Amplified Fragment Length Polymorphism Of Tilletia Foetida And Development Of A Specific SCAR Marker For Detection Of The Fungus

wheat is one of the most important crops in the world, and it is main foodstuff in our country. Common bunt of wheat, caused by Tilletia foetida (Wallr.) Liro. (syn.T.lavis Kuhn) ,is a destructive disease on wheat. The traditional methods for diagnosis and detection of the disease was mostly based on the morphological characteristics, germination test of teliospores, and the symptom of disease in the field, resulting in a long procedure and low accuracy. So, it is imperative to develop a molecular assay for rapid identification and accurate detection of this disease.5 isolates of Tilletia foetida, 3 isolates of Tilletia caries (DC.) Tul.(syn.T. tritici(Bjerk)Wint.), the causal agent of common bunt, and several related species were collected from the United States and China, respectively. SDS method was compared with the modified CTAB, Benzyl chloride (BC) method in the genomic DNA extraction of dormant teliospores of Tilletia foetida.The results showed the DNA extracted by SDS buffer was better than that by the BC and CTAB method whatever on purity and quantity.Amplified fragment length polymorphism(AFLP) was regarded as the best techniques of DNA molecular markers. It is used in many fields. AFLP techniques were used to analyze the DNA polymorphism of the isolates in our study. After cut by Mse I and EcoR I ,ligated by adaptors, pre-amplified and at last 132 pairs of AFLP primers were used to sel-amplified. And the primers generated polymorphic bands and one specific DNA fragment was amplified by M09/E04 primer combination in Tilletia foetida. The specific DNA polymorphic fragment, a 286 base pair band, was excise, purified and cloned into T-easy vector. Based on the sequence of the polymorphic DNA fragment, one specific primer pair was designed with the software of Primer Premier 5.0(SC1:5'-ACAAGTTGGAGTCAGGAG -3'; SC2:5'- ATCGTATGGTTTCGTCTT -3')- The AFLP marker was transformed to a sequence characterized amplified region(SCAR) marker, and all isolates were tested with the SCAR marker. The results of detection using the specific primers SC1/SC2 in the pathogen isolates tested indicated an unique DNA fragment was amplified in all Tilletia foetida isolated, and no any DNA fragments was obtained in the other alien fungus species, displaying an accuracy of 100%. The results provide an useful aid to test species of Tilletia foetida.