Establishment and verification of ISSR-PCR reaction system for Malania oleifera

【Objective】The purpose of the study was to establish a stable ISSR-PCR reaction system for Malania oleifera and to screen appropriate polymorphic primers for M. oleifera， so as to provide a reference basis for the subsequent study on the genetic diversity of M. oleifera and the conservation of germplasm resources. 【Method】Single factor test combined with L₂₅（54） orthogonal test was applied to optimize the four main factors （template DNA amount， primer concentration， dNTPs concentration， Taq DNA polymerase） and the number of reaction cycles affecting the amplification effect of the ISSR-PCR reaction system and to determine the optimal reaction conditions. On this basis， the polymorphic primers suitable for M. oleifera were screened， the optimal annealing temperature was explored， and the optimized reaction system was verified by collecting ten M. oleifera samples from three wild populations in Yunnan. 【Result】The optimal ISSR-PCR reaction system for M. oleifera （in 20 μL reaction system）：30 ng of DNA， 0.3 μmol/L of primer， 0.250 mmol/L of dNTPs， 1.00 U of Taq DNA polymerase， and ddH₂O replenished to 20.0 μL. The optimal reaction program for ISSR was 30 cycles. The optimal annealing temperature of eight primers （UBC825， UBC826， UBC827， UBC836，UBC844， UBC861， UBC834 and UBC851） was screened out using the optimized reaction system at 50.2， 56.5， 50.2， 56.5， 50.2， 50.2， 50.2 and 54.0 ℃， respectively. The measured optimal annealing temperatures of some primers （such as UBC827， UBC836， UBC861） differed greatly from the predicted annealing temperatures of the software. Ten M. oleifera samples were tested for ISSR-PCR molecular markers using the optimized ISSR-PCR reaction system and primers. The results showed that the established ISSR-PCR reaction system was stable and reliable， and the samples of M. oleifera from different sampling sites were relatively conserved genetically. Additionally， the coefficient of genetic differentiation （G_st） of the ten M. oleifera samples was 0.74， indicating that 74% of the genetic diversity occurred among the populations， and the gene flow （N_m） was 0.18， which was much less than 1.00， indicating that there was a high probability of genetic drift， which made it prone to the decrease of genetic diversity and the differentiation of the populations. 【Conclusion】By optimizing the ISSR-PCR reaction system of M. oleifera， a stable reaction system for M. oleifera ISSR-PCR molecular markers has been established， which can be used in the research work of M. oleifera germplasm resources conservation and utilization.