol/hinesol content ratio is considered important. In A. lancea, the ratio could be considered to be influenced by genetic factors, geographical environment factors and these interactions. Few studies of a detail genetic analyses for ß-eudesmol/hinesol content ratio have been reported. Therefore, we evaluated the heritability and genotype-environment interaction on the ß-eudesmol/hinesol content ratio in A. lancea using clonal lines propagated with division of rhizome. RESULTS: The heritability of the ß-eudesmol/hinesol content ratio in A. lancea was evaluated through the cultivation of clonal lines of A. lancea in both different years (2016, 2017) and locations (Hokkaido, Ibaraki). Correlations between ß-eudesmol and hinesol contents were identified in all clonal lines, with high correlation coefficients (r = 0.73-0.99). The broad-sense heritability of the ß-eudesmol/hinesol content ratio was revealed to be high at 0.92. The effects of cultivation year were smaller than that of genotype, and few genotype-environment interactions were observed. In addition, the influence of cultivation location was also smaller than that of genotype, and the correlation between the two cultivation locations on the ß-eudesmol/hinesol content ratio was high. The results suggested that the ß-eudesmol/hinesol content ratio in A. lancea is highly dependent on genetic factors. CONCLUSION: We demonstrate that the heritability of ß-eudesmol/hinesol content ratio is high and that the effects of genetic factors were stronger than that of environmental factors such as cultivation location and year. Our findings suggested that selective breeding and clonal propagation are effective strategies for the production of A. lancea with stable qualities for use in the production of crude drugs.

[Morphology and AFLP analysis of tetraploid plantlets of Atractylodes macrocephala].

In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala, diploid plantlets were taken as experimental material, sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared, and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index, leaf length and width, the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers, the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala, and the differences in morphology of them were reflected in the DNA polymorphism.

[Flow Cytometric Analysis of Genome Size in Atractylodes lancea].

OBJECTIVE: To develop a rapid analytical method for the determination of genome size of Atractylodes lancea by flow cytometry (FCM), and to estimate genome size of five typical plant types of cultivated Atractylodes lancea. METHODS: The fresh young leaves of Atractylodes lancea were used for the preparation of nuclear suspension with two-step protocol. After staining with propidium iodide, the mixture was analyzed by flow cytometry. Zea mays 'CE-777' or Vicafaba 'Inorce' was used for DNA reference standard. RESULTS: The flow cytometric method was developed after screening internal standard, optimizing sample preparation and FCM setups. The narrow leaf type of Atractylodes lancea got the biggest genome size, whereas the smallest one was fasciated stem type. It was suggested that parted leaf might be caused by genome size decrease. CONCLUSION: The methodology presented in this study is suitable for measuring the genome size of Atractylodes lancea. This study also provides useful information on population variation, evaluation of germplasm resources, and breeding of Atractylodes lancea.

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

[Identification of atractylodis macrocephalae rhizoma and atractylodis rhizoma from their adulterants using DNA barcoding].

Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma were widely used in strengthening spleen under different disease conditions, and were easily and often misused each other. Therefore, DNA barcode was used to distinguish Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma from their adulterants to ensure the safe use. The sequence lengths of ITS2 of Atractylodes macrocephala, Atractylodis Rhizoma (A. lancea, A. japonica and A. coreana) were both 229 bp. Among the ITS2 sequences of A. macrocephala, only one G/C transversion was detected at site 98, and the average GC content was 69.42%. No variable site was detected in the ITS2 sequences of A. lancea. The maximum K2P intraspecific genetic distances of both A. japonica and A. coreana were 0.013. The maximum K2P intraspecific genetic distances of A. macrocephala, A. lancea, A. japonica and A. coreana were less than the minimum interspecific genetic distance of adulterants. The ITS2 sequences in each of these polytypic species were separated into pairs of divergent clusters in the NJ tree. DNA barcoding could be used as a fast and accurate identification method to distinguish Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, from their adulterants to ensure its safe use.

B chromosome and its non-Mendelian inheritance in Atractylodes lancea.

Supernumerary B chromosomes contribute to intraspecific karyotypic variation. B chromosomes have been detected in more than 2000 organisms; they possess unique and diverse features, including non-Mendelian inheritance. Here, we report one or more B chromosomes in the gynodioecious plant Atractylodes lancea. Among 54 A. lancea lines, 0-2 B chromosomes were detected in both hermaphroditic and female plants, with the B chromosomes appearing as DAPI-bright regions within the nuclei. Genomic in situ hybridization revealed that the B chromosomes had no conserved A chromosome DNA sequences, confirmed by fluorescence in situ hybridization probed with independently dissected B chromosomes. In male meiosis, the B chromosome did not pair with an A chromosome and was therefore eliminated; accordingly, only 20.1% and 18.6% of these univalent B chromosomes remained at the end of meiosis for the 1B lines of KY17-148 and KY17-118, respectively. However, we also found that B chromosomes were transmitted from male parents in 40.8%-44.2% and 47.2% of the next generation; although these transmission rates from male parents were not essentially different from Mendelian inheritance (0.5), the transmission of gametes carrying B chromosomes increased through fertilization or seed development. B chromosomes were transmitted from three of four 1B female parents to 64.3%-92.6% of the next generation, suggesting B chromosome accumulation. We propose that the B chromosome of A. lancea has a specific sequence and persists via non-Mendelian inheritance from female parents. Overall, A. lancea, with its unique characteristics, is a promising model for understanding the structure, evolution, and mechanism of non-Mendelian inheritance of B chromosomes.

Characterization of a group of germacrene A synthases involved in the biosynthesis of ß-elemene from Atractylodis macrocephala.

ß-Elemene, an important component of the volatile oil of Atractylodis macrocephala, has been widely utilized as an antitumor drug for over 20 years. However, the germacrene A synthase (GAS) genes responsible for the biosynthesis of ß-elemene in A. macrocephala were previously unidentified. In this study, two new AmGASs were identified from the A. macrocephala transcriptome, demonstrating their capability to convert farnesyl pyrophosphate into germacrene A, which subsequently synthesizes ß-elemene through Cope rearrangement. Additionally, two highly catalytic AmGAS1 mutations, I307A and E392A, resulted in a 2.23-fold and 1.57-fold increase in ß-elemene synthesis, respectively. Furthermore, precursor supply and fed-batch strategies were employed to enhance the precursor supply, resulting in ß-elemene yields of 7.3 mg/L and 33.3 mg/L, respectively. These findings identify a promising candidate GAS for ß-elemene biosynthesis and lay the foundation for further functional studies on terpene synthases in A. macrocephala.

[Authenticate of Atractylodes macrocephala seed by amplification refractory mutation system].

OBJECTIVE: To design specific primers and authenticate Atractylodes macrocephala from Atractylodes lancea and A. chinensis. METHOD: SNPs in the psbA-trnH sequences of Atractylodes were found by ClustulW program and Bioedit software. Primers for authentic A. macrocephala is designed according to the SNP site, and ITS sequence universal primers plus to the authentic primer to construct a multi-PCR reaction system, and then optimized the PCR reaction system. RESULT: 172 bp band special for A. macrocephala were found using multi-PCR reaction. CONCLUSION: The multi-PCR reaction system could be applied to identify A. macrocephala seed.