Characterization of Polymorphic Microsatellite Markers Isolated from Genomic DNA of Elaeocarpus decipiens Hemsly (Elaeocarpaceae)

The development of compound microsatellite markers was conducted in Elaeocarpus decipiens to investigate genetic diversity and population genetic structure of this species. Eighteen microsatellite markers that were successfully amplified showed polymorphism when tested on 35 individuals from three populations in Chinese mainland. Overall, the number of alleles per locus ranged from 4 to 11, with an average of 7.06 alleles per locus. These results indicate that these microsatellite markers are adequate for detecting and characterizing population genetic structure and genetic diversity in E. decipiens. Of these primers, only four could be successfully transferred to E. sylvestris and E. japonicus. Journal of Data Mining in Genomics & Proteomics J o u r n a l of D ata Mi ning in Gmics & rot e o m i c s ISSN: 2153-0602 Citation: Gong X, Ge G (2013) Characterization of Polymorphic Microsatellite Markers Isolated from Genomic DNA of Elaeocarpus decipiens Hemsly (Elaeocarpaceae). J Data Mining Genomics Proteomics 4: 141. doi:10.4172/2153-0602.1000141


Introduction
Elaeocarpus decipiens is an evergreen, broad-leaved, woody species of the Elaeocarpaceae family with a disjunct distribution in south of Chinese mainland, the Ryukyu Archipelago and Taiwan. Currently, most of the efforts have been focused on the germplasm, breeding and cultivation of this species [1]. The study of population genetic diversity, population genetic structure and population ecology of this species is insufficient and limited. However, population genetic analysis of this disjunct plant will potentially provide insights into the geographic structure of genetic diversity that reflects the evolutionary history of E. decipiens. To assess gene flow across the populations and to infer biogeographic patterns, we developed microsatellite markers for this species, for which none were available previously. Additionally, these loci were tested for cross-amplification in E. sylvestris and E. japonicas.

Materials and Methods
Genomic DNA of E. decipiens was extracted from fresh leaves using a modified CTAB (cetyltrimethyl ammonium bromide) method [2]. An adaptor-ligated DNA library was constructed following the protocol of Lian et al. [3]. Briefly, total genomic DNA (10 μg) was digested with a blunt-end restriction enzyme, EcoRV (Takara, Dalian, Liaoning, China), and the restricted fragments were ligated to an unequal-length adaptor, using DNA Ligation Kit Version 2.0 (Takara, Dalian, Liaoning, China). Then, fragments flanked by a microsatellite at one end were amplified from the EcoRV DNA library using compound SSR primer (AC) 6 (AG) 5 and an adaptor primer AP2 (5′-CTATAGGGCACGCGTGGT-3′). The recovered DNA was ligated into a pGEM-T vector (Promega, Madison, Wisconsin, USA), and transformed into DH5α competent cells (Takara, Dalian, Liaoning, China). Transformants were cultured on selective agar media with ampicillin, X-Gal and IPTG, for blue/white colony selection. After PCR-tested for insert size of the white colonies, a total of 144 clones were found to contain (AC) 6 (AG) n compound SSR motifs. 55 sequences were too short to design primer. And 89 clones proved suitable for primer design using PREMIER version 5.0 [4]. These primers were tested for polymorphism in E. decipiens. A total of 53 out of the 89 primer pairs tested successfully amplified the target fragments. PCR was performed in 10-μL reaction volumes containing 30-50 ng/ μL of template DNA, 0.25 unit Taq DNA polymerase (TaKaRa, Dalian, Liaoning, China), 1 μL 10×PCR buffer, 0.5 μL of 2.5 mM MgCl 2 ,1 μL of 2.5 mM dNTPs, 0.05 μL bovine serum albumin (BSA) (TaKaRa, Dalian, Liaoning, China), and 0.6 μL of each 10 μM primer. The thermal profile used was initial denaturing for 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 45 s of annealing at the optimized annealing temperature (Table 1), 1 min 30 s of elongation at 72°C, ending with a 10-min extension at 72°C. The forward primer of each pair was labeled with a fluorescent dye (6-FAM). Products were resolved using an ABI 3730 sequencer (Applied Biosystems), along with a fluorescently labeled internal size standard (GeneScan 500 LIZ Size Standard; Applied Biosystems), and the samples were genotyped using GENEMAPPER version 4.0 (Applied Biosystems).

Results
Eighteen out of the 53 loci were identified as polymorphisms and generated consistent amplification products of the expected size range (Table 1)

Conclusion
The approach used in this study substantially reduces time in comparison with the FIASCO (Fast Isolation by APLR of Sequences Containing Repeats) protocol. Because a common fluorescent compound SSR primer can be used in polymorphism analyses for different loci and different species and the fluorescent primer is rather expensive, this may save investigation costs [7]. These polymorphic microsatellite markers of E. decipiens should represent a useful tool to assess patterns of geographical molecular variation in E. decipiens at the population level, and across the species' ranges in south of Chinese mainland, Taiwan and the Ryukyu Archipelago. Moreover, studies have shown that microsatellite primers developed in one species could be cross-amplified in related taxa [8]. However, only three and four loci were successfully amplified in E. japonicus and E. sylvestris, respectively. Even so, cross-species amplification in E. sylvestris and E. japonicus has opened an opportunity for comparative studies among these species.
In addition, the use of these markers will facilitate the follow up introgression of favorable variation from E. sylvestris and E. japonicus into E. decipiens.