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11 September 2015 Development of 23 Novel Polymorphic EST-SSR Markers for the Endangered Relict Conifer Metasequoia glyptostroboides
Yuqing Jin, Quanxin Bi, Wenbin Guan, Jian-Feng Mao
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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.


The 97,565 unigene sequences were downloaded from (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).

Table 1.

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).

Table 2.

Genetic properties of 23 polymorphic EST-SSR loci developed for Metasequoia glyptostroboides.a



We developed the first set of EST-SSR markers for M. glyptostroboides. The 23 polymorphic loci reported here will facilitate finer estimation of the genetic diversity and population structure of this species, as well as the development of functional studies and conservation strategies.



J. R. Andersen , and T. Lübberstedt . 2003. Functional markers in plants. Trench in Plant Science 8: 554–560. Google Scholar


X. Y. Chen , Y. Y. Li , T. Y. Wu , X. Zhang , and H. P. Lu . 2003. Size-class differences in genetic structure of Metasequoia glyptostroboides Hu et Cheng (Taxodiaceae) plantations in Shanghai. Silvae Genetica 52: 107–109. Google Scholar


M. Y. Cui , S. Yu , M. Liu , and Y. Y. Li . 2010. Isolation and characterization of polymorphic microsatellite markers in Metasequoia glyptostroboides (Taxodiaceae). Conservation Genetics Resources 2: 19–21. Google Scholar


J. J. Doyle , and J. L. Doyle . 1987. A rapid DNA isolation procedure for small quantities of leaf tissue. Phytochemical Bulletin 19: 11–15. Google Scholar


A. Farjon 2001. World checklist and bibliography of conifers. Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom. Google Scholar


R. D. Finn , A. Bateman , J. Clements , P. Coggill , R. Y. Eberhardt , S. R. Eddy , A. Heger , et al. 2014. Pfam: The protein families database. Nucleic Acids Research 42: D222–D230. Google Scholar


S. T. Kalinowski , M. L. Taper , and T. C. Marshall . 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16: 1099–1106. Google Scholar


Y. Y. Li , X. Y. Chen , X. Zhang , T. Y. Wu , H. P. Lu , and Y. W. Cai . 2005. Genetic differences between wild and artificial populations of Metasequoia glyptostroboides Hu et Cheng (Taxodiaceae): Implications for species recovery. Conservation Biology 19: 224–231. Google Scholar


E. Meglécz , N. Pech , A. Gilles , V. Dubut , P. Hingamp , A. Trilles , R. Grenior , and J.-F. Martin . 2014. QDD version 3.1: A user-friendly computer program for microsatellite selection and primer design revisited: Experimental validation of variables determining genotyping success rate. Molecular Ecology Resources 14: 1302–1313. Google Scholar


H. Nybom 2004. Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Molecular Ecology 13: 1143–1155. Google Scholar


R. Peakall , and P. E. Smouse . 2012. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research—An update. Bioinformatics (Oxford, England) 20: 1–3. Google Scholar


M. Schuelke 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18: 233–234. Google Scholar


R. K. Varshney , K. Chabane , P. S. Hendre , R. K. Aggarwal , and A. Graner . 2007. Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys. Plant Science 173: 638–649. Google Scholar


J. E. Zalapa , H. Cuevas , H. Y. Zhu , S. Steffan , D. Senalik , E. Zeldin , B. McCown , et al. 2012. Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. American Journal of Botany 99: 193–208. Google Scholar


Y. Zhao , S. M. Thammannagowda , S. Tang , X. Xia , W. Yin , and H. Liang . 2013. An EST dataset for Metasequoia glyptostroboides buds: The first EST resource for molecular genomics studies in Metasequoia. Planta 237: 755–770. Google Scholar


[1] This work was supported by the Fundamental Research Funds for the Central Universities (no. YX2013-41).

Yuqing Jin, Quanxin Bi, Wenbin Guan, and Jian-Feng Mao "Development of 23 Novel Polymorphic EST-SSR Markers for the Endangered Relict Conifer Metasequoia glyptostroboides," Applications in Plant Sciences 3(9), (11 September 2015).
Received: 3 April 2015; Accepted: 1 June 2015; Published: 11 September 2015

454 pyrosequencing
Metasequoia glyptostroboides
relict plant
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