The second complete chloroplast genome sequence of Nymphaea alba L. (Nymphaeaceae) to investigate inner-species variations

Abstract

Nymphaea alba L. which belongs to Nymphaeaceae has been used as gardening plants, foods, and medicines. In this study, we completed whole chloroplast genome of N. alba of which length is 151,925 bp consisting four subregions: 90,001 bp of large single copy (LSC) and 19,562 bp of small single copy (SSC) regions are separated by 25,181 bp of inverted repeat (IR) regions. One hundred and twenty-eight genes (84 protein-coding genes, 8 rRNAs, and 36 tRNAs) were annotated successfully. The overall GC content of the chloroplast genome is 39.2% and those in the LSC, SSC, and IR regions are 37.8%, 34.4%, and 43.4%, respectively. Eleven single nucleotide polymorphisms and six insertion and deletions are identified between two N. alba chloroplast genomes. This genome will be used for developing molecular markers and for understating micro-evolutionary events of this species.

Keywords:

• Nymphaea
• chloroplast genome
• Nymphaea alba
• Nymphaceae
• inner-species variation

Nymphaea alba L., called as European white water lily, is native species in North Africa, temperate and tropical Asia, and Europe. It has been used as gardening plants because of beautiful flowers. Nymphaea alba var. rubra, a hybrid of N. alba and N. odorata (Dkhar et al. 2011), has red flowers. In addition, its cooked root and seeds have utilized as foods for several thousand years (Mabey and Blamey 1972; Chiej 1984). N. alba contains several toxic alkaloids, such as nupharin and nymphaeine, which can affect nerve system (Wrobel 1967).

Its chloroplast genome was sequenced in 2004 to understand basal angiosperm families (Goremykin et al. 2004). Consequently, five more chloroplast genomes of Nymphaea have been sequenced. Here we sequenced another individual of N. alba deposited in the InfoBoss Cyber Herbarium (IN; IBS-00010) to dissect inner-species variations on the chloroplast genome. Total DNA was extracted from fresh leaves of N. alba by using a DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Genome sequencing was performed using HiSeq2000 at Macrogen Inc., Seoul, Korea and de novo assembly was conducted by Velvet 1.2.10 (Zerbino and Birney 2008). Assembled sequences were confirmed by sequence alignment by BWA 0.7.17 (Li 2013) and SAMtools 1.9 (Li et al. 2009). Geneious R11 11.1.5 (Biomatters Ltd., Auckland, New Zealand) was used for annotation using N. alba chloroplast genome (NC_006050; Goremykin et al. 2004).

The chloroplast genome of N. alba (Genbank accession is MK040444) is 151,925 bp in length and has four subregions: 90,001 bp of large single copy (LSC) and 19,562 bp of small single copy (SSC) regions are separated by 25,181 bp of inverted repeat (IR). It contains 130 genes (85 protein-coding genes, 8 rRNAs, and 37 tRNAs). The overall GC content of N. alba is 39.2% and those in the LSC, SSC, and IR regions are 37.8%, 34.4%, and 43.4%, respectively.

Based on the alignment of two chloroplast genomes of N. alba, 11 single nucleotide polymorphisms (SNPs) and 6 insertion and deletions are identified. Three SNPs (27.3%) and one insertion (TTT; 16.7%) in our chloropast genome are found inside tRNA-Arg (UCU), psbC, rps4, and rpoC2 genes, respectively. Interestingly, four SNPs near to 3’ end of psbA gene are surrounded by 14 bp IRs. Number of sequence variations in  N. alba reflects low level of genetic diversity in comparison to other studies (Young et al. 2011; Myszczyński et al. 2017).

Seven Nymphaea and three neighbor complete chloroplast genomes in Nymphaceae were used for constructing phylogenetic trees using maximum likelihood (bootstrap repeat is 1,000) and neighbor joining (bootstrap repeat is 10,000) methods with MAFFT 7.388 (Katoh and Standley 2013) and MEGA X (Kumar et al. 2018). The trees present that N. alba is clustered with N. maxicana belonging to subgenus Nymphaea. (Figure 1). In addition, Euryale ferox was clustered together with other Nymphaea chloroplast genomes, agreeing with previous studies (Figure 1). Taken together, this chloroplast genome shows inner-species sequence variations in N. alba, which will be used for developing molecular markers as well as for understanding micro-evolutionary events of this species.

Figure 1. Maximum likelihood and neighbor joining phylogenetic trees of Numphaeceae based on 10 complete chloroplast genomes: Nymphaea alba [MK040444 (this study) and NC_006050], Nymphaea capensis (MK111409), Nymphaea lotus (MK040443), Nymphaea ampla (NC_035680), Nymphaea jamesoniana (NC_031826), Nymphaea mexicana (NC_024542), Euryale ferox (NC_037719), Barclaya longifolia (NC_035633), and Nuphar advena (NC_008788). The numbers above branches indicate bootstrap support values of maximum likelihood and neighbor joining trees, respectively.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by both InfoBoss Research Grant (IBG-0007).