Establishment and optimization of ISSR reaction system for Anoectochilus formosanus / 中草药

ABSTRACT

Objective: To establish a stable, reproducible, and suitable reaction system of Anoectochilus formosanus for ISSR analysis of genetic differences according to the ISSR-PCR characters in plants of Anoectochilus Bl. Methods: The initial ISSR reaction system in plants of Anoectochilus Bl. was established and then the primers were screened. After that, PCR amplifications were carried out until the different concentrations of the factors in the ISSR reaction system were designed, and based on the principle of high stability and reproducibility, the stable and reliable ISSR reaction system was eventually established after a series of adjustment and optimization of the reaction system. Results: The optimal ISSR reaction system in plants of Anoectochilus Bl. was reported for the first time, and the volume of 25 μL contained approximately 20 ng template DNA, 1 U Taq DNA polymerase, 0.2 mmol/L deoxyribonucleotide triphosphate (dNTP), 2 mmol/L MgCl2, 200 umol/L dNTP Mixture, and 0.2 μmol/L primer within 1x reaction buffer [10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl]. The reaction mixtures were denatured at 94°C for 7 min and subjected to 45 cycles of 30 s at 94 °C, 45 s at 52 °C, 2 min at 72 °C, and a final extension step of 10 min at 72 °C and eventually stored at 4 °C. Conclusion: The ISSR-PCR systems established for studying on the plants of Anoectochilus Bl. show the characters of stable reaction system, better repeatability, clear marker site, and reliable abundant polymorphisms. It is suitable for the study on genetic diversity in plants of Anoectochilus Bl. in difference wild populations and genetic variation of its sterile seedlings after long time tissue culture.