Optimization of ISSR-PCR system for cultivar verification in watermelon (Citrullus lanatus var. lanatus)

摘要

Optimization of the inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) system is imperative to verification of watermelon (Cat-Wins barrows var. lanatus) cultivars. A uniform design was applied to optimize five factors influencing the ISSR-PCR system, including Mg2+, dNTP, primer, Tag DNA polymerase and template DNA concentration. The amplification procedure, including the annealing temperature and the thermal cycling profile, was also optimized based on the optimal ISSR-PCR system. The optimal ISSR-PCR system for watermelon was 20 mu L ISSR-PCR amplification reaction solution containing 1 x PCR Buffer, 2.00 mmol.L-1 Mg2+, 0.1 mmol.L-1 dNTP, 0.20 mu mol.L-1 primer, 0.5 U.20 mu L-1 Tag DNA polymerase and 2.00 ng.mu L-1 template DNA. The suitable amplification procedure consisted of an initial denaturation at 94 degrees C for 5 min, followed by 33 cycles of 94 degrees C for 30 s, annealing between 48 degrees C and 60 degrees according to each primer for 30 s, 72 degrees C for I min, and a final extension step of 72 degrees C for 10 min. With application of the optimal ISSR-PCR system and amplification procedure, the number of polymorphic bands detected with six ISSR primers ranged from I to 4, 1.8 polymorphic bands per primer. The combination of four ISSR printers, Primer UBC 810, Primer UBC 811, Primer UBC 817, and Primer UBC 834, could distinguish eight watermelon cultivars. The optimal system and amplification procedure described is feasible for watermelon cultivar verification.