Optimized AFLP System And AFLP Analysis Of T5 High-protein Content Wheat Mutants

It has obtained some fine mutants from hereditary improvement of crop and microbe by ion beam-induced mutation and ion beam-mediated transformation in our lab. But the mechanism of mutation was seldom studied by using molecular biology techniques. In order to provide scientific basis for the research of molecular mechanism, our study has established the optimized AFLP analysis system, respectively using wheat, rice and yeast mutants as materials. First, AFLP system suitable for wheat analysis was established by using T5 high-protein content wheat line 182809, after the optimization of four limiting factors in AFLP selective amplification: concentration of Mg²⁺, dNTPs, selective amplification primers and template DNA. And then, using transformed rice and CK to verify the established AFLP system, it was also suitable for rice genomic analysis. Finally, the AFLP system was used to analyze microbe genomic DNA. The result suggested that it was also suitable for yeast except that the concentration of Mg²⁺ should turn to 1.0mM from 1.5 mM.On the basis of optimized AFLP system, T5 high-protein content wheat lines, CK1 (acceptor wheat) and CK2(donor soybean) were analyzed. First, 64 couples of selective amplification primer were used to analyze two high-protein content wheat lines 204502,182808 and CK1. 34 couples of primer could produce different bands, and 15 primer combinations could produce the same different bands of 204502, 182808 compared with CK1. So the 15 primer combinations were selected out from 64 primer combinations and used to analyze eight high-protein content wheat lines, CK1 and CK2. As a result, the different bands between eight high-protein wheat lines and CK1 varied from15 to 29, and 23 on average; The rates of different bands varied from 2.54% to 4.92%, and 3.72% on average. According to the bands variety in DNA fingerprint, the result indicated that T5 high-protein content wheat lines have many differences at DNA level compared with control wheat. Now we are working for...