薄壳山核桃SSR-PCR体系优化及引物筛选

Optimization of SSR-PCR reaction system and selection of primers in Carya illinoinensis

【目的】为建立薄壳山核桃SSR-PCR的最佳反应体系,筛选薄壳山核桃高多态性SSR引物,为薄壳山核桃构建指纹图谱、亲缘关系分析、品种鉴定等后续的相关研究提供有力工具。【方法】采用单因素试验与L_(16)(4^(5))正交试验设计相结合的方法,以薄壳山核桃DNA作为模板,对影响薄壳山核桃SSR-PCR反应体系中的6个影响因素(DNA、Mg^(2+)、10×PCR Buffer、引物、Taq酶、dNTPs)进行单因素试验,根据单因素试验的结果确定各影响因素的适宜用量范围,再据此设计正交试验。【结果】根据正交试验扩增结果,确立薄壳山核桃的最佳反应体系(10μL)为:Taq酶0.15 U,Mg^(2+)2.0 mmol/L,dNTPs 0.1 mmol/L,引物1.0μmol/L,50 ng模板DNA 1.0μL,10×PCR Buffer 1.0μL,ddH_(2)O5.8μL。5个因素影响薄壳山核桃SSR-PCR反应体系的扩增效果,其影响程度大小依次为Taq酶>引物=DNA>Mg^(2+)>dNTPs。以8种薄壳山核桃DNA为模板,4对薄壳山核桃引物对优化后的薄壳山核桃SSR-PCR反应体系进行验证,均扩增出明亮清晰的条带,证明优化后的反应体系稳定可靠。利用优化后的反应体系在283对核桃及山核桃引物中筛选出高多态性薄壳山核桃引物12对,其多态性位点信息数均高于0.5。【结论】优化后的反应体系及筛选出的12对薄壳山核桃引物可直接用于后续的SSR分子标记试验,为薄壳山核桃亲缘关系鉴定、交配系统分析等研究奠定理论基础。

【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,Mg^(2+),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,Mg^(2+)2.0 mmol/L,dNTPs 0.1 mmol/L,primer 1.0μmol/L,50 ng DNA template 1.0μL,10×PCR Buffer 1.0μL,and ddH_(2)O 5.8μL.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>Mg^(2+)>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.