Efficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG box

Abstract

Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them. One of the pair of MAT genes, represented by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and mt a of Neurospora crassa (a pyrenomycete), encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. PCR with primer pairs corresponding to the borders of the C. heterostrophus and the N. crassa HMG boxes generated an ~ 0.3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not from MAT-1 and mt A strains. The C. heterostrophus primers amplified ~ 0.3-kb products from DNA of most loculoascomycete genera tested but not from DNA of pyrenomycete genera; this specificity was reversed with the N. crassa primers. The validity of the PCR procedure was documented by near sequence identity between the C. heterostrophus MAT-2 HMG box and PCR products from several Cochliobolus spp. and by cosegregation of the PCR product with mating type in progeny of Setosphaeria turcica and of Cryphonectria parasitica. Regions of the MAT locus flanking the HMG box were readily cloned using the TAIL-PCR procedure with a combination of random and specific primers.