Broadly applicable mating type primers for morels: a case study on life cycle and genotyping integrity detection

A comprehensive understanding of a species' life cycle is crucial for advancing genetic breeding and cultivation strategies. Currently, the life cycle of species within the genus Morchella remains a topic of debate, and detailed analysis of the mating type gene structures is urgently needed. Here, we report the development of primers (Con_Mat1F/R and Con_Mat2F/R) targeting evolutionarily conserved domains of the Mat1-1-1 and Mat1-2-1 genes in Morchella. These primers were validated across 12 strains representing the Rufobrunnea, Elata, and Esculenta clades, enabling efficient genotyping and facilitating studies of the morel life cycle. Genotyping of single-spore populations of Mes-6, M. sextelata, and M. importuna revealed that each strain carries only one mating type gene, supporting heterothallism. Using these primers, genotyping of stipe and pileus tissues from ten cultivated populations (M. importuna, M. sextelata and M. eximia) revealed that all 189 pileus tissues carried both mating types, while some stipes harbored only one, reinforcing the heterothallic nature of these species and highlighting existing genotype imbalances. Additionally, amplification tests on large-scale, multi-batch tissue isolated strains revealed that 47.34% of the mating type genes were incomplete, a trend also observed in commercial strains. The incompleteness of mating type genes (genotype loss) may contribute significantly to the instability of cultivated heterothallic Morchella species, underscoring the importance of genotyping integrity screening prior to production. Collectively, our findings provide critical insights into the life cycle of Morchella and offer practical guidance for improving the stability of their cultivation.