Molecular characterization of whole genome duplication in Panax ginseng

Abstract

Panax ginseng, belonging to Araliaceae, has been used as medicinal plant for centuries. Even though medicinal effect of ginseng is well-studied, the genomic information of ginseng is very limitedly available. Genome of P. ginseng has been considered to be experienced two rounds of whole genome duplication (WGD) based on expressed sequence tag (EST) sequences. However, there was no sign of the WGD at the genomic sequence level, except that multiple band patterns have been consistently observed during PCR amplification even though we used unique gene sequences in P. ginseng. Here, I tried to define the genome duplication at the sequence level. First, I analyzed the sequences of multiple bands derived from unique EST-SSR markers and at second, I deeply investigated four paralogous gene-rich genome blocks using whole genome draft sequence of P. ginseng. Re-amplification and sequencing of the individual bands for EST-SSR markers revealed that two bands around the expected size were genuine amplicons derived from two paralogous loci. Sequences derived from the two loci showed high similarity including the same primer-binding site, but each locus could be distinguished by the based on SSR copy number variations and additional SNPs or InDels. The comparison of the paralogous blocks in the draft sequence revealed that sequences are highly conserved between two recent duplicated paralagous scaffolds although there was some InDels derived from transposable elements. However, paleo duplicated paraloguous blocks showed little conservation only in genic regions. The paralogous gene pairs were identified and molecular clocks were calculated in four genome blocks. I estimated that first WGD happened at around 25.8 million years ago (MYA) which is common phenomena in all Panax species, and another recent WGD happened at around 2.54 MYA between P. ginseng and P. quinfuefolius only. Since high sequence homology between recent WGD blocks interrupts the amplification of single band in marker development, I developed the strategy of designing sequence tagged site (STS) primers, one for dCAPS based on single nucleotide polymorphism (SNP) site between cultivars, and another complementary primer for locus-specific primer based on SNP between paralogs. Genetic mapping using two STS markers derived from recent paralog blocks, Block 1 and Block 2, showed different segregation pattern in F2 population between Yunpoong and Chunpoong, which indicates that two recent duplicated paralog blocks are located independently at chromosomal locus and those were derived from WGDs. This study provide the first insight for WGD-derived genome structure in sequence level. The information and finding will be helpful for further ginseng genomics study for improving draft sequence quality and chromosome anchoring of scaffolds, and genetic mapping.