Lancea tibetica Hook. f. & Thomson (Phrymaceae) is an herb endemic to the Qinghai–Tibet Plateau. It usually grows in grasslands, sparse forests, or ravines at altitudes of 2000–4500 m. As a traditional Tibetan medicinal plant, it has been used in the treatment of leukemia, intestinal angina, heart disease, and cough (Hong et al., 1998). Investigations into the chemical constituents of L. tibetica have resulted in the isolation of phenylpropanoid glycosides and lignans, which contribute to the species' antioxidant effects (Song et al., 2011). To increase production of traditional medicine from this species, the harvest of wild populations has been greatly expanded. The serious depletion of L. tibetica through over-collecting has led to a need for proper management and a conservation plan to ensure its sustainable use into the future. A thorough study at the population level is required to evaluate the extent of remaining genetic resources and to inform management plans.

Simple sequence repeat (SSR) markers are widely used for population genetic studies due to their codominant nature, polymorphisms, and reproducibility (Litt and Luty, 1989). The development of SSR markers for L. tibetica will enable us to assess genetic diversity and contribute to a conservation strategy. The restriction-site associated DNA (RAD) method was proposed by Miller et al. (2007) as a reliable approach that reduces genome complexity. RAD sequencing has been successfully applied in many organisms, including crop species like barley (Chutimanitsakun et al., 2011). In this paper, we describe the process of isolation and characterization of 10 polymorphic SSR markers from L. tibetica based on RAD sequencing.

METHODS AND RESULTS

Plant materials and DNA extraction—In total, 56 L. tibetica individuals from three natural populations (YD, QML, and MY) were sampled (Appendix 1). Fresh leaves were collected and dried in silica gel; the voucher specimens are deposited in the Herbarium of the Northwest Institute of Plateau Biology (HNWP), Chinese Academy of Sciences, Xining, Qinghai Province, China. Total genomic DNA was extracted from dried leaves with the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1987).

RAD library preparation and sequencing—We selected one individual from each of the populations and pooled them. Subsequently, the RAD library was constructed based on published methods (Barchi et al., 2011). The library was quantified with Qubit (Invitrogen, Eugene, Oregon, USA) and sequenced using the Illumina MiSeq platform (Illumina, San Diego, California, USA). Before doing any further analysis, quality control and filtering of raw data were performed to detect whether the raw reads were of high enough quality, following Zhang et al. (2014). After that, clean reads were clustered using CD-HIT-EST (Li and Godzik, 2006) and assembled de novo using VelvetOpt (Zerbino and Birney, 2008). Sequencing produced 2,764,204,500 bp of clean reads after quality control from 2,800,948,250 bp of raw reads. We obtained 1,417,277 cluster tags, but only 222,628 cut cluster tags. We obtained 401,203 high-quality contigs, with an average size of 265 bp (N50 = 361) through de novo assembly.

Table 1.

Characteristics of 38 microsatellite loci developed in Lancea tibetica.

Continued.

Subsequently, we identified the SSR repeats from the assembled contigs using Trimmomatic version 0.32 (Bolger et al., 2014) and set the parameters for detection of di-, tri-, tetra-, penta-, and hexanucleotide motifs with flanking regions in SSR pipeline version 0.951 (Miller et al., 2013). A total of 4441 perfect SSR repeats from the assembled contigs were obtained in the study. Among them, the numbers of di-, tri-, tetra-, penta-, and hexanucleotide repeats were 2026, 2081, 220, 73, and 41, respectively.

SSR primer design and genetic diversity analysis—SSR primers were designed using Primer3web (Untergasser et al., 2012) for the SSR sequences. Primers were designed according to the following criteria: amplified regions within a size range of 100–200 bp, primer annealing temperature range 55.0– 62.0°C, and GC content range 45–60%. Different repeat motifs of SSR sequences were arbitrarily selected to design primers to obtain 100 pairs of qualified SSR primers. PCRs were performed with all 56 samples, with a 30-µL reaction mixture: 20–30 ng of template DNA, 5 µL 10× PCR buffer (15 mM MgCl₂), 1.5 µL of each primer (5 pM), 1.0 µL Taq DNA polymerase (TaKaRa Biotechnology Co., Dalian, China), 0.5 µL dNTP mix (10 mM), and supplemented with ddH₂O. The PCR program included the following steps: 94°C for 5 min, one cycle; 94°C for 35 s at the appropriate annealing temperatures (annealing temperatures for each specific primer pairs are given in Table 1) for 35 s; 72°C for 30 s, 35 cycles; 72°C for 10 min, one cycle. PCR products were visualized on 1.0% agarose gels with ethidium bromide. Of the 100 pairs of SSR primers tested, 38 amplified successfully (Table 1). These 38 primer pairs were used for PCR amplification in all 56 samples to detect polymorphism. PCR conditions are the same as those described above. PCR products were applied on agarose and then separated on 12% w/v nondenaturing polyacrylamide gels (PAGE) following Wang et al. (2014), with DL500 DNA Marker (TaKaRa Biotechnology Co.). We calculated the inbreeding coefficient (F_IS), total number of alleles per locus (A), observed heterozygosity (H_o), expected heterozygosity (H_e), null allele frequency (r), and deviations from Hardy–Weinberg equilibrium (HWE) using GENEPOP version 4.4 (Rousset, 2008).

Table 2.

Results of initial primer screening of 10 polymorphic loci in three Lancea tibetica populations.

After PAGE analysis, 10 pairs of SSR primers were found to be highly polymorphic among the three populations of L. tibetica; the other 28 showed no significant difference. A ranged from three to eight. H_o and H_e ranged from 0.200 to 1.000 and from 0.683 to 0.879 (Table 2), respectively, which indicates that genetic diversity in this species is relatively high. Additionally, r ranged from 0.000 to 0.307. Some loci (LT25 in population YD, LT4 in population QML, LT7 and LT9 in population MY) showed a significant departure from HWE, which could be caused by the presence of null alleles (Chapuis and Estoup, 2007).

There are just two species in the genus Lancea Hook. f. & Thomson, L. tibetica and L. hirsuta Bonati. We tested cross-amplification in L. hirsuta for all of the polymorphic primers developed for L. tibetica. Lancea hirsuta is distributed in northwestern Sichuan and northwestern Yunnan, China. We sampled five individuals from Xinduqiao (voucher no. Zhang2015569; geographic coordinates: 30°04′N, 101°29′E; altitude: 3496 m), Sichuan, China. All of the polymorphic primers were successfully amplified in L. hirsuta with the same PCR conditions used for L. tibetica, except for marker LT28.

CONCLUSIONS

In this study, we present the first report on L. tibetica SSR marker development based on RAD sequences. A total of 4441 SSR markers were identified at the genome-wide level. The 10 SSR loci that displayed polymorphisms among L. tibetica populations also have the potential to be useful for population genetic studies on the closely related L. hirsuta.