Diversity and evolution of Panax ginseng and its relatives inferred from complete chloroplast genome and nrDNA sequences

Abstract

Chloroplast (cp) genome and nuclear ribosomal DNA (nrDNA) are the main sequences used for genetic diversity and evolution research in the plant kingdom. The cp genomes are 57- to 217-kb circular DNA molecules containing ~100 conserved genes. In this study, high-throughput method of de novo assembly and error correction was developed to simultaneously obtain complete sequences of the chloroplast genome and nuclear ribosomal DNA units using relatively small amounts of whole genome shotgun sequence (WGS) produced by next generation sequencing (NGS) platform and coined as de novo assembly of low coverage WGS (dnaLCW). The dnaLCW method was successfully performed to obtain both types of sequence for hundreds of plants with various genome sizes. This research opens a new era for practical application of NGS data to high-copy genomic components and represents a breakthrough technology for analyzing genetic diversity, barcoding at both inter and intra species levels, and for fundamental understanding of evolution in the plant kingdom. I applied dnaLCW for understanding evolution and genetic diversity inter and intra Panax species. Cp genomes and 45S DNAs of five Panax species and five related genus, including new sequences from seven species, have been investigated simultaneously. I studied for the genetic diversity and evolutionary history based on both cytoplasmic and nuclear genome representative sequences. All the cp genomes were determined from 155,993 bp to 156,730 bp size, and showed same structure with common 79 protein-coding, 30 tRNA and 4 rRNA genes. The complete 45S unit is ~ 11 kbp with 5.8 kbp of transcription region and varied intergenic spacer region. Ten cp genomes were 97.5-99.6% sequence homologies and 0.009 to 0.032 synonymous substitution rates. Nucleotide diversity varies among 73 cp protein coding genes, and the pabM, rps19 and rpl22 genes showed the highest Ks values. Three genes, atpF, ycf2 and clpP, showed the highest Ka/Ks values (>1) suggesting these three genes might play the positive pressure for speciation of ten Panax relative species. Sequence polymorphism rates in 45S DNAs is 0.2-1.5% and 26S rRNA gene showed the highest polymorphism. Based on phylogenomics analysis inferred from both cp genomes and nrDNAs, taxonomical positions of Panax relatives were clearly resolved into two monophyletic lineages as Panax-Aralia and Eleutherococcus-Dendropanax group. Molecular clock estimation based on the 79 chloroplast genes reveals 9-12.5 million years ago (MYA) for diversification of species in Araliaceae family and 3.9 MYA for diversification of Panax genus. Supposedly, divergence and speciation in Panax were occurred in period of uplift of the Himalaya-Tibetan plateau approximately 3.9 MYA and also followed by recent tetraploidization event and speciation between P. ginseng and P. quinquefolius 0.9-2.25 MYA. For comprehensive study of intra-species level diversity for cp genome and 45S nrDNA sequences of P. ginseng species, I obtained complete cp genome and nrDNA from 11 ginseng cultivars in Korea. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. I also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, I developed six reliable and valuable markers for practical application for analysis of ginseng diversity and cultivar authentication.