The Molecular Markers Of Puccinia Striiformis F.sp.Tritici In China

Yellow (stripe) rust ,caused by Puccinia striiformis f. sp. 7nYz'c/(PST), is one of the most important diseases of wheat in China. The current strategy for management of wheat stripe rust is use of resistant cultivars. However, this economic strategy has been hampered by the resistance breakdown of wheat cultivars. Previous research indicates this phenomenon was caused by the occurrence and development of new races of PST. As an obligate fungus, the race identification and the virulence analysis of PST were complicated and time consuming. The identification results were affected by the environmental condition. Because of the limited differential number, it is very difficult to find the low-frequency race and to understand population genetic structure of this pathogen in nature.In recent years, the molecular biology provides us new way to monitor the constitution and change of PST races, to analysis the population structure and genetic relationship. As the inheritance background of PST was indistinct, this paper is planning to open out the genetic diversity between or within PST races using RAPD technique. In order to establish a practical molecular identification system of PST, specific molecular markers of the prevailing races in China were searched on a large scale. The results were obtained as follows:1. The genomic DNA extration methods of the wheat stripe rust were improved and compared. With the procedure mentioned in this paper, approximately 300~700ug of total DNA can be obtained from Ig urediospores. The result indicats that the CTAB method is better than the PhCH2Cl method in searching the special molecule markers.2. In order to facilitate communion, a standard RAPD system is also developed in this research: 10 × Reaction Buffer 2.5μl, MgCl2 2mmol/L, dNTPs 0.15mmol/L, DNA template 40ng, primer10ng, Taq 1.5U, ddH2O 15.5μl. Amplification program: 5minat94℃ for initial denaturation, then 45 cycles that consisted of 30s at 94 ℃ , 40s at 36℃ and 90s at 72 ℃, followed by a final 7min extension at 72 ℃.3. Of the 210 random primers analyzed in this research, 134 primers can show us steady amplification profiles. The results obtained from 26 single-spore isolations of 5 prevailingraces in China indicate that the PST still has some consistent DNA sequence, but the genetic variations between races were abundance as a whole. The RAPD profiles of different single-spore isolations in each race were identical, so the inheritance background of single race was coherent.4. Five Chinese races of P. striiformis f. sp. tritici were analysed by RAPD technique in this paper. The special DNA fragments of race CY31, CY29, CY23 and pathotype Shuiyuan were founded. Two special DNA fragments of race CY31, CY29 were cloned and sequenced. Based on the sequencing result, two pairs of specific PCR primers were designed and the SCAR markers for race CY31, CY29 were obtained successfully.Rapid identification of the races of Puccinia striiformis f. sp. tritici by molecular technique was very important to monitor and control of wheat stripe rust in China. The results here indicates that a molecular identification system for the PST races could be established by means of cosmically searching special RAPD fragments of different races. Using such system, the identification time of PST would be cut to a few days. This would be essential for developing more effective strategies in management of wheat stripe rust in China.