Introduction
Epilobium, the largest genus of the family Onagraceae, is widely distributed at high altitudes and latitudes (Keating et al., Reference Keating, Hoch and Raven1982; Raven, Reference Raven, Gottlieb and Jain1988). Epilobium species have been used as medicinal plants owing to their benefits to human health. In the past few decades, there has been a great interest in the phytochemical composition of Epilobium species, which possess various pharmacological and therapeutic properties (Granica et al., Reference Granica, Piwowarski, Czerwinska and Kiss2014; Kadam et al., Reference Kadam, Patil and Yadav2018). Epilobium nankotaizanense Yamamoto (Onagraceae), an endemic herb in Taiwan, grows on mountain peaks above 3300 m in elevation, including the Xue, Nanhu, Cilai, Zhongyangjian and Guan Mountains. This species has special habitat preferences and commonly grows on loose rocky slopes in mountain peaks. Therefore, the distribution of this species is narrow, and the number of its population is small. This species is one of the four plant species protected by the Cultural Heritage Preservation Act in Taiwan and is listed as a vulnerable species (Editorial Committee of the Red List of Taiwan Plants, 2017). Climate change is expected to have severe effects on herbaceous species inhabiting alpine ecosystems, thereby reducing their habitats and population size, and consequently influencing their genetic diversity and population genetic structure (Huang et al., Reference Huang, Hsu, Wang, Liu, Chen, Chiu, Huang, Hung and Chiang2016). When species distribution ranges are reduced, the species that are sensitive to the loss of genetic diversity consequently lose their evolutionary potential to survive (Schierenbeck, Reference Schierenbeck2017). However, information related to genetic diversity in terms of the upper altitudinal limits of herbaceous species is limited, and more studies on this topic in Taiwan are urgently required.
Microsatellites or simple sequence repeats (SSRs) are a type of genetic marker suitable for this kind of research, as they have been extensively employed for assessing the population genetic structure of endangered species. They have become essential tools for germplasm collection and authentication of medicinal plants. To date, only one study using isozyme markers has been carried out to determine the level of genetic variation and differentiation within E. nankotaizanense, and the results showed a low level of heterozygosity and moderate genetic differentiation between different mountains (Chen, Reference Chen1997). In the present study, we developed a set of microsatellite markers for E. nankotaizanense using high-throughput genome sequencing data with the goal of facilitating future studies on the conservation genetics of E. nankotaizanense, as well as the increased application values of other Epilobium species in the future.
Experimental
Total genomic DNA was extracted from freeze-dried leaf material using the CTAB procedure (Doyle and Doyle, Reference Doyle and Doyle1987). A single high-quality DNA sample was selected for microsatellite development. Ten micrograms of DNA aliquot was used for library preparation. DNA was sonicated using a Misonix Sonicator 3000 (Misonix, NY, USA) to generate fragments of 400–500 bp, and the library was size-selected by gel electrophoresis. The Illumina MiSeq platform (Illumina, CA, USA) was used for sequencing and generating 250-bp paired-end reads. After removing adapters and low-quality reads, we obtained approximately 2,672,042 clean reads. SSR Locator software (Da Maia et al., Reference Da Maia, Palmieri, de Souza, Kopp, de Carvalho and Costa de Oliveira2008) was used to screen for SSR regions, and the parameters were adjusted to identify the di-, tri- and tetra-motifs with a minimum of five repeats. Microsatellite primers were designed using Primer3 software (Untergrasser et al., Reference Untergrasser, Cutcutache, Koressaar, Ye, Faircloth, Remm and Rozen2012) with the following settings: primer length 17–22 bp and product size 100–400 bp. Twenty microsatellites were randomly selected to determine polymorphisms within the species.
To confirm the polymorphisms of these 20 primer pairs, we tested 30 samples collected from the Xue, Nanhu and Cilai Mountains. PCR amplification was carried out with a reaction mixture of total volume 25 μl, containing 10 ng genomic DNA, 0.2 mM dNTP, 2 mM MgCl2 and 5 pmol of each primer. The PCR conditions were as follows: 3 min at 94°C, 25 cycles for 30 s at 94°C, 30 s at primer-specific annealing temperatures (online Supplementary Table S1), 30 s at 72°C and a final extension step of 5 min at 72°C. Electrophoresis was performed by denaturing 6% polyacrylamide gels with a 10-bp DNA ladder (Invitrogen, CA, USA) to estimate allele sizes. GenAlEx version 6.5 (Peakall and Smouse, Reference Peakall and Smouse2012) was used to calculate the following genetic parameters: number of alleles per locus (A), observed heterozygosity (Ho) and expected level of heterozygosity (He). We also evaluated the polymorphic information content (PIC) and carried out Hardy–Weinberg equilibrium (HWE) analysis using PowerMarker version 3.25 (Liu and Muse, Reference Liu and Muse2005) and GENEPOP version 3.4 (Raymond and Rousset, Reference Raymond and Rousset1995).
Results and discussion
The 20 primer pairs were successfully amplified to match the expected size, and they showed substantial levels of polymorphisms within E. nankotaizanense. These polymorphic microsatellite markers produced 2.000 to 3.000 alleles per locus, with an average of 2.300 (Table 1). The Ho and He levels ranged from 0.000 to 0.929 and from 0.034 to 0.631, with averages of 0.284 and 0.352, respectively. At the population level, 11 loci were fixed on the Cilai Mountain (EP05, EP08, EP12, EP53, EP60, EP68, EP70, EP75, EP80, EP81 and EP90), six loci were fixed on the Xue Mountain (EP53, EP68, EP70, EP80, EP81 and EP90), and two loci were fixed on the Nanhu Mountain (EP08 and EP75). Lower average Ho and He values were recorded in the Cilai Mountain than in the Xue and Nanhu Mountains. This implied a severe loss of genetic variation in the population in the Cilai Mountain, resulting from the lower population size. Significant deviations from the HWE were detected in EP05, EP48, EP86 and EP92 (P < 0.001). The PIC value for primer pairs ranged from 0.033 (EP90) to 0.556 (EP30), with an average of 0.290. Of the 20 microsatellites, two exhibited high polymorphism (PIC > 0.50), 10 exhibited medium polymorphism (0.50 > PIC > 0.25), and eight exhibited low polymorphism (PIC < 0.25). This study reported the successful development of 20 polymorphic microsatellite markers from E. nankotaizanense. These markers will be useful for studying the conservation genetics of E. nankotaizanense, with the aim of conservation and management of this vulnerable species, as well as identification of alternative sources of cross-species transferable markers for other Epilobium species.
Table 1. Detailed genetic diversity parameters for Epilobium nankotaizanense evaluated using the 20 developed microsatellite markers
For each locus, the number of alleles (A), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and P values of deviation from the Hardy–Weinberg equilibrium (HWE) were evaluated. A significant P values are indicated in bold (P < 0.01).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262121000289.
Acknowledgements
We thank the Ministry of Science and Technology of Taiwan (MOST 103-2313-B-020-005) for their financial support of this study.
