均匀设计优化杨属的SRAP—PCR反应体系

采用均匀设计方法,对影响杨属SRAP-PCR体系的MgCl2、dNTPs、引物、TaqDNA聚合酶和模板浓度等因素分别进行了U16(45)和U12(35)两轮优化,建立了适用于杨属的SRAP-PCR反应体系。在25μL反应体系中,MgCl23.5mmol/L、dNTPs0.20mmol/L、引物0.44μmol/L、TaqDNA聚合酶1.50U、模板浓度28ng/L为最适条件。在此基础上又对退火温度进行了摸索,结果发现,扩增结果对退火温度变化不敏感。最后运用优化体系对杨属3派10个种共16个单株的DNA进行扩增验证,结果获得的DNA条带清晰,多态性比较丰富。说明这一优化的SRAP-PCR反应体系可用于杨属不同种间亲缘关系、系统进化和遗传多样性等方面的研究。

Optimization of SRAP-PCR amplification system for Populus by uniform design

A uniform design was applied to optimize the SRAP-PCR (sequence-related amplified polymorphism-polymerase chain reaction) system in Populus.Five factors in this SRAP-PCR,including MgCl_2,primers,dNTPs,TaqDNA polymerase and a DNA template,were tested using two uniform designs U_ (16) (4~5) and U_ (12) (3~5) .A suitable SRAP-PCR system for Populus was established.To 25 μL SRAP-PCR amplification reaction solution,3.5 mmol/L MgCl2,0.20 mmol/L dNTPs,0.44 μmol/L primer,1.50 U TaqDNA polymerase and a 28 ng/L template were added.The annealing temperature had no clear effect on the amplification results.By using this optimal system,genomic DNAs from 10 species of Populus were tested and DNA bands were clear and showed high polymorphism.It suggests that the improved SRAP-PCR system can be used to study genetic relationships,systematic evolution and genetic diversity in Populus.