摘要
以软条白沙枇杷三倍体株系为试材,采用L25(56)正交设计方法,对影响枇杷SCoT-PCR反应的Mg2+、dNTPs、引物、Taq DNA聚合酶及模板DNA用量等因素进行优化筛选,并对最适退火温度进行了探讨。建立了适于不同倍性枇杷分析的SCoT-PCR扩增体系,即20μL的优化体系中含有:Mg2+1.5 mmol·L-1,dNTPs 0.35 mmol·L-1,引物0.5μmol·L-1,Taq酶0.5 U,模板DNA 50 ng。引物最适退火温度为51.8℃。应用优化的反应体系,对32个SCoT引物和3个枇杷品种(泸州六号:2x,3x,4x,5x;龙泉1号:2x,3x,4x;早红3号:2x,3x,4x)的10个不同倍性株系进行扩增,获得了条带清晰,稳定可靠的电泳结果。
Triploid ruantiaobasha loquat was used as materials,orthogonal design method was applied to optimize SCoT-PCR amplification system of loquat in five factors such as Mg2＋,dNTPs,primers,Taq DNA polymerases and template DNA at five levels,and the annealing temperature was also optimized.An optimal SCoT-PCR amplification system（20 μL） of polyploidy loquat was established,i.e.dNTPs 0.35 mmol·L-1,Mg2＋ 1.5 mmol·L-1,primer 0.5 μmol·L-1,Taq polymerase 0.5 U,template DNA 50 ng.51.8 ℃was the most suitable annealing temperature.32 SCoT primers and 10 different ploidy cultivars of Luzhouliuhao（2x,3x,4x,5x）,Longquanyihao（2x,3x,4x） and Zaohongsanhao（2x,3x,4x,） were used to test the optimized reaction system.Ideal results with high stability and clear polymorphic bands were obtained.
出处
《果树学报》
CAS
CSCD
北大核心
2011年第3期433-437,共5页
Journal of Fruit Science
基金
国家自然科学基金(30971986)
重庆市科委重点攻关项目(CSTC2009AB1183)
重庆市科委自然基金项目(CSTC2008BB1122)
关键词
枇杷
多倍体
目标起始密码子多态性
正交优化
Loquat
Polyploidy
Start codon targeted polymorphism
Orthogonal design
