Herbal Medicine: Open Access

Keywords

Secondary metabolites; Genome annotation; Repeat elements; Assembly

INTRODUCTION

Next-generation sequencing (NGS) technologies have permitted the rapid and efficient development of genomic resources for non-model or orphan plant species [1-3]. The investigation of indigenous plant genomes to decipher the genomic characterization and the genetic basis of its active compounds with proven medicinal efficacy is a relatively new area of exploration. Plant secondary metabolites from a large repertoire of plants are implicated in a wide range of human diseases, including cancer, diabetes, bacterial, parasitic and fungal infections. The pathways involved in the production of the metabolites need to be deciphered at a molecular level using genome information. Whole genome analysis of important plant species, native to India, is the first big step in achieving a complete molecular understanding of their medicinal value. The genomic information is very limited in the area of medicinal plants and provides us with a golden opportunity to investigate the genetic makeup and embark on a high-throughput whole genome analysis.

Cissus quadrangularis, belonging to the family Vitaceae, is commonly found in tropical regions of India, Ceylon, East Africa, Malaysia and Thailand [4]. It is an annual or perennial herb, entire leaves, buff colored with greenish ting and requires warm tropical climate and propagated by stem cuttings. It is commonly referred to as the “bone setter,” “Asthisamdhani” in Sanskrit and “Hadjod” in Hindi. It has been used by common folk in India for promoting the fracture healing process [5]. All parts of plants are used for medicinal purpose in a form of extracts and powders [6]. Traditionally, Cissus quadrangularis have been used for many years to promote bone and tissues healing, as an analgesic, to treat infections, as an anabolic, and to promote weight loss and weight management [7]. In addition to bone healing property, Cissus quadrangularis has been shown to have antiosteoporotic [8], anti-oxidant [9], anti-microbial [10], analgesic and antiinflammatory [11], insulin-resistant [12], anti-ulcer and gastro protective properties [13] (Table 1).

Table 1: Features of Cissus quadrangularis genome assembly and annotation.

Phytochemical analysis of Cissus quadrangularis revealed the presence of several medicinally important plant secondary metabolites including α - and β -amyrin, β -sitosterol, Quercetin, kaempferol, stilbene derivatives, quadrangularins A, B, C, resveratrol and large amounts of Calcium and Vitamin-C [14-16]. All the literature discuss about the information of particular plant tissues and specific secondary metabolites for their implicated role in the cure of diseases. Till now, there has been no clear information of the presence of secondary metabolites at the genetic level and at the level of metabolic pathways for Cissus quadrangularis. Here, we report the draft genome of Cissus quadrangularis and these data will facilitate more comprehensive analysis to decipher genes and pathways involved in the production of medicinally important secondary metabolites.

Methods

The genomic DNA of Cissus quadrangularis was extracted from the leaf samples using Qiagen Plant genomic DNA extraction kit. The final DNA sample was eluted with nuclease free water and the quality of the samples were tested using Nanodrop (GE NanoVue). The concentration of the extracted samples was ~ 50 ng/μL as measured by the Qubit Fluorometer. The draft genome was sequenced using paired end (PE) library with an insert size of 350 bp by Illumina HiSeq 1000 platform. We obtained 336,794 reads from the PE library and the FASTX-Toolkit (https://hannonlab.cshl.edu/fastx_toolkit/) was used for the quality check of reads. Low quality reads were trimmed and quality score below Q20 were rejected. The good quality reads were further used for de novo genome assembly using SOAP denovo2 [17]. Different K-mers from 23 to 59, with a step size of two, were applied for the genome assembly. The assembled draft genome was further utilized for functional annotation using the MAKER program [18] with AUGUSTUS gene prediction tool [19] which was trained on Arabidopsis thaliana.

Results

We obtained the best assembly of 312 Mb at a K-mer of 31. The scaffold N50 is 5.7 Kb, 6206 scaffolds which are more than 100 bp in length and with the longest scaffold length of 111 Kb. Repeat elements in the assembled genome were identified using Repeat Scout (version 1.0.5) [20] and Repeat-Masker (https://www. repeatmasker.org/) (version 4.0.3). 18.20% of the assembled genome was masked due to the existence of repeat elements.

A total of 62,719 genes were initially predicted through the MAKER pipeline. After redundancy removal at 100% sequence identity using CD-HIT, 36,325 proteins were recognized as the final proteome. The CEGMA program [21] was implemented to identify the core set of essential eukaryotic genes. Around 53% (132 out of 248) of these essential genes were identified from our genome assembly. The draft genome of Cissus quadrangularis is deposited at NCBI (Bio sample accession number SAMN03223842). Further works, on the characterization of metabolite pathway-related genes, to elucidate their functional context and the orthology relationship of the predicted proteins, are in progress.

References

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