Cloning and characterization of the mating type ( MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp

• Published in: Fungal genetics and biology Vol. 39; no. 2; pp. 151 - 167
• Main Authors: Barve, M.P., Arie, T., Salimath, S.S., Muehlbauer, F.J., Peever, T.L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-07-2003
• Subjects: Amino Acid Sequence; Ascochyta; Ascochyta blight; Ascomycota - genetics; Ascomycota - growth & development; Cloning, Molecular; Didymella; DNA Primers; Fabaceae - microbiology; Fungal Proteins - genetics; Genes, Fungal; Genes, Mating Type, Fungal; HMG-Box Domains - genetics; MAT; Mating genes; Molecular Sequence Data; Open Reading Frames; Phylogeny; Polymerase Chain Reaction - methods; Sequence Alignment; Species Specificity; Mating genes; MAT; Phylogeny; Ascochyta blight; Ascochyta; Didymella
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/S1087-1845(03)00015-X

Degenerate primers designed to correspond to conserved regions of the high mobility group (HMG) protein encoded by the
MAT1-2 gene of
Cochliobolus heterostrophus,
Cochliobolus sativus, and
Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from
Ascochyta rabiei (teleomorph:
Didymella rabiei). A combination of TAIL and inverse PCR extended the
MAT1-2 sequence in both directions, then primers designed to
MAT1-2 flanking DNA were used to amplify the entire
MAT1-1 idiomorph.
MAT1-1 and
MAT1-2 idiomorphs were 2294 and 2693
bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to
MAT loci of other loculoascomycete fungi.
MAT genes were expressed at high levels in rich medium.
MAT-specific PCR primers were designed for use in a multiplex PCR assay and
MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore progeny from a cross of
MAT1-1 by
MAT1-2 isolates and to the mating phenotype of field-collected isolates from diverse geographic locations.
MAT-specific PCR was used to rapidly determine the mating type of isolates of
A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from 1:1 among isolates sampled from two commercial chickpea fields consistent with the hypothesis that these
A. rabiei populations were randomly mating. The mating type ratio among isolates sampled from an experimental chickpea field where asexual reproduction was enforced differed significantly from 1:1. A phylogeny estimated among legume-associated
Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal internal transcribed spacer (ITS) demonstrated the monophyly of
Ascochyta/Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of
MAT1-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation.
A. rabiei on chickpea is phylogenetically distant from other legume-associated
Ascochyta spp. and the specific status of
A. rabiei,
A. lentis,
A. pisi, and
A. fabae was confirmed by the HMG phylogeny