Genome Size Estimation Of Rhizoctonia Cerealis And Phylogenetic Analysis

The genus Rhizoctonia consists of a wide variety of fungi and is distributed worldwide, among which most of them are phytopathogen, which is responsible for many serious plant diseases. Rhizoctonia cerealis is the causal pathogen of sharp eyespot in wheat.The most classical classifiation method of Rhizoctonia is anastomosis group method based on hyphal fusion interaction. Currently, multinucleate Rhizoctonia is divided into 14 anastomosis groups (AG-1 to AG-13 and AG-BI), The binucleate Rhizoctonia is divided into 21 anastomosis groups (AG-A-AG-U). The binucleate species Rhizoctonia cerealis belongs to the AG-D anastomosis group. The genome information of Rhizoctonia cerealis has not been reported yet, and the phylogenetic analysis of it needs further research. Accordingly, our research estimated the genome size of Rhizoctonia cerealis R0301, made a phylogenetic analysis using amplified translation elongation factor gene of Rhizoctonia cerealis and other anastomosis group Rhizoctonia, which lay the foundation for the analysis of genomy of Rhizoctonia cerealis.Our study amplified the genome of Rhizoctonia cerealis R0301 virulent strain isolated from Nanjing Jiangsu province, using fungal translation elongation factor universal primers, made a Blast analysis of the obtained sequence, identified the sequence as tefA gene of Rhizoctonia cerealis. Southern blot analysis indicated tefA was a single copy in the genome of Rhizoctonia cerealis.We used the quantitative real-time PCR method to calculate the genome size of the sequenced mutinucleat Rhizoctonia solani AG-1-IA strain GD118, AG-1-IB strain 7/3/14. Using the real-time quantitative PCR amplified the tefA gene of standard strains, then quantified the gene based on absolute quantification standard calibration curve, calculated their genome size according to the formula proposed by Wilhelm. The estimated results by this method are almost in accordance with the genome size established by genome sequencing. The results show that real-time PCR method is applicable to a variety of fungal genome size estimation, which can also be used to estimate the genome size of Rhizoctonia.We estimated the genome size of Rhizoctonia cerealis R0301 via the quantitative real-time PCR method and reported the genome size of R. cerealis R0301 was between 32.2-36.6 Mb firstly. Quantitative real-time PCR was a fast, highly accurate and reliable method for the genome size estimation of Rhizoctonia. Our research first reported the genome size of Rhizoctonia cerealis via the quantitative real-time PCR method, which is of great importance for the species classification, identification, phylogenetic analysis and its pathogenic mechanism, but also the foundation for whole genome sequencing and assembly.We cloned and sequenced the partial translation elongation factor 1 gene (tefA) of R. cerealis strain R0301 and other anastomosis group strains isolated from Shandong、HenanN Hebei and Anhui wheat field,and made a phylogenetic analysis of these strains. The cloned sequence is in accordance with the sequence obtained by PCR product, and there is no polymorphism between different clones. The phylogenetic tree shows that tefA gene can well distinguish Rhizoctonia anastomosis group and subgroup and there is no heterogeneity between the same anatomosis group and the same strain, which can be used for Rhizoctonia classification and identification. We also cloned and sequenced the partial translation elongation factor 2 gene (tefB) of R. cerealis strain R0301 and other anastomosis group strains and made a phylogenetic analysis of these strains. The phylogenetic analysis showed that, tefB can well distinguish Rhizoctonia and other AG aric classes, but the same anastomosis group have the genetic diversity, and tefA is more conservative than tefB gene.Finally, we analyzed the structure of the gene and the amino acid sequences translated based on the tefA gene sequence and translated amino acid sequence, from the secondary structure and tertiary structure, we can see that the main secondary structure of tefA is a-helix and crimped elements, no P-sheet and P-turn structure were found; There is a high similarity between the target protein sequence and the model sequence, It can be considered that the three-dimensional structure of the target protein and the three-dimensional structure of the model are similar.Our study indicated tefA gene was a single copy in the genome through Southern blot analysis, estimated the genome size of Rhizoctonia cerealis by real-time quatitative PCR. The phylogenetic analysis of different anastomosis group of Rhizoctonia shows that tefA gene can well distinguish Rhizoctonia anastomosis group and subgroup and there is no heterogeneity between the same anatomosis group and the same strain, te/B can well distinguish Rhizoctonia and other AG aric classes, but the same anastomosis group have the genetic diversity. Our research laid an important foundation for exploring tef gene as molecular markers for Rhizoctonia classification and identification.