Developments Of Molecular Markers To Identify Wheat Stem Rust Races And Multi-PCR To Detect Three Biotrophic Diseases On Wheat

Wheat stem rust, caused by Puccinia graminis Pers. f. sp. tritici, is one of important worldwide airborne diseases. It has caused complete annihilation of wheat crops over wide areas with a long history, strong epidemic occurred in large areas causing severe damage. It is a serious threat to grain production safety in the world. In the long process of disease prevention and curation, it is the most popular method to control the disease through breeding and releasing resistant wheat varieties. However, the loss of wheat resistance is the prominent issue in deploying the disease resistant cultivars. Studies have shown that the breakdown of resistantace in wheat cultivars results from the variation and spread new of physiological races of the pathogen. So detecting the races and virulence of P. graminis f. sp. tritici is quite of importance for wheat breeding for resistance and preventing the disease.P. graminis f. sp. tritici is an obligate biotrophic pathogen. The traditional identification and virulence analysis of races are complicated and time consuming, and accurace is susceptible to the conditions. Moreover, the identification progress for the races with lower occurrence frequency was difficult owing to the limited hosts availiable. The advance of the researches about the population structure of P. graminis f. sp. tritici was affected by all those limitations.Lots of new approaches are rapidly supplied in various studies with the modern molecular methods. Molecular marker shows great advantages in detecting the population and the variation of pathogen races in different areas. In this study, three techniques were applied, inclueding AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat) and RAPD (Random Amplified Polymorphic DNA), for identifing the specific DNA fragments of the epidemic races so that to figure out the popular races of wheat stem rust (21C3CTH,21C3CPH,21C3CFH,34MKG,34C2MKK,34C2MKR, Ug99). This study was aiming at constructing the technology basis for efficiently monitoring the physiological races of the rust. In addition, it was the first time to establish the triplex PCR system for detecting P. graminis f. sp. tritici, P. triticina and Blumeria graminis f. sp. tritici in infected plant tissues simultaneously. The PCR-based method developed here could provide technical support for early rapid diagnosis and identification of the three kinds of diseases. The results of the study were summarized as following:1. Orthogonal experiment design was performed with 5 factors of the concentration of DNA, Mg2+, Taq DNA polymerase, dNTPs and random primer for the RAPD reaction system in P. graminis f. sp. tritici. On the basis of this, annealing temperature and circulation times were optimized. The results indicated that the RAPD reaction system of P. graminis f. sp. tritici was 1×Buffer,40 ng template DNA,1.5 mmol·L-1 Mg2+,2.0 U Taq DNA polymerase,0.25 mmol·L-1 dNTPs, and 0.40μmol·L-1 random primer in the 25μL reaction solution; 36℃ annealing temperature and 43 cycles.2. Six major physiological races of P. graminis f. sp. tritici were analyzed using 156 random RAPD primers. The results showed that among the 156 primers, stable and clear amplification patterns could be obtained by 121 ones. Vast genetic variation was found in the population of physiological races. A specific DNA fragment with about 1200 bp length was amplified from 21C3CTH with primer S8; an about 1700 bp specific DNA fragment was amplified from 21C3CFH DNA with primer S31; from 21C3CTH, we obtained an about 1100 bp specific fragment using primer S36; from 21C3CTH, a 782 bp specific DNA fragment was amplified with primer S92. Then, according to the sequence of the specific RAPD fragment from 21C3CTH by using primer S92, a new pair of PCR primers for sequence-characterized amplified region (SCAR) marker of 21C3CTH were designed, whereafter, it was validated that the marker was highly specific.3 A total of 7 physiological races of P. graminis f. sp. tritici were investigated for the DNA polymorphism by AFLP. Sixty-four pairs of AFLP primers were used to generate polymorphic bands. A 279 bp DNA fragment was amplified using E2/M2 primers in race 21C3CPH, a 171 bp DNA fragment by E7/M7 primers from race 34MKG, a 263 bp DNA fragment by E4/M6 primer combination in race 34C2MKK and a 388 bp DNA fragment by E7/M3 primer combination in race Ug99. The specific fragments were excised and then cloned into T-easy vector for sequencing. According to sequencing results, the SCAR markers were constructed by way of AFLP-markers. Great assistance was supplied for the molecular methods to detect the P. graminis f. sp. tritici races.4. The DNA polymorphism of 7 prevalent P. graminis f. sp. tritici races was analyzed by using 25 pairs of specific primers. The results showed that all tested primers were availiable for amplifing DNA fragments with polymorphism. Additionally, variations were found to exist among races. A 205 bp specific DNA fragment was amplified and obtained from 21C3CPH with primer set SSR180. A 170 bp length specific DNA fragment was amplified and obtained from Ug99 with primer set SSR6. All those fragments are repeatable.5. In this study, single-step triplex PCR was developed for the simultaneous detection of the three pathogens. Three sets of specific primers were selected, followed by optimization of the annealing temperature and the dNTP, Taq DNA polymerase, and primer concentrations for multiplex PCR to establish a method for the simultaneous detection of P. graminis f. sp. tritici, P. triticina, and B. graminis f. sp. tritici at an early stage of infection. The specificity and sensitivity of the triplex PCR were tested. We observed three specific bands of 395 bp,151 bp, and 464 bp on amplification with a three-reaction system. The detection sensitivity of the triplex PCR was 1 ng DNA for P. graminis f. sp. tritici,10 pg DNA for P. triticina, and 10 pg DNA for B. graminis f. sp. tritici.As summary, the above results indicated that the orthogonal method is an ideal method for optimization of RAPD amplification system because the optimization program is simplified, the reagent dosage is reduced, and the test cost is low, otherwise amplification bands are rich and clear; an attempt was made to develop a typical molecular method to specifically identify and monitor the most prevalent race in China. With the advantages of simplifying the rigmarole of conventional identification and shortening the period of identification, it is of important theoretical and practical significance for monitoring and controlling of physiological races, breeding and rational distribution of resistant varieties; this study pioneered the use of the triplex PCR system for simultaneous detection of P. graminis f. sp. tritici, P. triticina, and B. graminis f. sp. tritici in infected plant tissues. This novel PCR-based method will assist in the simultaneous diagnosis and monitoring of multiple plant diseases.