| Summary: | An inherent issue in high-throughput sequencing applications is that they provide compositional data for relative abundance. This often obscures the true biomass and potential functions of fungi in the community. Therefore, we presented a high-throughput absolute quantification (HAQ) method to quantitatively estimate the fungal abundance in Daqu. In this study, five internal standard plasmids (ISPs) were designed for the fungal ITS2 subregion with high length variations. Five ISPs were then utilised to establish standard curves with a quantitative concentration range of 10<sup>3</sup>–10<sup>7</sup> cells/g, and this was used to quantify the core fungi, including Basidiomycota, Ascomycota, and Mucoromycota. Using three types of mature Daqu from different regions, we demonstrated that the HAQ method yielded community profiles substantially different from those derived using relative abundances. Then, the HAQ method was applied to the Daqu during fermentation. The initial formation of the Daqu surface occurred in the fourth stage, which was mainly driven by moisture. The key fungi that caused the initial formation of the Daqu surface included <i>Hyphopichia burtonii</i>, <i>Saccharomycopsis fibuligera</i>, and <i>Pichia kudriavzevii</i>. The initial formation of the Daqu core occurred in the fifth stage, which was mainly affected by moisture and reducing the sugar content. The key fungi that cause the initial formation of the Daqu core included <i>S. fibuligera</i> and <i>Paecilomyces verrucosus</i>. We conclude that the HAQ method, when applied to ITS2 gene fungal community profiling, is quantitative and that its use will greatly improve our understanding of the fungal ecosystem in Daqu.
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