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Late blight, caused by Phytophthora infestans Montagne de Bary, is the most devastating disease of potato worldwide. Heilongjiang is one of the provinces in China well known for its production of seed potato, ware potato, and the potato for processing, and is also a province where late blight occurs frequently. Late blight has become one of the most important factors limiting the production of potato in Heilongjiang. Therefore, it is essential to improve the abilities of controlling the late blight in Heilongjiang. At present, research focuses just on phenotypic structure of P. infestans population in Heilongjiang, however, few reports can be found in the literature about the genetic diversity of P. infestans population using the genetic markers, especially the molecular markers. In this research, the genetic diversity among 103 isolates of P. infestans was assessed with 21 SSR primer pairs. The aims of the work were to monitor the change in the P. infestans population, to reveal the genetic relationship among the P. infestans isolates and to detect the abundance of the genetic diversity in P. infestans population. These may lay a sound basis for evaluating the risk of sexual recombination, understanding the interaction between the host and pathogen, employing the effective control methods and supporting the late blight resistant breeding program. The main results were summarized as followings.1. A total of 93 isolates of P. infestans were collected from different potato fields in Heilongjiang province in 2004, 2005, 2006 and 2008, and 10 isolates from the United States, Netherlands, and Sichuan, Yun’nan and Fujian province of China were also included in this research.2. A total of 1414 ESTs (Expressed of Sequence Tags) of P. infestans covering 896 kb were mined by SSRIT (SSR Identification Tool), and a total of 31 SSR loci for 31 P. infestans ESTs (2.19%) with the frequency of 1/28.90 kb were identified. Trinucleotide repeat was the dominant motif among the EST-SSR loci, and its percentage was 35.48%. In addition, 31 SSR motifs were found and there was only one SSR in every 31 ESTs. For all of the nucleotide repeats, CAG is the most abundant motif.3. A total of 13 SSR primer pairs were developed according to the ESTs strategy and all primer pairs amplified target fragment successfully. Of these, 9 pairs of primer exhibited polymorphism, and the rest of 4 primer pairs were monomorphic.4. The parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The optimal annealing temperature was 63℃, and the optimum PCR system of EST-SSR was: 25 ng template DNA, 0.5 mmol·L-1 dNTPs, 2μL 10×Buffer (Mg2+ free), 1.75 mmol·L-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20μL reaction system. The PCR program was initial denaturation at 94℃for 2 min; followed by 35 cycles of 94℃for 30s, 63℃for 30s, and 72℃for 30s; a final extension step was 72℃for 7 min, and then held at 4℃. The clarity and abundant polymorphism indicated that this system was stable and suitable for study of the genetic diversity of P. infestans population.5. A total of 29 SSR primer pairs, including 13 pairs of EST-SSR primers developed in this research, were tested against a set of P. infestans isolates and 21 pairs of primers (72.4%), including 9 pairs of EST-SSR primers developed in this research, showed to be polymorphic and therefore appropriate for further testing and statistic analysis. The resulting 21 SSR primer pairs were used to characterize the collection of 103 P. infestans isolates. The genetic diversity of P. infestans population was abundant, and DNA polymorphism of P. infestans was significantly associated with geographical origin, however no association was found with the year when the isolate was collected.6. Genetic similarities were calculated using three different methods, SM method, Jaccard’s method and Nei & Li’s method. The ranges of similarity coefficient based on the three genetic similarities were 0.337-1.000,0.153-1.000 and 0.265-1.000,respectively. All of the 103 isolates were separated into 4 groups by cluster analysis and 3 groups by the method of principal coordinate analysis with the SM method. The results based on the two analyses are consilient. Comparison of the data obtained by three genetic similarity matrixes was made by the method of Mantel test. The correlation coefficient r was all highly significant, with r correlation value being above 0.90.7. Seventeen isolates collected in Harbin in 2005, 2006 and 2008 were characterized by cluster analysis based on SM method, and the P. infestans was, to some extent, associated with year when the isolate was collected. In addition, forty six isolates collected from 8 counties (cities) in 2006 were characterized by cluster analysis based on SM method, and the P. infestans was also, to some extent, associated with geographical origin.
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