Development And Application Of Microsatellite Marker In Phytophthora Infestans Genome

Potato late blight caused by Phytophthora infestans was one of the most destructivediseases. Microsatellite marker was the best marker of biometrical genetics, populationdynamic succession and biological diversity for better disease surveillance and control.The exploration of microsatellite loci and mass-produce deveolpment of microsatellitemarker were provided by the decipher of P. infestans genome released in2010. A series ofwork were developed for microsatellite of P. infestans, aiming at providing references forgenetic diversity among population of P. infestans. The main work were listed as follows:1. The amount and distribution of pure and compound microsatellite in the wholegenome and its different regions of P. infestans were identified by using software SciRoKo.A total of5010loci for perfect microsatellites were detected in the genome, whose densitywas26.35microsatellites/Mb, and the preferent motifs were ACGG, AT and AGG. Onlymotif for3to6bases occurred in the exons, in which the percent of microsatellites with3and6bases motif accounted for93.68%. The number of compound microsatellites was275,of which82.18%was interrupted perfect microsatellites.2. Theoretically3conditions were presumed to be suit for exploringloci ofmicrosatellite marker, according to the sequence characteristics and marker theory. Therequired microsatelite marker should be a single copy and single variable with highpolymorphism. The loci suitable for development of microsatellite markers and its flankingsequences were identified using software SciRoKo, Clustal X and Primer Pair SpecificityChecking from Primer-BLAST. At last,430loci suitable for the development ofmicrosatellites were acquired. Compared with the intergenic regions in the P. infestansgenome, the frequencies of both pure and single copy microsatellites in the genic oneswere higher, which was more suitable for the development of microsatellites.3. The loci with L≥25bp, which were suitable for the development of highpolymorphism microsatellite marker, were selected. Microsatellite primers were designedbased on the selected loci and its flanking sequences, and tested by PCR amplificationusing9P. infestans strains. A total of115loci for development of microsatellite markerswere found in the genome of P. infestans with length of more than25bp.64pairs of primers were developed, of which33primer pairs with polymorphism occupied51.56%, incontrast to other31pairs without polymorphism.33markers with polymorphism based on40isolates of P. infestans were analysed by using UPGMA.40isolates of P. infestanswere analysed by the different microsatellite markers, finding that the least number ofmarkers to distinguish those isolates was9.4. The number of alleles of polymorphic microsatellites and their PIC values weredetermined and calculated by40isolates of P. infestans from diverse geographical regionsand years respectively. The PIC values of33microsatellite loci ranged from0.164to0.614,of which PIC≥0.5for7ones, which were E-04958, I-00408, F5-22735, I-10840, I-06861,I-01408and I-07111.0.25＜PIC＜0.5for25ones. PIC≤0.25for1locus.5.9selected P. infestans microsatellite markers were used to PCR amplification for9loci of100isolates collected from Hebei, Heilongjiang, Jilin, Yunnan, Sichuan, Ningxia,Inner Mongolia and Fujian and5isolates collected from Holland, Russia and America.34alleles and29genetypes were found. The analysis of genetic diversity showed that thegenetic diversity was high in P. infestans population in some areas of China.