Optimization of SSR-PCR reaction system and selection of primers in Carya illinoinensis
[Objective]In order to optimize the SSR-PCR reaction system of Carya illinoinensis,we conducted a screening of highly polymorphic SSR primers specific to C.illinoinensis.This screening process has provided a valuable tool for various subsequent research activities,including fingerprint construction,genetic relationship analysis,and variety identification of C.illinoinensis.[Method]A combination of univariate experiments and L16(45)orthogonal experimental designs was used.The DNA of C.illinoinensis was used as a template.Firstly,the effects of six factors(DNA,Mg2+,10×PCR Buffer,primer,Taq enzyme,dNTPs)on the SSR-PCR reaction system of C.illinoinensis were tested individually.Based on the results of the single factor tests,the appropriate dosage range for each factor was determined,and an orthogonal test was designed accordingly.[Result]Based on the results amplified by orthogonal test,the optimal reaction system of C.illinoinensis was determined as follows:Taq enzyme 0.15 U,Mg2+2.0 mmol/L,dNTPs 0.1 mmol/L,primer 1.0 μmol/L,50 ng DNA template 1.0 μL,10×PCR Buffer 1.0 μL,and ddH2O 5.8 μL.The extremum difference analysis of orthogonal test revealed that the five factors affecting the amplification effect of the SSR-PCR reaction system of C.illinoinensis ranked in the following order:Taq enzyme＞primer=DNA＞Mg2+＞dNTPs.To validate the optimized SSR-PCR reaction system of C.illinoinensis,four pairs of C.illinoinensis primers were used with the DNA of eight species of C.illinoinensis as a template.The results showed the successful amplification of bright and clear bands,confirming the stability and reliability of the optimized reaction system.Out of the 283 pairs of walnut and C.cathayensis primers,12 pairs of highly polymorphic primers were selected using the optimized reaction system.The number of polymorphic sites exceeded 0.5.[Conclusion]The optimized reaction system and 12 pairs of primers are screened and can be directly utilized for subsequent SSR molecular marker experiments.This lays a solid foundation for the identification of genetic relationships and mating system analysis of C.illinoinensis.
Carya illinoinensisSSR-PCR reaction systemselection of primerssingle factor testorthogonal design experiment
万方数据
维普
