The phylogenetic relationships of the genus Atractylodes DC. was analyzed using the nuclear ribosomal ITS and three chloroplast fragments, including atpB-rbcL, psbB-psbF and trnL-trnF intergenic spacer (IGS) sequences. Phylogenetic analyses revealed that A. lancea subsp. luotianensis and A. lancea, A. chinensis var. liaotungensis and A. coreana form monophyletic terminal clade, separately. The trees, within each the pairwise genetic distances, did not support Hu's classification. Authors suggested that A. lancea Subsp. luotianensis should be included in A. lancea rather than be treated as a separate subspecies. A. carlinoides was placed in the basal position of Atractylodes, which had a distant relationship with the others of the genus. The results lead us to suggest that A. chinensis var. liaotungensis be put into A. coreana, A. chinensis as a subspecies of A. lancea.
Atractylodes ; classification ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Genes, Plant ; Phylogeny

OBJECTIVETo design specific primers and authenticate Atractylodes macrocephala from Atractylodes lancea and A. chinensis.

METHODSNPs 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.

RESULT172 bp band special for A. macrocephala were found using multi-PCR reaction.

CONCLUSIONThe multi-PCR reaction system could be applied to identify A. macrocephala seed.

Atractylodes ; genetics ; DNA Primers ; genetics ; Mutation ; Polymerase Chain Reaction ; methods ; Quality Control ; Seeds ; genetics

OBJECTIVETo investigate the genetic diversity of main germplasm of Atractylodes macrocephala in China and the genetic differentiation of the germplasm of A. macrocephala.

METHODA molecular marker ISSR was used to analyze the genetic diversity of 7 populations of A. macrocephala and a population of A. lancea.

RESULTTwelve primers were used in the PCR amplification of 86 samples of A. macrocephala and 5 samples of A. lancea. Sixty-three bands with sizes ranged from 100 to 2500 bp were generated from 12 primers. Of all the 63 bands, 55 bands were polymorphic among 86 individuals of A. macrocephala, the percentage of polymorphic bands were 87.30% at the species level. The percentage of polymorphic bands (PPL) for a single population ranged from 58.73% to 71.43% (mean, 64.85%). Among the 7 populations, a population from Panan, GM exhibited highest variability (PPL =71.43%; HE = 0.2835; I = 0.4267). A dendrogram constructed by an unweighted pair group method of cluster analysis showed that populations from Panan constructed one branch and separated from other populations. In the AMOVA analysis, low level of genetic differentiation among populations was detected, 90.52% of the variability existed in population.

CONCLUSIONThe genetic diversity of cultivated A. macrocephala in China is high, which is good for the production of high quality herb medicine.

Atractylodes ; classification ; genetics ; Genetic Variation ; Phylogeny ; Plants, Medicinal ; genetics ; Polymorphism, Genetic

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-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 β-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 β-oxidation in A. lancea.
Amino Acid Sequence ; Atractylodes/genetics* ; Cloning, Molecular ; Coenzyme A ; Escherichia coli/genetics* ; Phylogeny

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.
Atractylodes/genetics* ; Mevalonic Acid/metabolism* ; Rhizome/genetics* ; Sesquiterpenes/metabolism* ; Transcriptome ; Triterpenes/metabolism*

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.
Amplified Fragment Length Polymorphism Analysis ; methods ; Atractylodes ; genetics ; Sequence Analysis, DNA ; Tetraploidy

We described chemical composition in Cangzhu in recent years, volatile oil is the important chemical composition, The beta-eudesmol, hinesol are active ingredient in volatile oil and there are 38 kind of glycosides. At the same time, we overview the applying of RAPD technology in atractylodes lancea. The results is that there are correlation in chemical composition, genetic differentiation and geographical distribution, there is some truth in bounded by a territorial division of the north-south Cangzhu, and genetic differentiation has been happened in atractylodes lancea to adapting the environment. We described advances of pharmacological in dampness spleen, cardiovascular system, genitourinary system, nervous system, and the results show that there are pharmacological activity in digestive system, cardiovascular system, genitourinary system of atractylodes lancea.
Animals ; Atractylodes ; chemistry ; genetics ; China ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Molecular Biology

OBJECTIVETo assess the genetic diversity of Atractylodes macrocephala in 12 cultivated populations and 3 wild populations.

METHODThe molecular marker technique ISSR was used to investigate the genetic diversity of 365 samples from 15 populations in Zhejiang, Anhui and Hebei Provinces. Data was analyzed by POPGEN 32, and a cluster diagram was presented by UPGMA.

RESULTOne hundred and two amplified fragments were obtained using 13 ISSR primers. 94 polymorphic loci were identified as 92.16%. Nei's genetic diversity index (He) was 0.406 5, Shannon diversity index (I) was 0.590 3, and the coefficient of gene differentiation (Gst) was 0.202 5. The genetic similarity coefficient among the populations ranged from 0.690 7 to 0.960 5 in an average of 0. 825 6. No significant difference was found among the populations based on the clustering analysis.

CONCLUSIONThere is a low level of genetic differentiation in higher genetic diversity of the A. macrocephala populations. No obvious regional or species diversities were identified. This is consistent with current status of cultivation and gerplasm circulation in A. macrocephala.

Amplified Fragment Length Polymorphism Analysis ; Atractylodes ; classification ; genetics ; China ; Genetic Variation ; Microsatellite Repeats ; Phylogeny

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.
Phylogeny ; Atractylodes/genetics* ; Genome, Chloroplast ; Whole Genome Sequencing ; Microsatellite Repeats ; Lamiales

In order to transform main active ingredient of volatile oil, endophytic fungi were screened from the root of Atractylodes lancea. Transformation method was used in vitro. The changes of volatile oil were traced by gas chromatography. One endophytic fungus (strain ALG-13) which could uitilize volatile oil selectively was screened. Single factor experiment were conducted for exploring the effects of various factors that including kinds of carbon source, speed, liquid volume, pH and concentration of plant tissue on degradation by this strain. Subsequently, the main affecting factors carbon source, speed, pH and liquid volume were optimized using orthogonal array design. Results showed that endophytic fungus ALG-13 selectively used the volatile oil, change the relative percentage of the main components of volatile oil, Atractylon and Atractydin were increased, While, beta-eudesmol and Atractylol decreased. After selectively degradation by fungus, volatile oil components percentage were closer to the geo-herbs. Strain ALG-13 was identified as Bionectria ochroleuca according to its morphological characteristics and systematic analysis of ITS sequence. The optimal conditions were as follows: sucrose used as carbon source, rotating speed was 200 r x min(-1), initial pH for medium was 4.5, 50 mL liquid was added in 250 mL flask. The endophytic fungus ALG-13 could degrade the volatile oil selectively, which was benefit for forming geoherbs A. lancea volatile oil composition.
Atractylodes ; chemistry ; microbiology ; Fungi ; classification ; genetics ; isolation & purification ; metabolism ; Oils, Volatile ; chemistry ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; genetics