Metabolomics and Transcriptomics Reveal that Diarylheptanoids Vary in Amomum tsao-ko Fruit Development.

Amomum tsao-ko is an important spice and medicinal plant that has received extensive attention in recent years for its high content of bioactive constituents with the potential for food additives and drug development. Diarylheptanoids are major and characteristic compounds in A. tsao-ko; however, the biochemical and molecular foundation of diarylheptanoids in fruit is unknown. We performed comparative metabolomics and transcriptomics studies in the ripening stages of A. tsao-ko fruit. The chemical constituents of fruit vary in different harvest periods, and the diarylheptanoids have a trend to decrease or increase with fruit development. GO enrichment analysis revealed that plant hormone signaling pathways including the ethylene-activated signaling pathway, salicylic acid, jasmonic acid, abscisic acid, and response to hydrogen peroxide were associated with fruit ripening. The biosynthetic pathways including phenylpropanoid, flavonoids, and diarylheptanoids biosynthesis were displayed in high enrichment levels in ripening fruit. The molecular networking and phytochemistry investigation of A. tsao-ko fruit has isolated and identified 10 diarylheptanoids including three new compounds. The candidate genes related to diarylheptanoids were obtained by coexpression network analysis and phylogenetic analysis. Two key genes have been verified to biosynthesize linear diarylheptanoids. This integrative approach provides gene regulation and networking associated with the biosynthesis of characteristic diarylheptanoids, which can be used to improve the quality of A. tsao-ko as food and medicine.

[Genetic diversity and population structure of germplasm resources of Amomum villosum based on SSR markers].

Amomum villosum, serving as an important medicinal material, is complex in the genetic background of germplasm resources. Exploring the genetic diversity and genetic relationship of germplasm resources is conducive to clarifying the germplasm source and genetic background of A. villosum, so as to improve the efficiency of parent selection and variety breeding of A. villosum. Seventy-one pairs of SSR primers were used for PCR amplification of 84 A. villosum samples by polyacrylamide gel electrophoresis. Fifty-four pairs of SSR primers with high polymorphism were screened out for the analysis of genetic diversity. The results showed that 293 alleles were detected from 84 germplasm resources by 54 pairs of SSR primers, with an average of 5.32 alleles for each pair of primers, and a variation range of 3-8, and the primer AVL12 marked the highest number of alleles. The PIC value of each locus varied from 0.068 7 to 0.828 9, with an average of 0.529 9, and the highest was marked by AVL24. The genetic diversity of A. villosum was the highest in Yunnan, followed by Guangxi, and the lowest was found in Guangdong. The population structure analysis and cluster analysis showed that the samples were classified into two groups. In terms of origin, samples from Yunnan and Guangxi had a close genetic relationship, and there was no obvious differentiation of A, villosum resources from different origins. In this study, 54 pairs of SSR markers were used to analyze the genetic diversity and population structure of 84 germplasm resources, which can reflect the genetic relationship between A. villosum samples from different germplasm sources and different populations, thus providing a theoretical basis for the collection, research, and breeding of A. villosum resources.

Research on germplasm diversity of Amomum villosum. Lour in genuine producing area.

BACKGROUND: Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum. METHODS: The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations. RESULTS: The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565. CONCLUSION: There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.

Essential oil components in the seed masses of Amomum xanthioides and its related species from Southeast Asia and China.

Previously, to develop an objective identification method for Amomi Semen (AS), the nucleotide sequences of nrDNA ITS region and two cpDNA regions of nine Amomum taxa specimens from Southeast Asia and China were determined, and the generated phylogenetic tree showed six taxa specimens were divided into four groups. In this study, 51 crude drug samples of AS in Japanese markets were classified into four groups or species based on their ITS sequences. Approximately 67% of samples were derived from A. villosum var. xanthioides or A. xanthioides, A. villosum var. villosum and A. longiligulare prescribed in Japanese Pharmacopoeia, and the rest were mixed with A. uliginosum and A. microcarpum. Subsequently, the essential oil compositions of Amomum taxa specimens and AS samples were determined by GC-MS to characterize each group or species. Group 1(A. xanthioides) samples were characterized by containing higher amount of camphor(6) than bornyl acetate(9), and a specific germacrene D-4-ol; group 2(Chinese A. villosum var. villosum and var. xanthioides) by containing higher amount of 9 than 6, a specific isobornyl acetate; group 3(Laotian A. villosum var. villosum and A. longiligulare) by containing higher amount of 6 than 9, and a characteristic neointermedeol, except for A. longiligulare specimen from Hainan, China; group 4(A. uliginosum) by containing equivalent amount of 6 and 9, and the specific (E,E)-farnesyl acetate and (E,E)-farnesol. A. microcarpum samples were discriminated from the above groups by absence of 6 and 9, and with higher amount of (E)-nerolidol. There was a good correlation between genetic classification and chemical discrimination.

Genetic diversity of Amomum xanthioides and its related species from Southeast Asia and China.

Amomum Semen, the seed mass of Amomum xanthioides, has been imported from Southeast Asia and China and used for the treatment of gastric and intestinal disorders. A. xanthioides has been treated as a synonym of A. villosum var. xanthioides. Furthermore, A. villosum var. villosum, A. villosum var. xanthioides, or A. longiligulare have been described as the botanical origin of Amomi Fructus, which is a similar crude drug in Chinese Pharmacopoeia. Under these circumstances, the botanical origin of Amomum Semen was changed to A. villosum var. xanthioides, A. villosum var. villosum, or A. longiligulare in Supplement II to the 17th edition of the Japanese Pharmacopoeia. To develop an objective identification method for Amomum Semen and to confirm the phylogenetic relationship among Amomum taxa, the nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer region and chloroplast DNA partial matK-trnK and trnH-psbA intergenic spacer regions were determined in specimens collected from Southeast Asia and China, including those from the type localities of each taxon. Six taxa were divided into four groups. A. xanthioides from Myanmar belonging to group 1 was discriminated from A. villosum var. xanthioides from China of group 2. A. villosum and its varieties were divided into two groups: group 2 included those from China, and group 3 consisted of A. villosum from Laos. A. longiligulare from China and Laos and A. uliginosum from Laos belonged to group 3 and group 4, respectively. These findings illustrate the phylogenetic basis for the need for taxonomical reorganization among the Amomum species.

RNA sequencing on Amomum villosum Lour. induced by MeJA identifies the genes of WRKY and terpene synthases involved in terpene biosynthesis.

Amomum villosum Lour. is an important Chinese medicinal plant that has diverse medicinal functions, and mainly contains volatile terpenes. This study aims to explore the WRKY transcription factors (TFs) and terpene synthase (TPS) unigenes that might be involved in terpene biosynthesis in A. villosum, and thus providing some new information on the regulation of terpenes in plants. RNA sequencing of A. villosum induced by methyl jasmonate (MeJA) revealed that the WRKY family was the second largest TF family in the transcriptome. Thirty-six complete WRKY domain sequences were expressed in response to MeJA. Further, six WRKY unigenes were highly correlated with eight deduced TPS unigenes. Ultimately, we combined the terpene abundance with the expression of candidate WRKY TFs and TPS unigenes to presume a possible model wherein AvWRKY61, AvWRKY28, and AvWRKY40 might coordinately trans-activate the AvNeoD promoter. We propose an approach to further investigate TF unigenes that might be involved in terpenoid biosynthesis, and identified four unigenes for further analyses.

A comparison study on polysaccharides from novel hybrids of Amomum villosum and its female parent.

The present study is a comparative study on preliminary characterizations and immunostimulatory activities of water-soluble polysaccharides (WSP) from newly hybrid Amomum villosum and its female parent. First, two kinds of WSP were extracted, respectively from the newly hybrid A. villosum called Spring No.1 (WSPH) and its female parent-Longfruit No.2 (WSPP). Then, the differences of preliminary characterizations and immunostimulatory activities in vivo for these two WSP samples were compared. Experimental results showed that WSPH and WSPP had the same monosaccharide composition, and similar ultraviolet and infrared spectra characteristics of polysaccharides, while their immunostimulatory activities, in terms of the weights of spleen and thymus, pinocytic activity and the level of serum hemolysin, showed no significant differences between the groups treated with WSPH and WSPP, respectively. Combined with the findings from other studies in our research group, these results suggested that this novel hybrid could be an acceptable alternative for cultivation of A. villosum.

SNP typing for germplasm identification of Amomum villosum Lour. Based on DNA barcoding markers.

Amomum villosum Lour., produced from Yangchun, Guangdong Province, China, is a Daodi medicinal material of Amomi Fructus in traditional Chinese medicine. This herb germplasm should be accurately identified and collected to ensure its quality and safety in medication. In the present study, single nucleotide polymorphism typing method was evaluated on the basis of DNA barcoding markers to identify the germplasm of Amomi Fructus. Genomic DNA was extracted from the leaves of 29 landraces representing three Amomum species (A. villosum Lour., A. xanthioides Wall. ex Baker and A. longiligulare T. L. Wu) by using the CTAB method. Six barcoding markers (ITS, ITS2, LSU D1-D3, matK, rbcL and trnH-psbA) were PCR amplified and sequenced; SNP typing and phylogenetic analysis were performed to differentiate the landraces. Results showed that high-quality bidirectional sequences were acquired for five candidate regions (ITS, ITS2, LSU D1-D3, matK, and rbcL) except trnH-psbA. Three ribosomal regions, namely, ITS, ITS2, and LSU D1-D3, contained more SNP genotypes (STs) than the plastid genes rbcL and matK. In the 29 specimens, 19 STs were detected from the combination of four regions (ITS, LSU D1-D3, rbcL, and matK). Phylogenetic analysis results further revealed two clades. Minimum-spanning tree demonstrated the existence of two main groups: group I was consisting of 9 STs (ST1-8 and ST11) of A. villosum Lour., and group II was composed of 3 STs (ST16-18) of A. longiligulare T.L. Wu. Our results suggested that ITS and LSU D1-D3 should be incorporated with the core barcodes rbcL and matK. The four combined regions could be used as a multiregional DNA barcode to precisely differentiate the Amomi Fructus landraces in different producing areas.

[Identification of Amomum villosum, Amomum villosum var. xanthioides and Amomum longiligulare on ITS-1 sequence].

OBJECTIVE: To identify Amomum villosum Lour. and some their adulterants on molecular biology. METHOD: The DNA of Amomum villosum Lour. and some their adulterants were extracted, and amplified using ITS-1 primer. The amplificed DNA were purified and then sequenced by direct PCR sequencing method. RESULT: The ITS-sequence of all of the samples are 248 bp in size. But there are 7 bases in Amomum villosum Lour var. xanthioides (Wall.ex Bak) T.L. Wu et Senjen and 12 hases in Amomum longiligulare T.L. Wu. differing from Amomum villosum Lour. CONCLUSION: The ITS-1 sequence can be used to identify effectively Amomum villosum Lour. and their adulterants.

[Analysis of Amomun villosum species and some adulterants of zingiberaceae by RAPD].

Different Amomum villosum Lour. species and some their adulterants of Zingiberaceae were researched using RAPD. The PCR indicates favourable differentiation of the reaction. The Amomum villosum Lour. species have the similar DNA fingerprints while it is obviously differ from adulterants. There are 12.17 percent of primers which appear polymorphism. We can identify different species according to these dissimilarity. We also construct the tree of the molecular evolution through NJ software, the hereditary distance of the graph shows different relationships between Amomum villosum Lour. species and their adulterants. The result created by software is analogous the traditional methods.