Development of EST‐SSR markers in Saxifraga sinomontana (Saxifragaceae) and cross‐amplification in three related species

Premise Saxifraga sinomontana (Saxifragaceae) is a widespread alpine species in the Qinghai–Tibetan Plateau and its flanking mountains. We developed a set of expressed sequence tag–simple sequence repeat (EST‐SSR) markers to investigate the genetic diversity and evolutionary history of the species. Methods and Results We initially designed 50 EST‐SSR markers based on transcriptome data of S. sinomontana. Nineteen of 50 loci (38%) were successfully amplified, 13 of which were polymorphic. These were tested on 71 individuals from four populations. Three to 18 alleles per locus were detected, and the levels of observed and expected heterozygosity ranged from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively. In addition, cross‐amplification was successful for all 13 loci in three congeneric species, S. tangutica, S. heleonastes, and S. congestiflora. Conclusions These EST‐SSR markers will be useful for studying the genetic diversity of S. sinomontana and disentangling the phylogenetic relationships of related species.

We performed a preliminary screen of 50 EST-SSR primers using three individuals each from four populations of S. sinomontana (Appendix 1). Total genomic DNA was extracted from silica-dried leaves using the modified cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1987). PCR reac tions were carried out in a total volume of 25 μL containing 2.0 μL of total genomic DNA (10-20 ng), 0.3 μL of each primer (10 pM), 0.3 μL (1.5 units) of Taq polymerase, 2.5 μL of 10× PCR buffer (with Mg 2+ ), and 2.5 μL of 10 mM dNTPs. The PCR profile included an initial pretreatment of 10 min at 94°C; followed by 35 cycles of 1 min denaturation at 94°C, 50 s at locus-specific annealing temperatures (Table 1), and 1 min elongation at 72°C; and a final extension at 72°C for 10 min. The PCR products were screened using 1% agarose electrophoresis to determine whether amplifications were successful for the expected sizes and then sep arated on 6% polyacrylamide gels. Overall, 19 of 50 (38%) EST-SSR primer pairs produced clear, unique amplification products of the expected size. Of these, 13 loci were polymorphic across populations. Characteristics of all 19 loci are listed in Table 1. For all 13 polymorphic SSR loci, the 5′ end of each forward primer was labeled with one of three fluorescent dyes (FAM, HEX, or ROX; Table 1). PCR amplifications were then performed using 71 individuals from four populations of S. sinomontana with the same protocol described above ( Table 2). The fluorescently tagged PCR products were analyzed on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA) with a GeneScan 500 LIZ Size Standard (Applied Biosystems), and allele sizes were scored with GeneMapper version 3.2 (Applied Biosystems). Number of alleles per locus (A), observed het erozygosity, and expected heterozygosity were calculated with POPGENE version 1.32 (Yeh et al., 1999). Hardy-Weinberg equi librium was tested for each population using GENEPOP version 4.2 (Rousset, 2008). Using MICRO-CHECKER version 2.2 (van Oosterhout et al., 2004), we found no evidence of null alleles across all loci.
Among the 13 polymorphic loci, A ranged from three to 18 (mean = 8). The Dingri population had the lowest mean values (A = 2) among the four populations. Levels of observed and ex pected heterozygosity varied from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively, which indicates that genetic diversity is rela tively high in this species. Additionally, a few loci showed signif icant deviations from Hardy-Weinberg equilibrium: three in the Aba population, two in the Chengduo population, and seven in the Dingri population (P < 0.05; Table 2).
All 13 EST-SSR markers also amplified successfully in S. tangutica, S. heleonastes, and S. congestiflora, using the same PCR proto col as for S. sinomontana (Table 3).

CONCLUSIONS
The 13 EST-SSR markers developed here showed high polymor phism in S. sinomontana and high cross-species amplification success. Hence, these are valuable loci for investigating genetic di versity, population structure, and evolutionary history in S. sinomontana and throughout Saxifraga.

DATA ACCESSIBILITY
Raw sequencing reads were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (BioProject SRR8365238). Sequence information for the developed primers has been deposited in NCBI's GenBank, and accession numbers are provided in Table 1.