Efficient PCR - based Approach for Rapid Identification of Earth Star Mushrooms Employing Species-Specific Primers

<i>Astraeus</i> species are valuable edible ectomycorrhizal mushrooms, particularly in Asia. The partial hypogenous nature of <i>Astraeus</i> fruit bodies in soil present challenges for sample collection and studying species distribution. In this study, we developed a PCR-based approach for identifying <i>Astraeus</i>. Two universal primers (AUPF1/AUPR1 and AUPF3/AUPR3) and specific primers for <i>A</i>. <i>asiaticus</i> (AAF4/AAR4), <i>A</i>. <i>odoratus</i> (AOF4/AOR4), and <i>A</i>. <i>sirindhorniae</i> (ASF2/ASR4) were designed based on the alignment of internal transcribed spacer sequences from various <i>Astraeus</i> species. Primer verification was performed by generating amplicons from extracted DNA of <i>Astraeus</i> fruit bodies and 130 soil samples collected from beneath various host plants of <i>Astraeus</i> spp. These novel primers were efficient and precise in identifying <i>Astraeus</i> species. Our results have implications for multi-sample assays for <i>Astraeus</i> identification and investigations into species distribution through large-scale inventories.