Sequencing, De novo Assembly, Functional Annotation and Analysis of Phyllanthus amarus Leaf Transcriptome Using the Illumina Platform

Abstract

Phyllanthus amarus Schum. and Thonn., a widely distributed annual medicinal herb has a long history of use in the traditional system of medicine for over 2000 years. However, the lack of genomic data for P. amarus, a non-model organism hinders research at the molecular level. In the present study, high-throughput sequencing technology has been employed to enhance better understanding of this herb and provide comprehensive genomic information for future work. Here P. amarus leaf transcriptome was sequenced using the Illumina Miseq platform. We assembled 85,927 non-redundant (nr) “unitranscript” sequences with an average length of 1548 bp, from 18,060,997 raw reads. Sequence similarity analyses and annotation of these unitranscripts were performed against databases like green plants nr protein database, Gene Ontology (GO), Clusters of Orthologous Groups (COG), PlnTFDB, KEGG databases. As a result, 69,394 GO terms, 583 enzyme codes (EC), 134 KEGG maps, and 59 Transcription Factor (TF) families were generated. Functional and comparative analyses of assembled unitranscripts were also performed with the most closely related species like Populus trichocarpa and Ricinus communis using TRAPID. KEGG analysis showed that a number of assembled unitranscripts were involved in secondary metabolites, mainly phenylpropanoid, flavonoid, terpenoids, alkaloids, and lignan biosynthetic pathways that have significant medicinal attributes. Further, Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values of the identified secondary metabolite pathway genes were determined and Reverse Transcription PCR (RT-PCR) of a few of these genes were performed to validate the de novo assembled leaf transcriptome dataset. In addition 65,273 simple sequence repeats (SSRs) were also identified. To the best of our knowledge, this is the first transcriptomic dataset of P. amarus till date. Our study provides the largest genetic resource that will lead to drug development and pave the way in deciphering various secondary metabolite biosynthetic pathways in P. amarus, especially those conferring the medicinal attributes of this potent herb.

De novo assembly, functional annotation, and analysis of the giant reed (Arundo donax L.) leaf transcriptome provide tools for the development of a biofuel feedstock

“…Genome-wide SSR analyses in nine species of the Poaceae family [82] revealed that the most abundant repeat type was a hexamer (58.82%). Conversely, our results were consistent with those obtained in licorice ( Glycyrrhiza uralensis ) in which the most frequent repeat motif was a mono-nucleotide, followed by tri-nucleotides and di-nucleotide motifs [83], and partially to those obtained in bhumyamalaki ( Phyllanthus amarus ), in which the most abundant repeat type was a mono-nucleotide, followed by di- and tri-nucleotides [84]. Although natural genetic variation in A. donax was reported to be low for the Mediterranean basin and slightly higher for the Asian area [85], and genetic uniformity was also observed in ecotypes in the United States [86], here 53 Candidate PolySSRs were predicted using CandiSSR software, suggesting a likely high level of genetic diversity among the analyzed ecotypes.…”

Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

“…In present study, multiple transcripts which encoded some of the known enzymes participated in the MVA pathway and the saponin biosynthesis pathway were found in traditional herbs, e.g., Panax ginseng (Cao et al, 2015), Phyllanthus amarus (Mazumdar and Chattopadhyay, 2016), Panax japonicus (Rai et al, 2016), Astragalus membranaceus Bge (Chen et al, 2015), and Eleutherococcus senticosus (Hwang et al, 2015). Genes involved in the MVA pathway in the A. bidentata transcriptome included 3-hydroxy-3-methylglutaryl CoA synthase (HMGS), HMG-CoA reductase (HMGR), phosphomevalonate kinase (PMK), mevalonate-5-diphosphate decarboxylase (MDD), isopentenyl diphosphate isomerase (IDI), geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPS), geranylgeranyl diphosphate synthase (GGPPS), squalene synthase (SS), squalene epoxidase(SE), beta-amyrin synthase (β-AS), and CAS.…”

Integrative analysis of genome‐wide lncRNA and mRNA expression in newly synthesized Brassica hexaploids

“…WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) was used to functionally classify the target genes and graphically represent the target gene functions (Mazumdar & Chattopadhyay, ). It is a useful tool for plotting GO annotation results and shows the categorization into three main ontologies: “cellular component,” “molecular function’,” and “biological process.”…”

“…WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) was used to functionally classify the target genes and graphically represent the target gene functions (Mazumdar & Chattopadhyay, 2016).…”