Full Length Research Paper

Optimization of RAPD-PCR reaction system for genetic relationships analysis of 15 camellia cultivars

X. F. Wang^1,2*, W. H. Zheng¹, H. X. Zheng¹, Q. Q. Xie¹, H. Y. Zheng¹, H. Tang¹ and Y. L. Tao¹

¹Life and Environmental Science College, Wenzhou University, Wenzhou, Zhejiang province, People Republic of China, 325027.

²Life Science College, Zhejiang University, Hangzhou, Zhejiang province, People Republic of China, 310058.

*Corresponding author. E-mail: wxf8080@126.com.

Abbreeviations: RAPD, Random amplified polymorphic DNA; PCR, polymerase chain reaction; dNTP, deoxynucleoside triphosphates; UPGMA, unweighed pair group method with arithmetic average.

Accepted 31 December, 2009

   Abstract

With orthogonal analysis by L₂₇(3⁶), the random amplified polymorphic DNA (RAPD)-PCR optimization reaction system for camellia were obtained. Results showed that the optimization system was 10×PCR Buffer (2.5 μL), 25 mM MgCl₂ (2.5 μL), 2.5 mM dNTPs (2.0 μL), 20 μM primer (1.0 μL), Tag (1.5 U), temple DNA (40 ng or so) and added ddH₂O to the total volume 25 uL; suitable annealing temperature was 36°C. With the optimized system and fifteen 10 nt random primers, we analyzed 15 camellia cultivars and observed 102 clear amplified loci, in which polymorphic loci were 79 while the percentage of polymorphic loci were 77.54%. Cluster analysis showed that the four groups were divided at the point 0.75 of similarity coefficient, indicating relatively high genetic diversity. We also found that the gene controlling petal color may play an important role in RAPD analysis. Moreover, genetic diversities based on RAPD analysis could be clearly reflected by morphological traits among 15 camellia cultivars. This study showed the RAPD optimization system was suitable and RAPD molecular marker was effective and useful tool for detection of genetic relationships among camellia cultivars.

Key words: Optimization, RAPD, Camellia cultivars, genetic relationships.