Comparative analysis of the complete chloroplast genomes and 45S nrDNAs of six Lonicera species

Abstract

The genus Lonicera belongs to the family Caprifoliaceae and comprises approximately 210 species distributed in East Asia. Many Lonicera species such as L. japonica and L. maackii are ornamental shrub plants, and used as herbal medicines. Despite their usefulness, its genetic, genomics and molecular phylogenetics are rarely reported. Here, we collected six Lonicera species from Medicinal Plant Garden, College of Pharmacy, Seoul National University, and produced 2.7 - 4.1 Gbp of whole genome sequencing (WGS) data. We obtained complete sequences of chloroplast genome and 45S nuclear ribosomal DNA (45S nrDNA) sequences using de novo assembly of Low-Coverage Whole genome sequence (dnaLCW). The chloroplast genome of the six Lonicera species ranged from 154,892 to 155,318 bp and showed high similarities (97.4%) each other. There were 114 genes in L. insularis, L. sachalinensis, L. praeflorens and L. maackii, 113 in L. vesicaria, and 112 in L. japonica. Comparative analysis of chloroplast genome and 45S nrDNA sequences revealed 17~2,261 single nucleotide polymorphisms (SNPs) and 5~278 insertion and deletions (InDels) between species in chloroplast, and a total of 45 SNPs and 4 InDels in 45S nrDNA. Furthermore, 266 large repetitive sequences and 288 simple sequence repeats (SSRs) were detected among six chloroplast genomes. In addition, we found several chloroplast protein-coding genes that showed high Ka/Ks values or highly conserved among six Lonicera species. Four genes, psaJ, rbcL, rps18 and ycf2 genes that showed high Ka/Ks values, might be positively selected in genus Lonicera. On the other hand, four genes in large single copy (LSC), psbI, psbZ, psbL and petZ genes, were highly conserved in six Lonicera species. Estimation of divergence time and phylogenetic relationship of six Lonicera species revealed that L. japonica was diverged first from the common ancestor of six Lonicera species (9.19-10.74 MYA), and L. insularis and L. sachalinensis were recently diverged (0-0.03 MYA). Phylogenetic trees based on chloroplast genomes and 45S nrDNA sequences showed a similar topology that L. insularis and L. sachalinensis were the closest, and they consist a clade with L. maackii. Moreover, phylogenetic analysis with related species in Dipsacales revealed that Lonicera species clustered with genera Patrinia and Kolkwitzia of the same family Caprifoliaceae, as expected. A total of seven molecular markers were developed from polymorphic sites such as SNPs, InDels and copy number variation (CNV) of tandem repeat (TR) in chloroplast genome. We could successfully discriminate six Lonicera species using these developed markers. The chloroplast genome and 45S nrDNA sequences of six Lonicera species along with DNA markers produced in this study will provide valuable information for further genetic diversity studies and authentication of six Lonicera species.