Rapid establishment of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) system for chloroplast DNA in tea [Camellia sinensis (L.) O. Kuntze]

Abstract

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) optimization reaction system for cpDNA in tea [Camellia sinensis (L.) O. Kuntze] was rapidly established. Results show that the optimal PCR reaction system was 100 ng template DNA, 200 μmolL^-1 dNTPs, 1.5 mmolL^-1 MgCl₂, 50 ng primer, 3U Taq DNA polymerase, and ddH₂O to the total volume of 25 μl; the optimal digestion system was 6 μl amplification product, 2 U endonuclease, 1×endonuclease buffer in digestion solution, and ddH₂O to the total volume of 15 μl; digestion time was 6 h at 37°C. With the optimized system, genetic diversity among 30 tea cultivars was investigated. Seven sets of chloroplast primers could produce one or more distinct bands. After the amplified products were digested by 10 restriction enzymes, a total of 135 bands were detected, among which 98 bands (72.59%) were polymorphic. The cpDNA PCR-RFLP based genetic distance (GD) among 30 tea accessions ranged from 0 to 0.071, with the mean of 0.049. This study suggests that the optimization system was suitable for PCR-RFLP analysis of cpDNA in tea.

Keywords: Camellia sinensis, PCR-RFLP, chloroplast DNA, establishment