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    马铃薯晚疫病菌线粒体DNA多态性的研究

      

    J Microbiol Methods. 2013 Nov;95(2):117-21. doi: 10.1016/j.mimet.2013.08.001. Epub 2013 Aug 13.

    Mitochondrial DNA polymorphisms in Phytophthora infestans: new haplotypes are identified and re-defined by PCR.

    Yang ZH1, Qi MX, Qin YX, Zhu JH, Gui XM, Tao B, Xu XH, Zhang FG.

    Author information
    1College of Plant Protection, Agricultural University of Hebei, Baoding 071001, China.

    Abstract
    Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~2 kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2 kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n = 1, 2, or 3) of a 36 bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as R(n = 1, 2, or 3). Finally, the P. infestans mt-DNA haplotypes were re-defined as IR(1) or IIR(2) according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR(1), IR(2), IR(3), IIR(2) and IIR(3)) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR-RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.


    发布时间: 2013-12-01 12:01 浏览次数:   【 打 印 】【 关 闭
     
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