Optimization of ISSR-PCR reaction system and selection of primers in <i>Bryum argenteum</i>

• Main Authors: Ma Xiaoying, Wei Wei, Zang Cheng, Yu Jing
• Format: Article
• Language: English
• Published: Academic Journals Center of Shanghai Normal University 2017-02-01
• Series: Journal of Shanghai Normal University (Natural Sciences)
• Subjects: <i>Bryum argenteum</i>; ISSR; orthogonal design; primer screening
• Online Access: http://qktg.shnu.edu.cn/zrb/shsfqkszrb/ch/reader/view_abstract.aspx?file_no=20170509&flag=1
• ISSN: 1000-5137; 1000-5137

In order to determine optimum ISSR-PCR reaction system for moss <i>Bryum argenteum</i>,the concentrations of template DNA primers,dNTPs,Mg²⁺ and Taq DNA polymerase were optimized in four levels by PCR orthogonal experimental method. The appropriate primers were screened from 100 primers by temperature gradient PCR,and the optimal anneal temperature of the screened primers were determined. The results showed that the optimized 20 μL ISSR-PCR reaction system was as follows:template DNA 20 ng/20 μL,primers 0.45 μmol/L,Mg²⁺2.65 mmol/L,Taq DNA polymerase 0.4 U/20 μL,dNTPs 0.45 mmol/L. Using this system,50 primers with clear bands,repeatability well and polymorphism highly were selected from 100 primers. The establishment of this system,the screened primers and the annealing temperature could provide a theoretical basis for further research on the genetic diversity of bryophytes using ISSR molecular markers.