Study On Molecular Markers Of Color Gene Of Flammulina Velutipes

Color is one of the most important traits of Flammulina velutipes, and it is controlled by a pair of alleles. In our studies, the strain FL19(yellow),8801(white), their monokaryotics and their offspring (F1, F2 generations),as well as the F1 generations' monokaryotics were used. A reaction system which accommodated RAPD reaction was established by optimizing DNA extracting and RAPD conditions. Through the combination of RAPD and BSA, a molecular marker S3751800 linked tightly to the white gene was found, and was converted into SCAR marker, which performed better than RAPD marker at repetition and particularity. Major results are as follows:1. optimization of DNA extraction condition Experiment was designed by using table of orthogonal arrays for the extraction DNA with Benzyl chloride, at three levels , by four factors (mycelium cultivation, mycelium treating, concentration of Benzyl chloride, concentration of SDS) , nine treatments in total. The OD260 and electrophoresis results on the DNA extracted showed that treatment No.3 (mycelium was cultivated statically and abraded with liquid nitrogen, concentration of Benzyl chloride was 5ml/g mycelium, concentration of SDS was 20%) has better results than others.2. Optimization of RAPD amplifying conditions different concentration of Mg2+, dNTP, primer, template DNA, Taq DNA polymerase and different annealing temprature were tested respectively by momofactorial screening. Eventually we obtained the optimal reaction system as follows: 2.5 ul Buffer, 2mM MgCl2, 50ng DNA, 0.211M Primer, 100 u M dNTP, 1. 25U Taq DNA polymerase, and add ddH2O1 to 25 u1 as terminal volume.3. screening of RAPD markers linked to color gene By the principle of Bulked Segregant Analysis,five white color DNA samples and five yellow DNA samples were prepared and mixed into two DNA pools: one white, and the other yellow, 200 RAPD primers were screened to identify the markers linked to thecolor gene of Flammulina velutipes. A RAPD marker S3751800 which was tightly linked to the color gene was obtained, which was confirmed by testing 75 monokaryotics and 13 homokaryotics.4. Conversion of the RAPD marker into SCAR marker In order to convert the RAPD marker into SCAR marker, a 20-mer specific primer was constructed and used for PCR amplifying. The white color-linked SCAR marker was obtained. The test on the offspring of F1 generation, as well as 32 homokaryotics from different sources and 90 monokaryotics about 6 homokaryotics showed that monokaryotics and homokaryotics with different color could be identified with the SCAR marker. The molecular marker offered a powerful tool for selection of white color strains and could speed up the process of Flammulina velutipes Breeding programmes.