SSR markers for a critically endangered species Euryodendron excelsum and a distantly related species Ternstroemia gymnanthera (Ternstroemiaceae)

Development and characterization of 23 polymorphic microsatellite loci for Amentotaxus argotaenia (Taxaceae), a relict vulnerable species

“…This enrichment procedure was repeated once, and the purified PCR fragments, enriched for microsatellites, were ligated into the pMD18T vector (TaKaRa Biotechnology Co., Dalian, China) at 16°C for 16 h and transformed into E. coli DH5α competent cells by transient thermal stimulation (ice bath for 30 min, 42°C water bath for 90 s, followed by ice bath for 2 min). Recombinant positive clones were selected by blue–white screening according to Cui and Su () and identified by PCR using universal M13F/M13R primers with the following conditions: initial denaturation at 94°C for 10 min; followed by 25 cycles at 94°C for 30 s, 53°C for 45 s, and 72°C for 60 s; and a final extension step at 72°C for 7 min. The positive PCR products were sequenced with primer M13 + /M13 − on an ABI 3730xL sequencer (Applied Biosystems, Foster City, California, USA), and SSRs were selected using SSRHunter software version 1.3 with parameters set to more than four repetitions for di‐, tri‐, and tetranucleotide repeats (Li and Wan, ).…”