利用多重PCR反应同时筛选番茄Ty-1和cf-5基因

Identification of Ty-1 and cf-5 genes of tomato by multiplex PCR

利用同一PCR反应体系同时扩增、筛选分别与番茄抗黄化曲叶病毒病的Ty-1基因和番茄抗叶霉病的cf-5基因紧密连锁的SCAR标记,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TaqI酶切位点,酶切后分别产生了303bp和95bp以及398、303bp和95bp的特异性片段,而感病基因型无此酶切位点,酶切后仍呈现398bp的特异带。与cf-5基因紧密连锁的SCAR2标记扩增后抗感材料均产生960bp的特异片段,用限制性内切酶TaqI酶切后,含cf-5基因的材料产生一条256bp的特异带,而不含cf-5基因的材料产生一条225bp的特异带。

The two SCAR markers tightly linked respectively with the Ty-1 gene resistant to toma- to yellow leaf curl virus and the cf-5 gene resistant to tomato leaf mold were amplified and screened by the same PCR system. The specific fragment amplified was identical with one amplified by a single SCAR primer. The SCAR1 marker tightly linked with Ty-1 gene was codominant and all the materials produced a specific fragment of 398 bp. After digested by TaqI, the homozygous Ty-1 genotype could produce 303 bp and 95 bp specific bands,and the heterozygous one could produce 398,303 bp and 95 bp spe- cific bands, but the disease susceptible genotype still presented a 398 bp specific band. After amplified, the SCAR2 marker tightly linked with cf-5 gene produced 960 bp specific fragment in both resistant and susceptible tomato lines. After digested by TaqI, the material with cf-5 gene could produce a 256 bp spe- cific band,but the material without cf-5 gene produced a 225 bp specific band. The replicated tests proved that the two resistant genes could be identified simultaneously by the same PCR system.