Optimization of SRAP-PCR reaction system，primer screening and verification for Lithocarpus litseifolius

【Objective】To establish an optimal reaction system applicable to SRAP-PCR of Lithocarpus litseifolius， and screen out polymorphic primers for the genetic diversity analysis of L. litseifolius germplasm resources，thereby providing scientific basis for the subsequent studies on conservation genetics and its development and utilization.【Method】 The combination of single-factor experiment and orthogonal experiment was adopted to optimize the main influencing factors（template DNA amount，dNTPs concentration，Taq DNA polymerase amount and primer concentration）affecting the amplification effect of SRAP-PCR. Using optimal reaction system，SRAP polymorphic primers suitable for L. litseifolius were screened.【Result】The optimal reaction system for SRAP-PCR of L. litseifolius（20.00 μL）was obtained as follows： 10×PCR Buffer 2.00 μL，Taq DNA polymerase 1.50 U，dNTPs 0.25 mmol/L，primer concentration 0.600 μmol/L and template DNA 30.00 ng. The degree of influence of each factor on the PCR amplification effect of L. litseifolius was ranked as follows：primer concentration > dNTPs concentration > Taq DNA polymerase amount > template DNA amount. Using the optimized SRAP-PCR optimal reaction system and the selected 20 pairs of polymorphic primers for SRAP-PCR amplification of 24 samples from 3 populations of L. litseifolius，it was found that 4 pairs of SRAP primers amplified a total of 38 loci from the 24 samples of the 3 wild populations，with an average of 9.5 loci per pair of primers. Among them，21 loci were polymorphic，with a polymorphic rate of 55.26%. The average number of alleles（Na），average effective number of alleles（Ne），Nei’s gene diversity（H'），and Shannon’s information index（I）of the 3 populations of L. litseifolius were 1.55，1.30，0.18 and 0.27 respectively. The total genetic diversity（Ht），within-population genetic diversity（Hs），and genetic differentiation coefficient（Gst）were 0.18，0.11 and 0.36 respectively. The gene flow（Nm）was 0.90（<1.00），indicating that there was a certain degree of gene exchange among the 3 populations，but the degree of gene exchange was limited.【Conclusion】The amplification effect of SRAP-PCR of L. litseifolius is most sensitive to primer concentration. The established optimal reaction system of SRAP-PCR of L. litseifolius and the selected SRAP primers produce clear and stable amplification bands with rich polymorphism，which can better reflect the genetic relationships among different individuals and populations，and can be applied to the analysis of genetic diversity and genetic structure of L. litseifolius germplasm resources.