The genus Ephedra L. (Ephedraceae), also referred to as ma huang in Chinese, contains species that are sources of important Chinese traditional and Tibetan medicines (Konno et al., 1985). Ephedra gerardiana Wall. ex C. A. Mey. is distributed at altitudes above 3900 m in the Himalayan ranges (Editorial Committee of Chinese Flora, 1978). Because it is both a drought-resistant plant species (Shen, 1995) and an important Tibetan medicine (Pandey, 2006), E. gerardiana is useful for the study of the adaptive evolution and maintenance of genetic diversity in Ephedra. Relatively high genetic differentiation and variation are found for plants distributed on the Qinghai–Tibet Plateau due to its great geographical variability, in addition to frequent natural hybridization and polyploidization events (Wen et al., 2014). Chloroplast fingerprints revealed a high level of genetic differentiation as reflected by high levels of genetic diversity (FST) among populations of Ephedra species on the Qinghai–Tibet Plateau, such as in E. gerardiana (0.98) and E. saxatilis (Stapf) Royle ex Florin (0.86) (Qin et al., 2013).
Microsatellite (also referred to as simple sequence repeat [SSR]) markers are widely used in studies of plant population genetics and genetic diversity because of their high levels of polymorphism, stability, and codominance. However, traditional methods to develop microsatellite markers are time-consuming and complex. High-throughput and low-cost next-generation sequencing has accelerated the identification of large numbers of microsatellite markers (Rico et al., 2013). In this paper, we report the development of microsatellite markers for Ephedra species collected from the Qinghai–Tibet Plateau using Illumina MiSeq genome sequencing technology.
METHODS AND RESULTS
We collected a total of 106 individuals representing three E. gerardiana populations, two E. saxatilis populations, and one E. monosperma C. A. Mey. population from various locations on the Qinghai–Tibet Plateau. Vouchers of the sampled population materials were deposited in the Herbarium of Tibet University, Lhasa, China. The materials and their locality information are listed in Appendix 1.
The high-quality DNA sample for Illumina MiSeq sequencing (Illumina, San Diego, California, USA) was isolated from a randomly selected individual in an E. gerardiana population (Eg_QM, Appendix 1) using a DNA extraction kit (Tiangen Biotech, Beijing, China). DNA samples of all other Ephedra individuals used for the validation of the identified microsatellite markers were extracted using the cetyltrimethylammonium bromide (CTAB) method following Qin et al. (2013). The quality of extracted DNA samples was monitored on 1% agarose gels. The purity and concentration of the DNA samples were determined using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
For the development of microsatellite markers, an Illumina paired-end library was constructed using the TruSeq DNA Sample Prep Kit (Illumina) following the manufacturer's instructions and sequenced using the Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) to generate 250-bp paired-end reads. A total of 3,306,253,832 high-quality reads, consisting of 5,492,116 bp (Q20 = 95.22%), were generated for the E. gerardiana samples (GenBank BioProject number: PRJNA354648). Raw reads were trimmed using SeqPrep ( https://github.com/jstjohn/SeqPrep) and Sickle software (Joshi and Fass, 2011). Those reads with quality scores of <20 and lengths <20 bp were removed, leaving only the clean paired-end sequences. The high-quality filtered reads were assembled into 71,461 contigs by the GS De Novo Assembler version 2.8 (Roche Applied Science, Mannheim, Baden-Württemberg, Germany) with a length of 46,547,889 bp, N50 scaffold size of 1478 bp, N90 scaffold size of 1070 bp, and 32.78% GC content. Consequently, 3442 microsatellite sequences were identified using the MicroSAtellite Identification Tool (MISA; Thiel et al., 2003). Frequencies of each repeat type were classified as follows: di-, tri-, tetra-, penta-, and hexanucleotide repeats of 34.1% (1176 loci), 14.12% (486 loci), 0.67% (23 loci), 0.12% (4 loci), and 0.11% (2 loci), respectively. Of these, 2655 loci were designed for primer pairs using Primer3 software (Rozen and Skaletsky, 1999), and 135 loci (mostly dinucleotide or pure nucleotide repeats) were determined suitable to use. Primer pairs were then synthesized at the 135 loci by Sangon Biotech Co. Ltd. (Shanghai, China), and we tested whether these primer pairs could produce PCR products in eight randomly selected individuals from two E. gerardiana populations (Eg_Chd and Eg_Qm; Appendix 1).
Characteristics of 29 microsatellite primer pairs developed in Ephedra gerardiana.
Allelic diversity of Ephedra gerardiana populations based on 15 microsatellite loci.a
PCR amplifications were performed in a 10-µL volume with 10 ng of template DNA, 5 µL of 2× Taq PCR MasterMix (Tiangen Biotech), 0.4 µM of forward primers, 0.4 µM of reverse primers, and 3.7 µL of ddH2O following the protocol by Xu et al. (2014), except for the annealing temperatures as indicated in Table 1 for 30 s. PCR products were visualized on a 6% polyacrylamide gel electrophoresis (PAGE) gel with a 10-bp DNA ladder marker. Consequently, 45 primer pairs could produce PCR products with clear bands on PAGE, and only 29 of these primer pairs produced polymorphic bands among the eight E. gerardiana individuals. To confirm the reproducibility of polymorphisms of the 29 primer pairs in the eight E. gerardiana individuals, PCR amplifications were repeated with fluorescent dye–labeled (Jiang et al., 2012) forward primers and the PCR products were analyzed using the ABI3710XL DNA analyzer (Applied Biosystems, Foster City, California, USA), using GeneScan 500 LIZ (Applied Biosystems) as the internal size standard. The peaks of the loci were read by Peak Scanner Software version 1.0 (Applied Biosystems). The PCR reaction was performed in a 10-µL reaction solution containing 10 ng of template DNA, 3.8 µL ddH2O, 5 µL 2× Taq PCR MasterMix (Tiangen Biotech), 0.4 µM reverse primers, 0.04 µM M13-tailed forward primers, and 0.36 µM M13 label (5′-CAC-GACGTTGTAAAACGAC-3′) following the program: initial denaturation at 94°C for 3 min; followed by 12 cycles of denaturation at 94°C for 30 s, annealing for 35 s at the temperatures described in Table 1, and extension at 72°C for 35 s; followed by another 24 cycles again at these conditions; and a final extension at 72°C for 7 min. The results showed perfect reproducibility of polymorphisms of the 29 primer pairs in the eight E. gerardiana individuals.
Allelic diversity of populations of Ephedra saxatilis and E. monosperma based on 15 microsatellite loci developed in E. gerardiana.a
Fifteen randomly selected loci from the 29 confirmed microsatellite markers were used to test their polymorphisms and transferability in a larger sample set of E. gerardiana (59 individuals in three populations) and two related Ephedra species (47 individuals in E. saxatilis and E. monosperma) (Appendix 1). The data matrices of the scored bands from all Ephedra species based on the 15 primer pairs were subject to analysis for genetic diversity estimates. Genetic parameters included the number of effective alleles per locus, observed heterozygosity, and Nei's unbiased expected heterozygosity (Nei, 1978). All the analyses were conducted using GenAlEx software version 6.0 (Peakall and Smouse, 2012). Our results showed moderate to high polymorphisms of the 15 primer pairs among E. gerardiana populations, with effective alleles ranging from two to six per locus and observed and expected heterozygosity ranging from 0.23–0.83 and 0.44–0.86, respectively (Table 2). Results further indicated high polymorphisms of the 15 loci in the populations of E. saxatilis and E. monosperma (Table 3). All the results suggest that these 15 microsatellite loci are suitable for use across species of the genus Ephedra in addition to the 14 loci that are suitable for use in E. gerardiana (Table 1).
We developed and confirmed polymorphic microsatellite markers at 29 loci based on E. gerardiana populations, using the Illumina MiSeq sequencing method. Fifteen of these microsatellite loci were highly transferable to two related species in the genus, E. saxatilis and E. monosperma. These microsatellite markers will be useful for the characterization of genetic diversity and analysis of genetic structure for Ephedra species.
This work was supported by the National Natural Science Foundation of China (91131901, 31300201), the Specimen Platform of China (Teaching Specimen sub-platform), and the Program for Changjiang Scholars and Innovative Research Team (PSCIRT). The authors thank Min Xu for identifying the Ephedra samples and Bao-Rong Lu for his critical comments on this manuscript.