光萼荷属植物SRAP-PCR反应体系建立与引物筛选

为了建立光萼荷属植物(Aechmea) SRAP-PCR反应体系，为今后光萼荷属植物种质资源研究提供技术支持，本研究通过L16(45)正交试验设计，对光萼荷属植物SRAP反应体系中的Mg2+、dNTPs、Taq DNA聚合酶、引物和模板DNA浓度等5个因素进行优化实验，并筛选多态性SRAP引物组合。结果表明，光萼荷属植物的最佳SRAP反应体系为1.50 mmol/L Mg2+、400 μmol/L dNTPs、1.5 U Taq DNA聚合酶、15 μmol/L引物、30 ng模板DNA及1×PCR buffer。各因素对SRAP-PCR扩增反应结果影响的差异较大，依次为模板DNA>Taq DNA聚合酶>dNTPs>引物>Mg2+。从56对SRAP引物组合中筛选出51对扩增条带清晰、多态性丰富的SRAP引物组合，多态性引物比率达90%以上。通过不同光萼荷属植物和不同引物组合对该反应体系进行验证，均获得了多态性丰富、条带清晰的扩增图谱，表明本研究建立的光萼荷属植物SRAP-PCR反应体系稳定可靠。

Establishment of SRAP-PCR reaction system of Aechmea species and screening for polymorphic primers

The aim was to establish SRAP-PCR reaction system and provide a new tool for future research on Aechmea germplasm.A orthogonal design of L16 （45） was used to optimize SRAP-PCR reaction system for Aechmea species with 5 factors,namely Mg2＋ ,dNTPs,primers,Taq DNA polymerase and template DNA,and SRAP primer combinations were screened for polymorphism.The result showed that a suitable SRAP-PCR reaction system for Aechmea species was 1.50 mmol/L Mg2＋,400 μmol/L dNTPs,1.5 U Taq DNA polymerase, 15 μmol/L primer combination,30 ng template DNA and 1 × PCR buffer.In addition,each factor in SRAP-PCR reaction system had different effects on the amplified patterns and the descending order was DNA Taq DNA polymerasedNTPsprimerMg2＋.A total of 51 polymorphic SRAP primer combinations were screened out from 56 SRAP primer combinations,with the polymorphic primer ratio more than 90%.The optimized SRAP-PCR reaction system was identified by different genomic DNAs of Aechmea species and different SRAP primer combinations;as a result,the amplified pattern with rich polymorphism and clear bands was obtained.It proved that the established SRAP-PCR reaction system for Aechmea species was steady and reliable.