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 pro-viding 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 fac-tors(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 tem-plate 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 amplifica-tion 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),in-dicating 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 indi-viduals and populations,and can be applied to the analysis of genetic diversity and genetic structure of L.litseifolius germ-plasm resources.
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