Genetic and molecular characterization of mating type genes in Cochliobolus sativus

Abstract

Genetic and molecular approaches were used to characterize the mating type (MAT) genes in Cochliobolus sativus. One hundred and four ascospore progeny derived from a cross of C. sativus isolates ND93-1 (MAT-1) X ND9OPr (MAT-2) were backcrossed with their parents to determine mating type, but only five progeny produced pseudothecia with asci and/or ascospores. When degenerate primers from the conserved high mobility group (HMG) protein domain encoded by the MAT-2 gene in Cochliobolus species were used in polymerase chain reaction (PCR) with genomic DNA of C. sativus as templates, an amplicon of predicted size was amplified only from MAT-2 isolates. The presence of a MAT-2 homolog in these MAT-2 isolates was confirmed by Southern hybridization with the HMG box as a probe. Additionally, the presence or absence of the HMG homolog in the progeny segregated in a 1:1 ratio, as expected for the single gene control of mating type. Using primers based on the conserved regions at the 5' and 3' flanks of the idiomorphs in the MAT genes of other Cochliobolus species, the full-length MAT-1 and MAT-2 idiomorphs were cloned by PCR from C. sativus isolates ND93-1 and ND9OPr, respectively. DNA sequence analysis indicated that these two idiomorphs are organized in a manner similar to their respective counterparts in other Cochliobolus species. DNA hybridization and PCR amplification analysis of 54 field isolates of C. sativus collected worldwide showed that both mating types exist in populations round the world. The low frequency of successful backcrosses of progeny to parents in the ND93-1 X ND9OPr cross, combined with the fact that many crosses between isolates of opposite mating type are unsuccessful, suggests that genetic factors other than MAT genes affect the fertility of the fungus.