Development of chloroplast microsatellite markers for Glyptostrobus pensilis (Cupressaceae)

Premise Glyptostrobus pensilis (Cupressaceae) is a critically endangered conifer native to China, Laos, and Vietnam, with only a few populations remaining in the wild. Methods and Results Using a complete chloroplast genome sequence, we designed 70 cpSSR loci and tested them for amplification success and polymorphism in 16 samples. Ten loci were found to be polymorphic and their genetic diversity was characterized using a total of 83 individuals from three populations in China. A total of 43 haplotypes were present, the effective number of haplotypes varied from 4.55 to 13.36, and the haplotypic richness ranged from 8.04 to 16.00. Gene diversity ranged from 0.81 to 0.97 (average 0.89). The number of alleles per locus and population ranged from one to eight, and the effective number of alleles ranged from 1.00 to 3.90. All polymorphic loci were successfully amplified in the related species Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata. Conclusions These newly developed chloroplast microsatellites will be useful for population genetic and phylogeographic analyses of G. pensilis and related species.

microsatellite loci exhibiting a minimum of eight repeats as these loci are likely to exhibit a higher level of polymorphism (Ueno et al., 2012). For loci with a minimum of eight repeats, primers were designed using the online software Primer3Plus (Untergasser et al., 2007) using default parameters. In total, 70 cpSSR loci were selected and evaluated for their amplification efficiency and level of polymorphism using 16 G. pensilis DNA samples from different populations (Appendix 1). DNA was extracted from G. pensilis leaves using a modification of the cetyltrimethylammonium bromide (CTAB) method (Tsumura et al., 1995).
PCR amplification was carried out in volumes of 15 μL using the following protocol: 7.5 μL of 2× Taq PCR Master Mix (Tiangen, Beijing, China), 0.75 μL of forward primer (10 μM), 0.75 μL of reverse primer (10 μM), 3 μL of 20-50 ng/μL DNA template, and 3 μL of ddH 2 O. The mixture was then cycled using the following profile: 94°C for 4 min; 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; with a final extension at 72°C for 30 min. PCR products were visualized on a 1.6% agarose gel. All loci that could be amplified successfully were tested individually using 16 G. pensilis samples to establish their polymorphism. These amplifications were carried out using fluorescently labeled primers (FAM, HEX, TAMRA, and ROX; Applied Biosystems, Foster City, California, USA) and the same PCR protocol as detailed above. PCR products were run on an ABI 3730xL DNA Analyzer adding a GeneScan 500 LIZ internal size standard (Applied Biosystems) to size fragments. The software GeneMarker version 1.9 was used to score the electropherograms of all samples (Hulce et al., 2011). Sixty-five of the 70 cpSSR loci amplified successfully across the 16 test individuals, but 55 loci were monomorphic, and only 10 loci were polymorphic (Table 1, Appendix 2). These polymorphic loci were used to investigate the genetic diversity of 83 individuals across three Chinese G. pensilis populations (Appendix 1).
The software Haplotype Analysis version 1.05 (Eliades and Eliades, 2009) was used to calculate the following statistics: number of haplotypes (A), number of private haplotypes (P), effective number of haplotypes (N e ), haplotypic richness (R h ), and gene diversity (H e ). The software GenAlEx6.5 (Peakall and Smouse, 2012) was used to calculate the following parameters: number of alleles (N a ), effective number of alleles (N e ), Shannon's information index (I), and diversity (H).
A total of 43 haplotypes were detected in the three assayed populations. The number of haplotypes per population ranged from 11 to 18, the number of private haplotypes ranged from nine to 16, the effective number of haplotypes ranged from 4.55 to 13.36, the haplotypic richness ranged from 8.04 to 16.00, and the gene diversity ranged from 0.81 to 0.97 ( Table 2). The number of alleles per locus ranged from one to eight per population, the effective number of alleles ranged from 1.00 to 3.90, Shannon's information index ranged from 0.00 to 1.52, and the diversity ranged from 0.00 to 0.74 (Table 3). The 10 polymorphic loci could also be successfully amplified in five individuals in each of the following four related species: Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata (Table 4, Appendix 1).

CONCLUSIONS
In this study, we developed 10 polymorphic cpSSRs (as well as 55 pairs of monomorphic primers) that can be used to assess the     Numbers shown represent the size in base pairs of the amplified fragments.