Accurate species-level identification of mushrooms is essential due to the extensive diversity in their morphology and ecological characteristics. Consequently, this study utilized the amplification of the internal transcribed spacer (ITS) region via polymerase chain reaction (PCR) to facilitate the molecular identification of mushroom species. The rDNA-ITS (ribosomal DNA internal transcribed spacer) region of genomic DNA from 10 wild mushroom accessions collected in Sulaimani Province, Iraq, succeeded amplification using ITS1 and ITS4 primers. The PCR amplification products ranged in size from 680 to 800 bp and underwent comparison with sequences in the National Center for Biotechnology Information (NCBI) database. When comparing with GenBank data, BLAST (Basic Local Alignment Search Tool) analysis revealed the identified mushrooms had sequence identities ranging from 93.8% to 100%. All identified mushroom accessions were at the species level, with all being newly documented species in Iraq. These include Volvariella bombycina (PP921334.10), Collybia nuda (PP921333.1), Amanita crocea (PP921336.1), Melanoleuca rasilis (PP921332.1), Stropharia coronilla (PP921331.1), Amanita lividopallescens (PP410315.1), Macrolepiota orientiexcoriata (PP410313.1), and Phallus hadriani (PP410312.1). Two of them (PP921329.1 and PP921330.1) belonged to the same species, Melanoleuca leucopoda. Phylogenetic analysis revealed a close evolutionary relationship among the identified mushrooms. This work discovered the originality of the mushroom species, which expands the Iraqi mushroom diversity.

Keywords: Wild mushrooms, species, internal transcribed spacer (ITS), macrofungi, phylogenetic analysis, rDNA-ITS, evolutionary relationship

Key findings: The molecular identification of wild mushrooms by internal transcribed spacer (ITS1 and ITS4 primers) facilitated precise taxonomic categorization and successfully distinguished the closely related species. Phylogenetic analysis elucidated genetic links, offering evolutionary insights and accurately confirming species identification.