Development of polymorphic simple sequence repeat markers in Huperzia serrata (Lycopodiaceae)

Premise The natural population size of Huperzia serrata (Lycopodiaceae) has dramatically decreased and the species has become endangered due to overexploitation. Here, we developed simple sequence repeat (SSR) markers for H. serrata to survey both its genetic diversity and population structure. Methods and Results Based on 177 individuals, 120 SSR primer pairs were developed and optimized from five regions of the H. serrata transcriptomic data. Of these primer pairs, 20 were successfully amplified and 10 showed obvious polymorphism. These polymorphic loci were investigated to study the genetic diversity of H. serrata. Two to 11 alleles per locus were identified, the level of observed heterozygosity ranged from 0.00 to 1.00, and the level of expected heterozygosity ranged from 0.19 to 0.79. All loci were successfully amplified in H. crispata, H. sutchueniana, and H. selago. Conclusions The 10 polymorphic primer pairs developed here will be valuable for studies of the endangered H. serrata and other related species.

Huperzia serrata (Thunb.) Trevis., also named qiancengta in China, is a member of the Lycopodiaceae (Almeida, 2016). More than 90% of H. serrata species are distributed east of the Hengduan Mountains and south of the Tsinling Mountains-Huai River line, in an area with a subtropical monsoon climate. Huperzia serrata is a valuable medicinal plant because it contains the alkaloid huperzine A, which has been shown to effectively attenuate cognitive deficits and has been used for the treatment of Alzheimer's disease (Lei et al., 2015). The rapidly growing demand for natural H. serrata and the unrestricted and continuous harvesting has led to the rapid extinction of wild resources. Therefore, many scientists have begun to conduct research on H. serrata, including the study of its cultivation and reproduction physiology. However, only a small number of markers (3459 expressed sequence tags) are currently available in the National Center for Biotechnology Information (NCBI), and the current research on H. serrata is still limited, especially with regard to molecular information.
Simple sequence repeat (SSR) markers have been widely used as effective genetic markers for plant breeding and genetic applications (Sharma et al., 2009), and have been applied to genetic diversity analysis, genetic map construction, molecular breeding, and germplasm conservation (Kumar et al., 2015). Luo et al. (2010) discovered thousands of SSR loci in H. serrata and selected 10 SSR sequences, including several candidate gene-encoding enzymes involved in bioactive compound biosyntheses, for further detection and verification. However, no optimum SSR loci for the study of genetic diversity and population structure in H. serrata have been reported. In this study, microsatellite markers were developed based on the H. serrata transcriptome, which will help to investigate the reproductive characteristics of H. serrata, evaluate its evolutionary potential, and develop a reasonable strategy for its protection, development, and utilization.

METHODS AND RESULTS
In this study, 177 H. serrata individuals were collected from the Chinese localities of Luan, Enshi, Jizhou, Hanzhong, and Jinping (Appendix 1). Total RNA was extracted from 100 mg of fresh leaves using TRIzol following the instructions of the manufacturer (TIANGEN, Beijing, China). To eliminate potential DNA contamination, we used DNase to purify total RNA following the manufacturer instructions (QIAGEN, Hilden, Germany). RNA purity and concentration were determined by NanoDrop Spectrophotometer (Qubit2.0, Agilent 2100; Shimadzu, Kyoto, Japan). mRNA was isolated using magnetic oligo (dT) beads, and then cut into short fragments using NEBNext Poly (  Biolabs, Ipswich, Massachusetts, USA). First-strand cDNA synthesis used random hexamer primers, buffer, dNTPs, and RNase H, and second-strand cDNA was synthesized by supernumerary DNA polymerase I. The total high-quality RNA was used to construct the cDNA library. Then, the cDNA library of H. serrata was sequenced based on synthesis by sequencing (SBS) technology using the Illumina HiSeq2500 Sequencing platform (Illumina, San Diego, California, USA). After trimming the sequencing linker and primer sequences in reads and after filtering low-quality data to ensure data quality, high-quality sequences were de novo assembled into transcript and unigenes. Furthermore, reads were divided into 25-bp (k-mer) segments using Trinity software (Grabherr et al., 2011). The final assembly was composed of 111,251 unigenes and had an N50 size of 997 bp.
To analyze the genetic diversity of H. serrata, annotated unigenes were used to identify SSRs. The identification and localization of SSRs were performed using the MIcroSAtellite Identification Tool (MISA; Thiel et al., 2003). A total of 4395 SSR loci were found by MISA in 3685 unigenes (24.7%), which was higher than previously reported for H. serrata (Luo et al., 2010) and for bryophytes such as Physcomitrella patens (Hedw.) Bruch & Schimp. (6.3%) (Kobayashi and Morita, 2005). A relatively high frequency of repeats with diand trinucleotides was detected in H. serrata (Appendix S1).
Due to short flanking sequences of the SSR loci or inappropriate sequences, only 2064 loci could be used for the design and validation of primer pairs. To investigate the genetic diversity of H. serrata, 120 SSR makers were randomly selected and synthesized. DNA amplification was performed with Ex Taq (TaKaRa Biotechnology Co., Beijing, China) following the manufacturer's instructions. PCR amplification conditions were as follows: 95°C for 2 min; 35 cycles at 95°C for 30 s, 45.8-66.8°C (depending on the primer pair) for 30 s, 72°C for 30 s; and a final extension for 5 min at 72°C. PCR products were detected by 1.5% (w/v) agarose gel electrophoresis and 8.0% (w/v) non-denaturing polyacrylamide gel electrophoresis. SSR primer pairs that produced clear and reproducible polymorphic bands were used to detect alleles via capillary electrophoresis.
There were 20 primer pairs with expected sizes as well as high specificity, amplification efficiency, and repeatability that were successfully amplified by PCR (Table 1). Furthermore, we used the NCBI database to align the selected SSR markers, and annotated their functions separately. Among these 20 primer pairs, 10 primer pairs exhibited monomorphism and were not studied further, and 10 polymorphic primer pairs were used to evaluate the polymorphism information in five populations of H. serrata, identifying a total of 72 alleles. The polymorphism information content (PIC) value for SSR primers ranged from 0.313 (primer c52211) to 0.730 (primer c51797) with an average of 0.568; this value was higher than that found in other plants (e.g., Sesamum indicum L. [Cho et al., 2011]). The level of expected heterozygosity of the genetic diversity ranged between 0.06 and 0.79 and the level of observed heterozygosity ranged from 0.00 to 1.00 (Table 2). Eight SSR markers had levels of expected heterozygosity above 0.5, indicating a high level of polymorphism in H. serrata.
Cross-species amplification of 10 microsatellite primers was tested on DNA extracts in three related species: H. crispata (Ching) Ching, H. sutchueniana (Herter) Ching, and H. selago (L.) Bernh. Ten loci were successfully amplified in three related species and were shown to be polymorphic (Table 3).

CONCLUSIONS
This study successfully developed 10 polymorphic primers from H. serrata and assessed their transferability in related species. The selected polymorphic microsatellites are valuable for the study of wild H. serrata resources with regard to its genetic diversity, population structure, and evolution.  Chi-square test for Hardy-Weinberg equilibrium. Locus showed significant deviations from Hardy-Weinberg equilibrium (P < 0.001).  graph_c0 193-197 188-197 193-197 c51797.graph_c0 194-207 197-200 193-209  Voucher and locality information are provided in Appendix 1.