Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Expression and DNA methylation of SERK, BBM, LEC2 and WUS genes in in vitro cultures of Boesenbergia rotunda (L.) Mansf.

The process of somatic embryogenesis and plant regeneration involve changes in gene expression and have been associated with changes in DNA methylation. Here, we report the expression and DNA methylation patterns of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), BABY BOOM (BBM), LEAFY COTYLEDON 2 (LEC2) and WUSCHEL (WUS) in meristematic block of newly emerged shoots from rhizome, embryogenic and non-embryogenic calli, prolonged cell suspension culture, ex vitro leaf, and in vitro leaf of regenerated plants of Boesenbergia rotunda. Among all seven samples, based on qRT-PCR, the highest level of expression of SERK, BBM and LEC2 was in embryogenic callus, while WUS was most highly expressed in meristematic block tissue followed by embryogenic callus. Relatively lower expression was observed in cell suspension culture and watery callus for SERK, LEC2 and WUS and in in vitro leaf for BBM. For gene specific methylation determined by bisulfite sequencing data, embryogenic callus samples had the lowest levels of DNA methylation at CG, CHG and CHH contexts of SERK, LEC2 and WUS. We observed negative correlation between DNA methylation at the CG and CHG contexts and the expression levels of SERK, BBM, LEC2 and WUS. Based on our results, we suggest that relatively higher expression and lower level of DNA methylation of SERK, BBM, LEC2 and WUS are associated with somatic embryogenesis and plant regeneration in B. rotunda.

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda).

Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue.