Molecular Characterization Of Pina Gene In Einkorn Wheat And Biocontrol Agents Of Wheat-pseudomonas Fluroscence

Wheat is one of the most important grain crops in the world with its strong adaptability, wide spreading and multi-purpose. Its total growth area, yield and trade took the first place among all the grain crops respectively. There is 1/3 of world population fed on wheat product. In China, wheat is the dominating crops just on the neck of rice. Nowadays, more and more researchers focused on the study of wheat seed quality and disease resistant. Our study is mainly about the molecular characterization of Pina gene of einkorn wheat and the potential biocontrol agent of wheat pathogens-Pseudomonas fluorescens. The following are key results:1. Fifty-six sequences encoding the pina protein were characterized from three species or subspecies of einkorn wheat. These sequences contained 1,595 nucleotides, including 1,270 conserved sites,21 single nucleotide polymorphisms (SNPs), and 16 indels. The average frequency of SNPs and indels was one out of 76.1 and 99.9 bases, respectively. Five SNPs and no indels were found in the translated sequences. Fourteen haplotypes were defined, and the accessions in each haplotype ranged froml to 18. There were nine haplotypes in Triticum monococcum ssp. aegilopoides, eight in T. monococcum ssp. monococcum, and two in T. urartu. Phylogenetic analysis showed that Pina genes from different species or subspecies could be clearly differentiated based on the open reading frame. Genes from T. urartu grouped together, whereas genes from T. monococcum ssp. aegilopoides and T. monococcum ssp. monococcum were shared by three and two clusters, respectively. Both the haplotype and phylogenetic analyses indicated that T. monococcum ssp. aegilopoides was more diverse. These results would contribute to the understanding of functional aspects and efficient utilization of Pina genes.2. In our study, a polyphasic molecular approach was used to study the biodiversity of close to 300 fluorescent Pseudomonas strains isolated from long-term (>30 years) mineral fertilization experimental agricultural plots.3. One hundred and forty-three pseudomonads were isolated from one of the field plot were chosen for more intensive studies. The strains were grouped into two distinct phenotypes (Form I and II) based on colony morphology and growth patterns on King's B medium. Further characterization was performed by electron microscopy, BOX, ERIC, Miniprimer PCR and 16S rRNA and gacA sequencing. The genomic fingerprints from ERIC and BOX were similar but not identical. As expected, fingerprints derived from miniprimer PCR exhibited comparatively lower correlations to ERIC and BOX. Sequencing and phylogenetic analyses of the 16S rRNA gene identified three major groups, consisting of P. fluorescens, P. koreensis and P. putida. Tajima's D statistics applied to the 16S rRNA sequences of the two subpopulations indicated an excess of low frequency polymorphisms within strains of form I, suggesting a population size expansion and or positive selection. This is corroborated by the 16S rRNA BLAST search in that the form I strains are more diverse. The methods employed allowed for the characterization of the bacterial strains leading to a tentative identification of potentially a new species within the genus Pseudomonas. The characterization also revealed the potential presence in Canadian soils of Pseudomonas koreensis, a recently described new species, leading to the conclusion that this new species could have a wider distribution.4. The characterized bacterial strains were evaluated in vitro for their ability to suppress major soilborne fungal pathogens of wheat, leading to the identification of 12 effective bacterial biocontrol agents. The potential biocontrol strains phenotypically and genotypically characterized relative to a well-known biological control strain pf-5. Phenotypic methods used included Biolog, API 20NE assays and FAMEs. Genotypic methods used included REP-PCR and gacA gene sequencing, the phylogenetic tree inferred from all the 52 type strains of P. fluorescens subgroup from flurescent Pseudomonas genus and the isolated strains. By using phenotypic tests, the cluster results were similar with each other except some small differences by dividing these strains into two groups with a close relation with Pf-5 together, which was supported by gacA sequence analysis. API 20NE, Biolog PM and FAME proved to be the best for characterization of isolates, whereas 16S rRNA sequence analysis were found to be the best resolution for preliminarily identification of these newly isolated strains.