Speranskia Baill. (Euphorbiaceae) is a small genus endemic to China, comprising three herbaceous perennial species: S. tuberculata (Bunge) Baill., S. cantonensis (Hance) Pax & K. Hoffm., and S. yunnanensis S. M. Hwang (Hwang, 1989). Speranskia tuberculata is endemic to northern China and occurs on grassy slopes, grasslands, and thickets. The entire plant is commonly used for Chinese traditional medicine (Mazzio et al., 2014). Although S. tuberculata is not listed in the IUCN Red List, it is exhibiting a general decreasing trend or even disappearing completely in many distributional areas because of agricultural intensification and over-exploitation of natural population resources. To explore the genetic consequences of recent habitat fragmentation for this medical plant and generate useful information to facilitate the conservation and sustainable use of wild genetic resources, we developed the first set of 18 polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers for S. tuberculata using high-throughput transcriptome sequencing. We also tested these developed markers in S. cantonensis, a closely related species (Hwang, 1989), to identify their cross-species utility.

METHODS AND RESULTS

Fresh leaves of S. tuberculata seedlings were gathered in Beijing (39°59′06″N, 116°02′04″E; voucher specimen accession no. TB2013079, deposited at the Herbarium of Southwest Forestry University [SWFC], Kunming, China) and immediately frozen in liquid nitrogen, and then stored at −80°C. RNA extraction, cDNA library construction, and transcriptome sequencing were conducted following the procedures previously described by Ju et al. (2015). After removing adapter sequences and low-quality sequences, a total of 86,138,489 nonredundant unigenes were assembled from 95,791,418 raw reads. A high-quality reference genome with nonredundant unigenes was then generated by performing de novo transcriptome assembly using Trinity with the parameter of full clean up (Grabherr et al., 2011) and clustering similar contigs using CD-HIT with default parameters (Fu et al., 2012). Furthermore, we used MISA Perl script (MIcroSAtellite identification tool; Thiel et al., 2003) to screen for SSR motifs from all unigenes, and the minimum numbers of repeats were set as seven, five, five, five, and five for di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. MISA recovered a total of 26,202 SSR motifs, of which 30 were randomly selected for primer design using Primer3 software (Rozen and Skaletsky, 1999). The major parameters for primer pair design were set as follows: primer length of 15–25 bases, PCR product size of 100–400 bp, and annealing temperatures of 55–60°C.

The 30 target EST-SSR markers were initially tested for amplification using DNA from 24 S. tuberculata individuals from four natural populations located in different provinces across the distributional range in northern China (populations YA, XZ, YT, and KQ; Appendix 1). Total genomic DNA was extracted from silica gel–dried leaves using the Ezup DNA Extraction Kit (Sangon Biotech, Shanghai, China) following the manufacturer's protocol. PCRs were performed using the S1000 Thermal Cycler (Applied Biosystems, Foster City, California, USA) in a 25-µL total volume with 1 µL (∼10 ng) of genomic DNA, 12.5 µL of Taq PCR Mix (Sangon Biotech), 9.5 µL of ddH₂O, and 1 µL (5 pmol) of each primer. The PCR program consisted of 10 min of initial denaturation at 95°C; followed by 35 cycles of denaturation at 94°C for 45 s, annealing at specific temperature (58– 60°C; Table 1) for 1 min, extension at 72°C for 1 min; and a final extension at 72°C for 10 min. All PCR products were run on 1% agarose gels to check for successful amplification. Twenty primer pairs produced clear amplicons of the expected size ranges. Multiplex-Ready PCR technology (Hayden et al., 2008) was then applied for fluorescence-based SSR genotyping. Forward primers for the 20 successfully amplified loci were labeled with three different fluorescent dyes (6-FAM, HEX, and NED; Applied Biosystems; Table 1) and used for amplifications with the same protocol. The labeled PCR products were analyzed on an ABI 3730 DNA Analyzer with a GeneScan 500 LIZ Size Standard (Applied Biosystems). Allele sizes were called using GeneMarker version 2.6.0 (SoftGenetics, State College, Pennsylvania, USA). Number of alleles per locus (A), observed heterozygosity (H_o), and expected heterozygosity (H_e) were calculated using GenAlEx version 6.2 (Peakall and Smouse, 2006).

Table 1.

Characteristics of the 18 polymorphic microsatellite markers developed for Speranskia tuberculata.

Table 2.

Genetic properties of the 18 novel polymorphic EST-SSR markers developed in four populations of Speranskia tuberculata.^a

Eighteen of the 20 candidate markers showed polymorphisms among the four populations of S. tuberculata. The corresponding sequences of these markers were deposited in GenBank (Table 1). The number of alleles per locus ranged from two to 11, H_e ranged from 0.187 to 0.827, and H_o ranged from 0.042 to 0.917 (Table 2).

Cross-species amplification of the 18 newly developed polymorphic markers was tested in 24 S. cantonensis individuals from a single population (Ruyuan, Guangdong; Appendix 1), using the same procedures described above. Thirteen loci (72.22%) were successfully amplified in all S. cantonensis individuals tested, of which six showed polymorphisms (Table 3).

CONCLUSIONS

These 18 novel polymorphic SSR markers will be used to evaluate impacts of recent habitat fragmentation on the genetic diversity and structure of S. tuberculata, and to develop suitable conservation strategies for the species. Of these SSR markers developed in S. tuberculata, 13 were successfully amplified in single population samples of the related species S. cantonensis, extending their potential usefulness for future research in the genus Speranskia (e.g., comparisons of genetic diversity).

Table 3.

Polymorphisms at the 13 successfully cross-amplified EST-SSR markers in single population samples of Speranskia cantonensis (N = 24).^a