The present study investigated the expression patterns of several candidate effector genes of Ascochyta rabiei using RNAseq data available in the Sequence Read Archive (SRA) database. Results revealed that the GSh302 gene exhibits a distinct expression profile during the fungus-plant interaction. Analysis of the gene sequence in genomic data from 250 isolates available in the SRA database showed that the sequence was highly conserved in A. rabiei isolates. Further comparative analyses across different Ascochyta species confirmed the presence of the sequence in all examined species. Multiple sequence alignment identified both conserved and variable regions of this gene, with the variable regions subsequently used to design specific primers. The distinct regions were used to design specific primers for the detection of A. rabiei isolates. The sequence of this gene was tracked in A. rabiei isolates collected from different farms in northeastern Iran during 2020–2022, as well as isolates related to pathotypes PI, PIII, and PVI. The findings demonstrated that a specific band could be amplified using the designed differential primers in all isolates of A. rabiei. Moreover, the fungus was detected in inoculated plants as early as 36 hours post-inoculation, while no amplification occurred in healthy controls. The findings of this study indicated that the gene, GSh302, which is involved in pathogenicity and shows expression in fungal isolates during interaction with host plant, can serve as a molecular marker for the identification and population diversity analysis of pathogens such as A. rabiei with closely related species like Ascochyta lentis, Ascochyta fabae and Ascochyta viciae.

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