Resequencing Research Of High And Low Virulent Isolates Of Rhizoctonia Solani AG-1IA

Rice is one of the most important crops in the world, while rice sheath blight disease is the worldwide rice disease, together with rice blast and rice bacterial leaf blight known as the three major diseases in China. Rhizoctonia solan is responsible for Rice sheath blight, characterized by wide distribution, extensive host range and no asexual spores. Besides lost caused by rice sheath blight, it is also harmful to cereal crops as well as a variety of vegetables, flowers and other cash crops. Diverse symptoms were often observed, such as standing dead, cataplexy, leaf blight, sheath blight and crop damage. In addition, Rhizoctonia solani possesses strong capability of saprophytic competence in soil and survived long. Thus, research on Rhizoctonia solani is greatly beneficial to pathology and economy. Pathogenic mechanism of Rhizoctonia solani is quite complex. With our dedicated work, sheath blight pathogen genome framework has been established, evolutionary history of pathogens was constructed, and pathogenic mechanism was clearly illustrated at genomic level. All the results facilitated a comprehensive understanding of multi-fusion group and multi-nucleus heterozygous. Based on complete genome sequence of Rhizoctonia solani AG1-IA, a comparative genomics was carried out between high virulent strain D23and low virulent D12by whole genome re-sequencing. Bioinformatics analysis was then used to verify pathogenic genes for the sake of further clarification of pathogenic mechanism. Main results of this work were listed as follows:1. Cytological observation:Rhizoctonia solani AG-1IA, low virulent strain D12and highly virulent strain D23are observed by cytological, the early mycelium is white, gradually turns to yellow-brown, sometimes easy to form a large number of aerial hyphae, post some small sclerotia formed at the edge of the Petri dish, the initial sclerotia is white, gradually grows to brown. Mycelial growth rate:D12grows the fastest, followed by the AG-1IA, and D23grows slowest. The sclerotia formation time:D12formed first, followed by the AG-1IA, D23at the latest. Mycelium is dye by DAPI, and we get the nucleus amount of data:the D12’s nucleus is4-7, AG-lIA’s nucleus is8-10, D23’s nucleus is13-16, the number of the nucleus and toxicity is proportional to the size. Dfferent virulent strains infect in fresh rice leaves, we found that the D12’s virulence minimum; the IA has strong virulence; D23has the strongest virulence.2. Karyotype analysis:Strains of Rhizoctonia solani AG-1IA’s chromosome tablet observation and its karyotype analysis have completed. select50good results, more than90%of the cells have the same chromosome number13, so make sure to stand Rhizoctonia solani AG-1IA chromosome number as2n=13. The relative length of chromosome n=L+5M2+7M1. Then, Rhizoctonia solani AG-1IA02888pathogenic genes and chromosomes by fluorescence in situ hybridization, the results clearly show that the distribution of the target gene on the chromosome, the genome of a total of three distinct signal loci, and we can determine that the three loci are located at the the chromosome end portion or proximal end portion.3. Resequencing data analysis:We using the Illumina Hiseq platform for re-sequencing the opposition highly virulent D23and low virulent D12, by means of bioinformatics analysis of the structural differences between the different individual genome, completed at the same time notes, and then verify the functional gene, position SNPs metabolic pathways, and then find out the associated SNPs which is connection with pathogenic genes. We can realize that the genomic structural variation of the outside of the sequence and the sequence deeply. In total, we acquired approximately16million paired-end reads of70-80bp with500bp insert size for each sample. We aligned all these short reads onto the reference AG1IA genome with command map of MAQ using the default parameter, and found about84%of the reads can be mapped to the genome, including about78%pair mapped reads. Our resequencing of two samples of AG1IA covered more than96%of the AG1IA genome, Then the consensus sequence of D12and D23were assembled by the command assemble of MAQ with default parameter according to the mapping result separately.4. Functional verification of SNPs loci and different gene loci on pathogenic(1) Select eight genes which contain with SNPs in each of the D23and D12strains randomly. After a series of experiments such as clones, connection, transformation, the identification and sequencing of the positive recombinants. The sanger sequencing results showed that the consistency of the various sequences in the three strains was more than97%. SNPs loci of Prediction has high reliability.(2) Prokaryotic expression the three Effector gene, then with protein to infect rice leaves, and analysis sequence structure, the results show that the three Effector gene can be expressed in the D12, IA and D23strains. With diluted expression protein infection9311rice leaves, we found that the effect protein of D23strains’pathogenic is strongest, IA Second, and D12weakest. Illustrate that the SNPs in the three Effectors gene is influential to the pathogenicity of strain. Effect factors on rice immune degree and effect factor SNPS loci were positively correlated, it is show that the three effectors as key effect factor of sheath blight trigger an immune response.5. Metabolomic analysis of High and Low Virulent Isolates:The metabolomics instrument detection and data analysis to the three groups of Rhizoctonia solani sample(high, middle and low virulent strains) has been completed by GC/MS instrument. PCA analysis of each group are divided in different regions proved that the metabolomics methods are reliable. Multidimensional statistical analysis methods such as PCA, PLS-DA and OPLS-DA are adopted. The pairwise comparison of each group have significant metabolic differences between the groups. Further OPLS-DA combining with the t-test method has screened to the difference between the two groups of metabolites.