摘要
以30个茶花品种为试材,采用5因素4水平正交实验以及单因素优化试验方法,研究Mg2+浓度、dNTP浓度、引物浓度、Taq DNA聚合酶浓度和模板DNA浓度对ISSR-PCR指纹图谱条带清晰度的影响,以进行茶花品种ISSR-PCR反应体系优化及引物筛选。结果表明:茶花品种ISSR-PCR最适扩增条件为25μL反应体系中,Mg2+3.0mmol/L、dNTPs 0.2mmol/L、引物0.3mmol/L、Taq DNA聚合酶0.5U、模板DNA 80ng以及52.1℃退火温度;试验从100个ISSR引物中筛选出12个适用引物;并对12个ISSR引物的多态性和稳定性进行了检验。
Taking 30 Camelliacultivars as test materials,the effects of concentrations of Mg2＋,dNTP,Primers,Taq DNA polymerase,template DNA,and the annealing temperature for primers on the ISSR-PCR molecular fingerprint map were examined by orthogonal design test and single factor modified.The results showed that the optimal ISSR-PCR conditions in the experiments were as following：in 25μL reaction system containing Mg2＋3.0 mmol/L、dNTPs 0.2 mmol/L、primers 0.3mmol/L、Taq DNA polymerase 0.5U、template DNA 80 ng and annealing temperature 52.1℃.Base on this optimal reaction system,100 ISSR primers were used to screen for the suitable primers with 1cultivar samples,of which12 ISSR primers with high resolution and multiple polymorphic bands were screened.Further amplification of 30 Camellia japonicacultivars using these 12 primers demonstrated that this optimized reaction system had specialties of good repeatability and stability.
出处
《北方园艺》
CAS
北大核心
2014年第22期84-88,共5页
Northern Horticulture
基金
广西科技攻关资助项目(桂科攻10100012-4)
关键词
茶花品种
分子指纹图谱
ISSR体系优化
引物筛选
Camelliacultivar
molecular fingerprint map
ISSR reaction system optimizing
primer screening
