Panax L. (Araliaceae) is a medicinally important genus, which consists of seven well-recognized species and one species complex, and is widely distributed in East Asia and North America (Lee and Wen, 2004). Panax ginseng C. A. Mey. is very popular in traditional Chinese medicine and has been used as an herbal remedy in East Asia for thousands of years (Liu and Xiao, 1992). Although the pharmacology and medical effects of P. ginseng have been investigated extensively, only a few studies have focused on the genetic diversity and population structure of this species (Li et al., 2015), primarily owing to the limitations of molecular markers. In our recent studies, we employed single-copy nuclear genes to investigate the genetic diversity of cultivated and wild ginseng (Li et al., 2015). However, the relatively low mutation rate of single-copy nuclear genes and the recent domestication of cultivated ginseng largely limited the application of our selected nuclear genes in assessment of its population structure and domestication history. In addition, we have recently discovered multiple rounds of whole-genome duplication within the genus Panax (Shi et al., 2015). Panax ginseng is an allotetraploid species and has undergone two rounds of whole-genome duplication, which makes it difficult to obtain all alleles through traditional Sanger sequencing. In this regard, as codominant markers with high mutation rates (Jarne and Lagoda, 1996), nuclear microsatellites might provide novel insights into the polyploidy of Panax and domestication of P. ginseng. In previous studies, other microsatellites have been developed (Kim et al., 2007; Yang et al., 2008; Ahn et al., 2009; Van Dan et al., 2010; Choi et al., 2011; Reunova et al., 2014). Nevertheless, most of these studies developed microsatellites based on traditional Sanger sequencing, and the transferability of most of these microsatellites to congeneric species remains unknown. We employed next-generation sequencing to develop microsatellites from whole-genome data and to test the transferability of these identified microsatellites in other Panax and Aralia L. species.
METHODS AND RESULTS
Plant material and DNA extraction— A total of 63 wild and cultivated ginseng accessions were collected from North Korea, eastern Russia, and northeastern China (Appendix 1). The related species P. notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng, P. stipuleanatus H. T. Tsai & K. M. Feng, P. bipinnatifidus Seem., P. quinquefolius L., and Aralia elata (Miq.) Seem. were sampled from Yunnan, Jilin, and Sichuan provinces of China and from Wisconsin, USA (Appendix 1). We collected cultivated ginseng accessions from multiple geographic localities, while wild P. ginseng accessions and related species were generously provided by our collaborators. Of these P. ginseng accessions, 18 were subjected to whole-genome sequencing and 45 were used to test the polymorphisms of the identified microsatellites. Total genomic DNA was extracted from silica gel–dried leaves for each accession separately using the TIANGEN Plant Mini Kit (TIANGEN Biotech, Beijing, China).
Microsatellite development— The construction of DNA libraries of the 18 ginseng accessions was conducted by Novogene Corporation (Beijing, China), and then the libraries were sequenced using an Illumina HiSeq 2000 system (Illumina, San Diego, California, USA). We also downloaded the whole-genome data of one South Korean ginseng accession from GenBank (GenBank accession no.: SRR1181600). Because P. ginseng is an allotetraploid species with a large genome size (ca. 3 Gb), we were not able to perform de novo assembly in this study. Instead, we downloaded transcriptome data of the diploid species P. notoginseng (GenBank accession no.: SRX378873, SRX378878, and SRX378880) and performed de novo assembly using Trinity (Grabherr et al., 2011). These assembled transcripts were then used as references to perform the short read alignment for P. ginseng using Burrows-Wheeler Aligner's Smith-Waterman Alignment (BWA-SW; Li and Durbin, 2010). The insertions/ deletions were reported using SAMtools (Li et al., 2009). Thereafter, we developed a series of Perl scripts to identify the polymorphic microsatellites from the obtained variant call format (VCF).
Characteristics of 21 microsatellite loci developed in Panax ginseng.
Microsatellite marker data analysis— A total of 60 multiallelic microsatellites were detected from the whole-genome data of the 19 ginseng accessions (including SRR1181600). To determine the function of microsatellite-associated unigenes, these assembled transcripts were searched against the GenBank nonredundant protein database using BLASTX (Altschul et al., 1997) with an expected value <10-7. The putative functions of these microsatellite-associated genes are listed in Table 1. We chose the candidate microsatellites according to the following criteria: (1) more than 20 bp at the flanking regions of the microsatellite repeat, and (2) no large intron (<500 bp in length) within the target region used to design primers. Forty-one microsatellites were selected to design the PCR primers, and 38 of them showed clean PCR amplifications in all five P. ginseng accessions tested. The PCR amplifications were conducted in a 25-µL volume with 2.5 mM MgCl2, 0.1 µM forward and reverse primers, 400 µM dNTPs, 1 unit rTaq (TaKaRa Biotechnology Co., Dalian, Liaoning, China), and 20–50 ng DNA. PCRs were performed for each microsatellite under the following conditions: an initial denaturation step of 5 min at 95°C; followed by 35 cycles of 94°C for 30 s, annealing temperature (Table 1) for 30 s, and 72°C for 30 s; and a final step of 8 min at 72°C. After amplification, the PCR products were sequenced on an ABI730 sequencer (Applied Biosystems, Foster City, California, USA). The obtained genomic DNA sequences of P. ginseng were then compared with the assembled transcripts of P. notoginseng. As expected, all microsatellites were confirmed in the genomic sequences of P. ginseng. All DNA sequences of P. ginseng obtained from this study were submitted to Gen-Bank (accession no.: KU879255–KU879294; Table 1).
To further evaluate the polymorphisms in cultivated ginseng, we amplified these microsatellites with 45 accessions of three major ginseng landraces. Twenty-one microsatellites yielded abundant PCR products across the 45 ginseng accessions. Fluorescently labeled PCR products were resolved to genotype on an ABI 3730 sequencer (Applied Biosystems). The number of alleles of these microsatellites varied from one to five, and the polymorphism information content ranged from 0.0000 to 0.6450 (Table 1). We also evaluated polymorphisms of these microsatellites in each of the three ginseng landraces (Table 2). We noted that the polymorphic microsatellite S25 was monomorphic in the landraces SHIZHU and BIANTIAO. Similarly, the polymorphic microsatellites S11 and S12 were monomorphic in the landrace COMMON. The transferability of these primers was tested with P. notoginseng, P. stipuleanatus, P. bipinnatifidus, P. quinquefolius, and A. elata. All 21 primers amplified successfully in the four congeneric species except P. bipinnatifidus (Table 3). All of these microsatellites also yielded clear PCR products in the closely related species A. elata, suggesting the high transferability of these primers (Table 3). Notably, we found that seven microsatellites were monomorphic across all 45 ginseng accessions, but five of them were polymorphic in related species (Table 3).
Allelic diversity in 21 microsatellites for three major ginseng landraces.a
Cross-species amplification information for 21 microsatellite loci in closely related Panax and Aralia species.a
Development of molecular markers from nonmodel species has been increasing in recent years. In this study, we identified polymorphic microsatellite markers from the nonmodel species P. ginseng using whole-genome data. These polymorphic microsatellites provide useful molecular markers to assess the germplasm resources of P. ginseng. In particular, the high transferability of these microsatellites provides reliable molecular markers to investigate the population genetics and polyploid evolution of Panax and Aralia.
The authors thank the editor and an anonymous reviewer for their constructive comments that greatly improved the manuscript, Dr. Zhenhui Wang for the de novo assembling of Panax notoginseng, and Dr. Hongxing Xiao and Dr. Yuezhi Pan for providing the wild P. ginseng samples and related Panax species. This work was supported by the National Natural Science Foundation of China (no. 31470010).