| Trifolium pratense cv. Minshan is a special local legume resource with abundant isoflavone cultivated in alpine pasture of Gansu province, China. It is a vital crop in the animal husbandry and pharmaceutic industry because of its medical and feeding value. However, in the long planting process, it was seriously infected by powdery mildew(Uncinula bicornis(Wallr.: Fr.) Lév) due to slow renovation and severe degeneration. In this study,F1 generation of red clover, obtained from the hybridization of T. pratense cv. Minshan, highly susceptible to powdery mildew, and new red clover line resistant to this disease, was used as the population to construct the AFLP system. Red clover genetic linkage map was constructed and the QTL related to the powdery mildew resistance was located based on AFLP marker. The main results were as follows,(1) A suitable system of red clover based on AFLP markers was established by optimizing the main factors by repeated experiment: the 9μL about 200 ng/μL red clover DNA was digested with 8 U Eco RⅠand 6 U MseⅠat 37℃ for 5 h and then 65℃ for 15 min, ligation mixture was kept at 16℃ for more than 22 h. Pre-selective amplification was set up with 4 μL restriction- ligation sample DNA, which diluted 10 times, 0.8 μL Eco RⅠand 0.6 μL MseⅠpre-selective amplification primers, 2×PCR Master Mix 15 μL, dd H2 O was added up to 30 μL. In 30 μL selective amplification, 2 μL diluted(20×) DNA preμselective amplification product, 0.8 μL Eco RⅠand 1.0 Mse I selective primers. 2×PCR Master Mix 10 μL, dd H2 O was added up to 20 μL.Diversity detection showed that the genetic similarity coefficient of red clover new line and Minshan red clover was only 0.39 and they had distant genetic relationships. Primers including E3M2, E5M2, E7M2, E3M3, E4M3, E7M4, E2M5, E6M5, E5M6, E7M7 and E6M8 had 24 amplification bands and were chosen to do further experiments.(2) The AFLP amplification to the F1 generation of red clover and their parents showed that 11 pair of prime combination could amplify 197 bands and 165 polymorphic bands were included. The averaged polymorphic bands were 15 which acounted for 83.75% of the total bands. The genetic similarity coefficient for the 30 F1 generations and their parents ranged from 0.67 to 0.91. When the threshold value was 0.718, 32 materials were divided into 3 categories. The first and second group included one material which were female parenta(red clover new line) and male parent(Minshan red clover), respectively. All F1 generations were divided into the third group.(3)Seven linkage groups(LG1, LG2, LG3, LG4, LG5, LG6, LG7) were constructed while based on 149 AFLP markers were integrated into 7 linkage groups. It The covered a total map distance was of 640.5 c M and the average distance between two markers was with an average interval of 4.3 c M. The Nnumber of markers on each in linkage map group varied from 10 to 33 and 21.3 markets were averagly included on each linkage genetic map. The largested number of marker(33) was found on LG6, while the smallest number(15) was obtained on LG1. Among all the linkage maps, LG1 had the largest genetic distance(140.6 c M), included 15 markers and had the largest average distance(9.4 c M) between two makers, while LG4 had the smallest genetic distance(55.2 c M), included 31 markers and had the smallest average distance(1.8 c M) between two makers. Therefor, LG4 was the most intensive linkage map in all 7 maps.Five QTLs related to the powdery mildew resistance was detected after using the interval mapping method. Five QTL were mapped in LG4 and LG5 linkage groups respectively, among which, 4 QTLs distributed on LG4 and 1 on LG5. The contribution rates of 5 QTLs to the resistance of powdery mildew varied from 29% to 90%. They all had positive effect on the resistance of powdery mildew. The maximum effect was-1.11 and the minimum value was-2.22. It was also found that the marker E5M2-07 was closely linked to the trait. |
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