Metasequoia glyptostroboides H. H. Hu & W. C. Cheng (Cupressaceae), the dawn redwood, is a relict conifer of the genus Metasequoia H. H. Hu & W. C. Cheng. Its natural population has been found only in highly restricted regions bordering Hubei, Hunan, and Chongqing provinces in China (Farjon, 2001). This tree species is valued for its essential oils and crude extracts, which have great potential for antifungal activity. Moreover, this conifer is widely used as an ornamental tree in eastern China. Given its limited natural population size, M. glyptostroboides has been listed as an endangered species by both the Chinese government and the International Union for Conservation of Nature (Li et al., 2005).
Low genetic diversity was previously found in both wild and artificial populations of M. glyptostroboides using random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs) (Chen et al., 2003). Nevertheless, dominant markers, including RAPDs and AFLPs, cannot provide reliable estimates of genetic diversity because they are unable to distinguish heterozygotes from homozygotes (Nybom, 2004). This drawback can be overcome using simple sequence repeats (SSRs). In contrast to RAPDs and AFLPs, SSRs are typically studied separately at each identified locus and can be regarded as codominant markers (Nybom, 2004). Cui et al. (2010) developed 11 polymorphic microsatellite markers with a high polymorphism information content (PIC) by sequencing a microsatellite-enriched library. In recent years, with increasing exploration of expressed sequence tags (ESTs), EST-derived SSRs (EST-SSRs) have emerged as useful tools for estimating functional variation (Andersen and Lübberstedt, 2003; Varshney et al., 2007; Zalapa et al., 2012). Additional genetic markers, especially codominant EST-SSRs, are valuable and will be critical for resolving finer genetic variation patterns and developing functional studies and conservation strategies for this endangered species.
Zhao et al. (2013) generated 1,571,764 high-quality reads (assembled into 97,565 unigenes) from vegetative and female buds by transcriptome sequencing using 454 pyrosequencing technology, which provides a large amount of sequence information for microsatellite mining. In this study, we referenced the transcriptome sequences and developed the first set of EST-SSR markers for M. glyptostroboides.
METHODS AND RESULTS
The 97,565 unigene sequences were downloaded from http://www.genome.clemson.edu/node/273 (presented by the transcriptome sequencing project) (Zhao et al., 2013) and used for subsequent microsatellite mining. We first detected the microsatellite sequences from the unigene data set and identified unique microsatellites through an all-against-all BLAST search. Finally, we successfully designed primer pairs for 2087 unique EST-SSR loci. A bioinformatics analysis was implemented for microsatellite development and primer design using QDD version 3.1 (Meglécz et al., 2014). We selected 96 di- or trinucleotide loci with large numbers of repeats for primer synthesis and tested them in eight mature individuals collected from the Beijing Botanical Garden with permission. The sampled trees were introduced separately from natural populations in three counties (Lichuan [108.56°N, 30.18°E], Shizhu [28.28°N, 108.58°E], and Longshan [29.59°N, 106.50°E]) in the 1950s. Voucher specimens were deposited in the herbarium at Beijing Forestry University (accession no.: BFU-shuishan201403). Genomic DNA was extracted from the leaves of eight individuals using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). Functional annotations were prepared for each EST-SSR locus with polymorphisms detected by querying the Pfam protein families database (Finn et al., 2014).
The 96 selected primer pairs were validated by PCR using the M13-tail technique (Schuelke, 2000) to test for polymorphisms. Three primers were synthesized for each genotyping experiment: a 5′ M13-tailed forward primer, a reverse primer, and a fluorescently labeled M13 primer. M13 primers carrying FAM, HEX, TAMRA, and ROX (BGI, Beijing, China) labels were used separately. Each PCR mixture contained 5 µL of 2× MIX (New Industry, Beijing, China), 10–20 ng of template DNA, 1.6 pmol of reverse primer, 1.6 pmol of single fluorescently labeled M13 primer, and 0.4 pmol of forward primer in a final volume of 10 µL. PCR was performed as follows in a thermal cycler: 94°C for 5 min; 28 cycles of 94°C for 40 s, annealing at 55°C for 40 s, and elongation at 72°C for 45 s; 10 cycles of 94°C for 40 s, annealing at 53°C for 40 s, and elongation at 72°C for 45 s; and a final extension at 72°C for 10 min. The products were analyzed using an ABI 3730 Prism Genetic Analyzer (Applied Biosystems, Foster City, California, USA).
Characteristics of 23 polymorphic EST-SSR loci developed for Metasequoia glyptostroboides.a
The raw data were analyzed using GeneMarker version 2.62 (SoftGenetics, State College, Pennsylvania, USA). The number of alleles, observed heterozygosity (Ho), and expected heterozygosity (He) were calculated using GenAlEx version 6.5 (Peakall and Smouse, 2012). The allelic PIC was also calculated for each locus using CERVUS version 3.0 (Kalinowski et al., 2007).
Of the 96 loci, 53 demonstrated the expected fragment sizes; the remaining loci failed to amplify any product. After capillary electrophoresis scoring, 42 of the 53 loci showed a clear, single peak for each allele, of which 23 (53.49%) were polymorphic and 19 were monomorphic. The EST-SSR loci evaluated did not overlap with putative polymorphic SSRs described in a previous study that detected SSR loci from the same transcriptome data set (Zhao et al., 2013). The primer sequences, repeat motifs, sizes, and functional annotations from Pfam are shown in Table 1. The number of alleles from these 23 polymorphic EST-SSRs varied from two to eight (average: 3). The Ho, He, and PIC ranged from 0 to 1.0, 0.117 to 0.813, and 0.110 to 0.789, respectively (Table 2). The mean He was 0.551, which is higher than that estimated using RAPDs (Chen et al., 2003). Indicators including the percent of polymorphic loci and He suggest considerable genetic variability for this endangered conifer. Target sequences for the 23 microsatellite loci are attached as Appendix S1 (apps.1500038_s1.docx).
Genetic properties of 23 polymorphic EST-SSR loci developed for Metasequoia glyptostroboides.a