Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers
Figure 3
Mapping a novel induced mutation.
(A) Southern blot of digested N. crassa genomic DNA with methylation-sensitive AvaII, showing loss of methylation in the AX7 mutant strain, as compared to the parental mutagenized N2977 strain. (B) Polymorphic RAD markers from N32 (red) and N2977 (green) parental strains were mapped along the seven N. crassa linkage groups. Red and green bars above the linkage groups are measures of linkage in the recombinant progeny, indicating the number of tightly linked markers in the local region. (C) A close-up view of linkage group II, showing the locations of confirmative RFLP markers (arrows). (D) RFLP marker confirmation. RFLP markers were designed using polymorphic RAD markers at 2.1 Mb and 3.1 Mb on LGII for N. crassa. The marker at 2.1 Mb confirmed the lack of recombinants in the wild-type pool, while a portion of individuals in the mutant pool have undergone recombination at this location. RFLP analysis at 3.1 Mb showed complete linkage in both wild-type and mutant pools to the methylation-deficient phenotype.