Develop Microsatellite Markers for Ginkgo biloba L. Clones

Gingko biloba is a tree species with high economic value and research potential. To better understand their molecular phylogeny among Ginkgo clones, we obtained 44068 ESTs from female strobili and 29779 ESTs from leaves and 157327 DNA sequences by 454GSFLX sequencing. A total of 562 ESTs containing SSRs in female strobili, 336 ESTs in leaves, and 3044 in the genome DNA with SSRs were discovered. We designed 556 pairs of EST-SSR primers with Primer 5.0 and 417 pairs of them could be steadily amplified. We designed 432 pairs of genomic SSR primers and obtained 132 pairs with steady amplification. Polymorphism analysis was carried on in 6 samples and 176 pairs of EST-SSR and 82 pairs of genomic SSR primers were polymorphism, accounting for 31.7% and 41.0% of the total designed primers. These primers with polymorphism could be used as molecular tools for future genetic research in Gingko biloba. With these polymorphic primers, we selected 30 pairs of EST-SSR and 10 pairs of genomic SSR primers and applied them for 48 seed produced Ginkgo clones. The results revealed that up to 7 alleles were detected with 40 pairs of primers in 48 clones, 3.4 markers on average at each locus. Phylogenetic relationships of 48 clones were constructed and specific bands were observed in 21 of them. All 48 clones could be distinguished completely with 5 pairs of EST-SSR and 3 pairs of genomic SSR primers selected from the above. Among these primers, Gb_gSSR38 and Gb_eSSR120 were high efficient and their combination could separate 25 clones.