Establishment Of SCAR Markers For Mycogone Perniciosa Magn

The method for fast identification of the species and subspecies of Mycogone perniciosa Magn were established by Molecular means in this research. Three kinds of molecular markers technology including random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP) were used to estimate genetic variation among 49 strains of M. perniciosa which come from Culture Collection Library of Mycological Research Center in Fujian agricultural and forest university. Transferred the specific fragments into SCAR markers and the SCAR markers were utilized to do multiple-PCR. DNA of M. pernicinsa from the cover soil of suscepted Agaricus bisporus were extracted to verify the accuracy of the SCAR markers.DNA of 49 strains of M. perniciosa were used to screen the primers of ISSR, RAPD and SRAP. The polymorphism fragments obtained with the selected primers were used to make presence versus absence (1/0) table and do cluster analysis by the unweighted pair-group method algorithm (UPGMA) of system clustering method.11 ISSR primers,4 RAPD primers and 4pairs of SRAP primers were obtained in the screen work.49 strains of M. perniciosa were divided into two big groups, and each big group was also divided into two small groups.The polymorphism bands were reclaimed, cloned and transferred into SCAR primers. There were 13 pairs of SCAR primers were synthesized successfully. The subgroups of each big group were distinguished according to the presence or absence of bands. So, the subgroup with dominant marker of the first group was named the third group, and the subgroup with dominant marker of the second group was named the fourth group. The first group got 4 pairs of SCAR primers, the second group got 3 pairs of SCAR primers, the third group got 1 pairs of SCAR primers and the fourth group got 3 pairs of SCAR primers. Two pairs of co-dominant SCAR primers were obtained.Multiple-PCR was constructed to detect the accuracy and repeatability of SCAR primers. Different groups of SCAR primers were mixed to do multiple-PCR and double-PCR which mixed with primers of the first and the second group and mixed with primers of the second and the fourth group were obtained. The cluster result was corresponded with the result of ISSR, RAPD and SRAP.The M. perniciosa were used to infect experiment for A. bisporus, DNA of M. perniciosa from the cover soil of suscepted Agaricus bisporus were extracted when infection was successfully. The SCAR primers were used to identify whether the DNA of M. perniciosa were existed in the total DNA and determine its group. The goals of further validation for the accuracy and repeatability of SCAR primers was also achieved.