Construction Of Whole Genome Sequencing Library And Preparation Of Protoplasts Of Puccinia Striiformis F.sp. Tritici

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Complete genome sequencing of Pst will help better understand its physiological characteristics and pathogenesis, and it is also very important for preventing and controlling the occurrence and epidemicity of this disease. CYR-32 was used for complete genome sequencing of Pst by Solexa sequencing of Whole-Genome shotgun and paired-end Sequencing of Fosmid plasmid with the Sanger method. This study is groundwork of the complete genome sequencing project and mainly includes five respects as follows:1. We separated and purified the monospore of a physiological race used for complete genome sequencing of Pst. Then we identified the toxicity of the monospore by the traditional method. Finally CYR32 monospore was propagated to extract DNA for genome sequencing.2. Extraction of large fragments of DNA and construction of Fosmid genomic library. We successfully obtained large fragments of DNA from the germ tubes of uredinospores of Pst by an improved method. Main band DNA was centralized between 35 and 48 kb by pulsed field gel electrophoresis. Then we recovered the large fragments of DNA and constructed the Fosmid plasmid library. The library contains 20,000 clones and the recombination is 96.88%. The average length of the inserted fragments is 35 kb. There was no difference in the clone number of this library at the first day and the sixth day, indicating that the library was much stable.3. Extraction of high-quality DNA and construction of the Solexa PE library. DNA was extracted from the uredinospores of Pst and then purified, OD260/280 is between 1.8 to 2.0, which could satisfy the requirement of Solexa PE library. The Solexa PE library was constructed by inserting fragments of 200 bp, 500 bp, 2000 bp and 6000 bp.4. Paired-end Sequencing of 20,000 Fosmid clones and Solexa sequencing have been completed. Total splicing length of Pst genome is 136 Mb and the N50 of scaffold is 119 kb by analyzing the data of Solexa sequencing with SOAPdenovo, combined with the Scaffold constructed on basis of the data of Fosmid plasmid sequencing.5. Establish and optimize and preparation conditions of the protoplasm of Pst. Uredinospores were treated with 10ml buffer , including 10 ml 1.2 M KCl, 400mg driselase, 15 mg chitinase, 400 mg catenase, 400 mg cellulase, at 30℃for 54 h. The protoplasm of Pst were successfully obtained. This work helps to analyze karyotype by pulsed field gel electrophoresis and Scaffold localization.