Computational protocol: Development of 16 Microsatellite Markers for Prince’s Pine, Chimaphila japonica (Pyroleae, Monotropoideae, Ericaceae)

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Protocol publication

[…] Genomic DNA samples of C. japonica were extracted from silica-gel-dried leaves of two different individuals using a modified hexadecyltrimethylammonium bromide (CTAB) method []. The extracted DNA was dissolved in ddH2O. The fast isolation by AFLP of Sequences Containing repeats (FIASCO) was performed in this study []. Total genomic DNA (approximate 250 ng) was completely digested with 2.5 U of MseI restriction enzyme (New England Biolabs, Beverly, MA, USA), and then ligated to an MseI AFLP adaptor (5′-TAC TCA GGA CTC AT-3′/5′-GAC GAT GAG TCC TGA G-3′) using T4 DNA ligase (Fermentas, Burlington, ON, Canada). The digested-ligated fragments were diluted in a ratio of 1:10, and 5 μL were used amplification reaction with MseI-N primer (5′-GAT GAG TCC TGA GTA AN-3′). The amplified DNA fragments (200–800 bp) were enriched by magnetic bead selection with 5-biotinylated (AG)15 and (AC)15 probes, respectively. The recovered DNA fragments were reamplified with MseI-N primers. The PCR products were purified using the EZNA Gel Extraction Kit (Omega Bio-Tek, Guangzhou, China), and were ligated into a pGM-T vector (Tiangen, Beijing, China), and then transformed into E. coli strain DH5α competent cells (TaKaRa, Dalian, Liaoning, China). The positive clones were picked out and tested using vector primers T7/SP6 and primer (AC)10/(AG)10 respectively to select appropriate fragments which contained SSRs. In other words, a set of tested PCRs included three reactions was performed using T7 and SP6, T7 and (AC)10, (AC)10 and SP6 as primers, respectively. The second set of tested PCRs was tested using T7 and SP6, T7 and (AG)10, (AG)10 and SP6 as primers, respectively. All of these PCR reactions had the same conditions: 94 °C for 4 min followed by 35 cycles at 94 °C for 45 s, 52 °C for 45 s, 72 °C for 1 min, and a final extension step at 72 °C for 3 min. The selected positive clones were sequenced using an ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences containing simple sequence repeats and enough flanking regions were selected for primer design using Primer Premier 5.0 [].The designed Primer pairs were assessed in 24 individuals of C. japonica pooled from two natural populations (KD, Kangding, Sichuan: 30°09′N, 102°09′E, 2857 m a.s.l. and KM, Kunming, Yunnan: 25°11′N, 102°44′E, 2345 m a.s.l.). Herbarium vouchers were deposited in the Kunming Institute of Botany, Chinese Academy of Science (code LZW 0128-0151). The PCR reactions were performed in 20 μL of reaction volume containing 30–50 ng genomic DNA, 0.35 μM of each primer, 10 μL 2× Taq PCR MasterMix (Tiangen; 0.1 U Taq Polymerase/μL, 0.5 mM dNTP each, 20 mM Tris-HCl (pH 8.3), 100 mM KCl and 3 mM MgCl2). PCR amplifications were conducted under the following conditions: 94 °C for 3 min followed by 32 cycles at 94 °C for 30 s, the annealing temperature for each specific primer (optimized for each locus, ) for 30 s and 72 °C for 45 s with a final extension step at 72 °C for 3 min. The amplification products were separated and visualized using a QIAxcel capillary gel electrophoresis system (QIAGEN, Irvine, CA, USA).The number of alleles per locus, observed heterozygosity (HO), and expected heterozygosity (HE) were estimated in POPGENE version 1.31 []. […]

Pipeline specifications

Software tools Primer Premier, POPGENE
Application Population genetic analysis
Organisms Cryptomeria japonica