First report of the chloroplast and mitochondrial genomes of the Indian pitcher plant, Nepenthes khasiana Hook.f.

Abstract

Objectives

Nepenthes, sometimes known as tropical pitcher plants or monkey cups, is a carnivorous plant genus that contains more than 160 species. Nepenthes khasiana, India's sole representative of the genus, is a rare and endangered dioecious plant endemic to North-east India. Despite the fact that it is a prominent insectivorous plant in the Nepenthaceae family, genomic resources for the species are limited, making genomic breeding and understanding the genetic basis of botanical carnivory difficult. Herein, we report the complete chloroplast (cp) and mitochondrial (mt) genomes of N. khasiana for the first time. These organelle genomes were assembled as part of a whole-genome sequencing project aimed at gaining deeper insights into their evolutionary relations with genomes of other carnivorous plants.

Data description

The complete cp genome (156,914 bp) and mt genome (900,031 bp) of N. khasiana are presented here. The cp genome contains two repeat regions and 131 genes (112 unique genes): 86 protein coding genes, 8 rRNA coding genes and 37 tRNA coding genes. The mt genome contains 84 genes (55 unique genes): 50 protein coding genes, 7 rRNA coding genes and 27 tRNA coding genes. The cp and mt genomic data generated will be useful for future molecular characterization and evolutionary research related to botanical carnivory.

Objective

Because of their distinctive adaptations, carnivorous plants have become a valuable model system for studying a variety of ecological and evolutionary issues. The cone-shaped leaves of carnivorous pitcher plants (CPPs) facilitate in trapping prey and deriving nutrients. The Indian pitcher plant Nepenthes khasiana, is endemic to West and South Garo Hills, West and East Khasi Hills and Jaintia Hills of Meghalaya, India [1]. It is categorized as endangered on the IUCN Red List of Threatened Species and is included in the Government of India's Negative List of Exports [2]. It is also included in Appendix I of CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora), which bans trade of this species [3]. The fluid from unopened pitcher has been in use by the local tribes for treating cataract and night blindness, skin infections, gastrointestinal and gynecological problems. The juice from leaf is used for treating diabetes and urinary disorders [4, 5]. The different adaptation strategies of pitcher plants serve as a source of inspiration for a wide range of applications, including therapeutic treatments, reagents for proteomics experiments and as biocontrol solutions in agriculture [6,7,8].

The genus Nepenthes contains more than 160 species and five incompletely diagnosed taxa [9]. The physical environment and plant-animal interactions are important drivers of species diversity. The genus Nepenthes, owing to its high diversity in certain biogeographic regions presents a challenge to taxonomists, who are still searching for reliable methods of characterizing members of this genus by applying species concept. The CPPs have a unique combination of biochemistry, morphology and physiology to enable prey capture and nutrient assimilation from digested prey [10]. We still do not fully understand if these adaptations are also reflected at the genome level. Previous studies have indicated that the chloroplast genomes of carnivorous plants have undergone distinct evolutionary routes compared to that of its non-carnivorous relatives [11]. Nevertheless, specifics like the extent of divergence and general characteristics remain unclear [12]. In this data note, we report the first complete chloroplast (cp) and mitochondria (mt) genomes of N. khasiana, which will be crucial for understanding its genetic diversity, and unique evolutionary adaptations.

Data description

Nepenthes khasiana leaf sample was collected from its natural habitat in Jaintia hills, Meghalaya. Plants were identified by Dr. Devendra Kumar Biswal. The Government of Meghalaya granted the necessary authorization to gather samples for research. An herbarium specimen was prepared and submitted to the herbarium at Eastern Regional Centre of Botanical Survey of India located in Shillong, Meghalaya, India where it was assigned the accession number 86834. Using Qiagen Genomic-tip 100/G, high-quality gDNA was extracted from the N. khasiana leaf sample; Agarose gel electrophoresis, Nanodrop (Thermo Fisher Scientific, NanoDrop One) and Qubit Fluorometer (Thermo Fisher Scientific, Qubit 3.0) were used to confirm the quantity and quality of the resulting gDNA. The paired-end sequencing library was prepared using TruSeq Nano DNA Library Prep Kit for Illumina. The mean fragment size of shotgun libraries ranged from 586 to 612 bp. The libraries were sequenced on Illumina NextSeq 500 using 2 × 150 bp chemistry, which generates two reads of 150 base pairs per DNA fragment, one in the forward direction and the other in reverse. After preprocessing i.e., removing low quality reads (reads with N > 5%) and adapter sequences, the filtered reads were used for assembling the organelle genomes. The cp and mt genomes were assembled as part of a whole genome sequencing project for N. khasiana, aimed at enhancing our understanding of the evolutionary relations among the genomes of carnivorous plants.

For the chloroplast genome assembly, rbcL gene sequence from N. khasiana was used as seed sequence for the de novo assembly with NOVOPlasty [13]. When the assembler was run, the K-mer length, i.e., the amount of overlap between matching reads was set to 39. The assembled cp genome has a length of 156,914 bp, with 37% GC content. It has a quadripartite structure that consisted of two inverted repeat regions of 25,193 bp each, one large single-copy region of 87,237 bp and one small single-copy region of 19,291 bp. The assembled genome was annotated using CpGAVAS [14], and GeSeq [15]. A total of 86 protein coding genes of which 79 are unique, 37 tRNAs of which 29 are unique and 8 rRNAs of which four are unique were annotated in the assembled cp genome. Twelve genes had introns: rps16, atpF, rpoc1, ycf3, clpP, ndhB, ccsA, ndhA and both copies of rps12 and rpl2. Repeat regions harbored 20 genes: ndhB, rpl2, rpl23, rps19, rps7, rrn16, rrn23, rrn4.5, rrn5, trnA-UGC, trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC, ycf1, ycf2 and rps12. The annotated cp genome (Fig. 1A) has been submitted to GenBank with accession number: MH923233. Furthermore, NCBI RefSeq Accession number NC_051455 has been added to this submitted cp genome. (Data file 1, Table 1).

Fig. 1

Organelle genomes of Nepenthes khasiana. A Whole chloroplast genome annotated, with GenBank accession number MH923233, containing 87 protein-coding genes, 37 tRNAs,and 8 rRNAs. B Assembled mitochondrial genome, with GenBank accession number MK330891 depicts a total of 84 genes–50 protein coding genes, 27 tRNAs and 7 rRNAs. Thegraph shows two copies of twelve genes: atp9, cob, cox2, nad2, nad3, nad5, nad6, nad9, rpl5,rps1, rps12, rps4. Genes functioning in related processes are coded with colors.

Table 1 Overview of data files/data sets

For the mitochondrial genome assembly, matR gene sequence of N. mirabilis was used as seed sequence for assembly using NOVOPlasty with K-mer length set to 39 and genome range of 20,000–2200000 bp to prevent premature circularization. The assembled mt genome was annotated using MITOFY [16] and BLAST (blastn, blastp and tblastx) [17]. The annotations generated by MITOFY were manually curated to check the genomic coordinates for start and stop codons. Manual curation is necessitated as automated annotation tools sometimes fail to identify the entire coding region leading to the annotation of a coding sequence as “misc_features”. Homology tools like BLAST were used to verify if complete coding region had been annotated. This approach reduced instances of partial annotations. The assembled mt genome had a length of 900,001bp with 43.8% GC content. The mt genome features a total of 84 genes, with 55 unique genes. The annotated genes include 50 protein coding genes (36 unique), 27 tRNA genes (16 unique) and 7 rRNA genes (3 unique). It has two copies of twelve protein coding genes: atp9, cob, cox2, nad2, nad3, nad5, nad6, nad9, rpl5, rps1, rps12, rps4; two rRNA genes: rrn18 and rrn5; and 9 tRNA genes: trnC, trnD-GTC, trnE-TTC, trnF-GAA, trnM-CAT, trnP-TGG, trnS-TGA, trnW-CCA and trnY-GTA. The assembled and annotated mt genome (Fig. 1B) of N. khasiana is deposited in GenBank with accession number MK330891 (Data file 2, Table 1).

The generated datasets will be used for comparative genomic analysis to identify variations in the cp and mt genomes of N. khasiana.

Limitations

The present study reports organelle genomes, which were assembled with short reads. Data from long read sequencer would have been an added advantage, as it would have enabled hybrid strategy in assembling the genomes. The mtDNA is not sufficient as a single source of information for species identification because of introgression, maternal inheritance, recombination, unequal mutation rates, and compounding evolutionary processes. Organelle genomes are less likely to undergo mutation than nuclear genomes, hence this information may not be enough to fully understand the population genetics of N. khasiana.