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.

Background Amomum villosum Lour. is the plant that produces the famous traditional Chinese medicine Amomi Fructus. Frequent habitat destruction seriously threatens A. villosum germplasm resources. Genetic diversity is very important to the optimization of germplasm resources and population protection, but the range of inherited traits within A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library as a resource for conservation efforts. Methods DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and used in the construction of a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity index (H), and Shannon’s polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by measuring Nei’s gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs), and gene flow (Nm). Analysis of molecular variance (AMOVA), Mantel tests, unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, and principal co-ordinates (PCoA) analysis were used to elucidate the genetic differentiation and relationship among populations. Results A total of 531 sequences were obtained from the five DNA barcodes with no variable sites from any of the barcode sequences. A total of 66 ISSR bands were generated from A. villosum populations using the selected six ISSR primers; 56 bands, 84.85% for all the seven A. villosum populations were polymorphic. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895), whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there is obvious genetic differentiation amongA. villosum populations and more genetic variations existed within each population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347.

Chloroplast genomes for 3 Bidens plants endemic to China (Bidens bipinnata Linn., Bidens pilosa Linn., and Bidens alba var. radiata) have been sequenced, assembled and annotated in this study to distinguish their molecular characterization and phylogenetic relationships. The chloroplast genomes are in typical quadripartite structure with two inverted repeat regions separating a large single copy region and a small single copy region, and ranged from 151,599 to 154,478 bp in length. Similar number of SSRs and long repeats were found in Bidens, wherein mononucleotide repeats (A/T), forward and palindromic repeats were the most in abundance. Gene loss of clpP and psbD, IR expansion and contraction were detected in these Bidens plants. It seems that ndhE, ndhF, ndhG, and rpl32 from the Bidens plants were under positive selection while the majority of chloroplast genes were under purifying selection. Phylogenetic analysis revealed that 3 Bidens plants clustered together and further formed molophyletic clade with other Bidens species, indicating Bidens plants might be under radiation adaptive selection to the changing environment world-widely. Moreover, mutation hotspot analysis and in silico PCR analysis indicated that inter-genic regions of ndhD-ccsA, ndhI-ndhG, ndhF-rpl32, trnL_UAG-rpl32, ndhE-psaC, matK-rps16, rps2-atpI, cemA-petA, petN-psbM were candidate markers of molecular identification for Bidens plants. This study may provide useful information for genetic diversity analysis and molecular identification for Bidens species.

Background: The Amomum villosum has the situation that it is inferior and other other varieties are used as A. villosum in the market. In order to develop and utilize the genuine medicinal materials A. villosum, this experiment aims to carry out the identification and research of variety of the A. villosum and analyze its genetic diversity, constructing the DNA barcode database of the genuine medicinal materials A. villosum in Guangdong Province and providing recommendations for populations planting, which will be critical to the further research of A. villosum. (2) Methods: A total of 141 samples of A. villosum were analyzed by DNA barcoding to construct DNA barcode database. The genetic diversity of A. villosum sampled from 7 populations in Guangdong Province was detected based on ISSR molecular marker technology. (3) Results: The success rates of PCR amplification and sequencing of five barcodes of A. villosumwas rbcL> ITS > ITS2 >psbA-trnH>matK. 141 samples of A. villosum from 7 populations in Guangdong Province were used to construct a reference DNA barcode database containing 531 sequences. The results of genetic diversity were as follow, the number of alleles Na ranged from 1.2879 to 1.7121, the effective number of alleles Ne ranged from 1.1848 to 1.4240, the gene diversity index (H) ranged from 0.2536 to 0.1117, and the Shannon index (I) ranged from 0.3816 to 0.1658, whichindicatedthegenetic diversity of A.Villosum was rich. The total genetic diversity among the 7 populations (Ht) was 0.3299, the genetic diversity within the populations (Hs) was 0.1819, and the gene differentiation coefficient (Gst) was 0.4487. AMOVA showed that the genetic variation within the populations and the genetic variation between the populations accounted for 68.74% (P<0.05) and 31.26% (P<0.05) respectively, indicating that the genetic variation of A. villosum was mainly within the populations. The gene flow Nm was 0.6143.The genetic distance of the 7 populations was 0.0844 - 0.3347, and the genetic identity was 0.7156 - 0.9191, confirming that the genetic relationship of each population was relatively close. The 7 populations were significantly grouped in the cluster analysis and the genetic level of each population from high to low was as follow: ZY (National Highway Roadside) > ZJD (Zhongjiaodong) > GY (Geopark) >MM (Dianbai) > YC (Dadong Village) > XFC (Xingfu Village) > TK (Tankui Village). There was no correlation between the geographic distance and the degree of genetic differentiation among populations. Conclusion: By amplifying and sequencing five barcodes of ITS2, psbA-trnH, ITS, matK and rbcL, a reference DNA barcode database of A. villosum with 531 sequences was constructed. The results of genetic diversity showed that it is necessary to take appropriate in situ protection measures for the populations of A. villosum in Yangchun City and increase the genetic exchange between populations to improve the genetic diversity of A. villosum.

Background Amomum villosum Lour. is the plant of a famous traditional Chinese medicine Amomi Fructus and its habitat has been frequently destroyed that seriously threatened its germplasm resources. The genetic diversity has great significance to the optimization of germplasm resources and protection of populations, but that of A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library for the purpose of more appropriate protecting measures. Methods DNA barcoding and ISSR markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and constructed a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei's gene diversity index (H) and Shannon's polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by Nei’s gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs) and gene flow (Nm). AMOVA analysis, Mantel tests, UPGMA dendrogram and PCoA analysis were used to elucidate the genetic differentiation and the relationship among populations. Results 531 sequences of five DNA barcodes were gained and all the sequences of the each barcode had no variation sites. A total of 66 ISSR bands were generated from A. villosum populations by the selected 6 ISSR primers. And 56 bands were amplified polymorphic with the percentage was 84.85% for all the seven A. villosum populations. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895) whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there’s obvious genetic differentiation among A. villosum populations and more genetic variations existed in within population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347. Conclusions Overall, this study confirmed a high genetic diversity but weak gene flow of A. villosum in Guangdong province, especially in Amomi Fructus’s Daodi producing area, Yangchun city. And a local DNA barcode reference library containing 531 sequences were subsequently constructed; facilitating the identification, conservation, breeding and cultivation of A. villosum.