多重PCR技术鉴定番茄Ty-1和Mi基因

Identification of Ty-1 Gene and Mi Gene by Multiplex PCR Reaction in Tomato

利用同一PCR反应体系,对分别与番茄(Lycopersion esculentum)抗黄化曲叶病毒(Tomato YellowLeaf Curl virus)病的Ty-1基因和番茄抗根结线虫(root-knot nematode)的Mi基因紧密连锁的SCAR标记进行同时扩增筛选,扩增的特异性片段与单引物扩增片段完全吻合。与Ty-1基因紧密连锁的SCAR1标记为共显性标记,抗感材料均产生398bp的特异片段,纯合和杂合抗病基因型存在Taq I酶切位点,酶切后分别产生了303bp和95bp以及398bp、303bp和95bp的特异性片段,而感病基因型无此酶切位点。与Mi基因紧密连锁的SCAR2标记也为共显性标记,抗感材料均产生750bp的特异片段,纯合和杂合抗病基因型存在Taq I酶切位点,酶切后分别产生了570bp和180bp以及750bp、570bp和180bp的特异性片段,而感病基因型无此酶切位点。酶切结果仍为750bp的产物。经反复验证,结果准确可靠,可以用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。

Ty-1 and Mi in tomato （Lycopersicon esculentum） tightly linked with two SCAR markers and respectively resistant to Tomato Yellow Leaf Curl virus and root-knot nematode were amplified and screened by using a single PCR reaction. The PCR products were completely correspond to the amplified bands produced by single SCAR primer. Among them, co-dominant SCAR1 marker tightly linked with Ty-1 gene produced 398 bp fragment in both resistant and susceptible tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq Ⅰ. Genotype with homozygous and heterozygous Ty-1 gene could produce respectively 303 bp, 95 bp and 398 bp, 303 bp and 95 bp bands. Susceptible genotypes still presented 398 bp fragment. The co-dominant SCAR2 marker tightly linked with Mi gene would produce 750 bp fragment in both resistant and susceptible tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq Ⅰ. Genotype with homozygous and heterozygous Mi gene could produce respectively 570 bp, 180 bp and 750 bp, 570 bp and 180 bp bands. Susceptible genotypes still presented 750 bp fragment. The replicated stable results proved that two resistant genes could be identified simultaneously by using corresponding PCR primer under adaptable condition.