Genome Sequencing And Developing Of Microsatellite Markers Of Alternaria Solani

Potato early blight caused by the pathogenic fungus Alternaria solani is one of the most important diseases worldwide. Based on the whole genome sequencing and genes annotation of A. solani, which were providing a large number of gene resource for functional genomics, the basic genetic characteristics, sporulation genes and pathogenesis related proteins were analysed. The main work were listed as follows:1. Genome sequencing and annotation: The whole genome sequence of A. solani HWC-168 using a Whole Genome Shotgun(WGS) method. Two DNA libraries with insert size of 500 bp and 5kb were constructed and sequenced by the Illumina Hiseq2000 sequencing platform. Altogether 21,909,421 and 33,720,474 reads were obtained from the two libraries, and the genome coverage is 200 and 308-fold, respectively. All reads were assembled into 61 scaffolds using SOAPdenove and the genome size was 32,838,780 bp with a GC content of 51.2%. This project has been deposited at Gen Bank under the accession number JRWV00000000.2. Comparative genomics: A. solani, A. arborescens and A. brassicicola three genera in almost all of the COG functional categories have a similar distribution. A large number of chromosomal proteins involved in cellular processes and signaling and metabolism. Comparatively, secondary metabolites biosynthesis, transport and catabolism was found to play a vital role in A. solani. Replication, recombination and repair and signal transduction mechanisms were reduced to inferiority. Such information has an important guiding significance in our future functional studies.3. Genes prediction: 15 genes associated with sporulation were aba A, wet A, tps A, rco-3, hsf2, mcb, Flu G, Flb A, Stu A, WC-1(Lre A), CRY2, NOP-1, SPO71, SPO5, Nii A. The central regulatory pathway genes(aba A and wet A), the central regulatory pathway modifiers(Stu A) and the central regulatory pathway activators(Flu G and Flb A) as well as the other related genes of conidiation can be used as references for researching asexual developmental mechanisms. Moreover, the softwares Signal P, Target P, TMHMM and Big-pi were combined to predict the sectetome of A. solani HWC-168. A total of 643 possible secreted proteins were identified in A. solani HWC-168 secretome. The secretome was equipped with 261 CAZymes enzymes, 138 putative pathogen-host interaction proteins, 119 Rx Lx motif containing proteins as well as 27 A.solani specifically secreted proteins. To some extent, these aspects revealed the complexity and diversity of A. solani secreted proteins.4. Development of microsatellite markers: 2738 perfect simple sequence repeats and 70 compound microsatellites in the whole genome were identified by using software Sci Ro Ko 3.3. 35 microsatellite primers were designed based on perfect microsatellite loci, and tested for specificity and polymorphism by PCR amplification using 12 A. solani strains. A total of 25 microsatellite makers with polymorphism were developed.5. Detectin of colony sectorization: Colony sectorization of a single-spore isolate of A. solani was studied and molecular differences were analysed between the wild type strain HQQ-2 and its three single-spore isolates HQQ 2-1, HQQ 2-2 and HQQ 2-H using 15 microsatellite markers. The result showed that the sectorizations tended to grow slower but sporulate more during subculturing. SSR showed that there were 3 different loci between the wild type strain and its three sectors, suggesting that genetic variations occurred in A. solani. We hypothesized parasexal reproduction might be a significant factor in the life-cycle of A. solani.