## 1 Introduction  
Sourdough is a mixture of cereal flour and water, fermented by a complex biological ecosystem, consisting of different genera, species and strains of lactic acid bacteria (LAB) and yeasts, which interact among themselves, often establishing stable associations. During fermentation, yeasts are primarily responsible for the leavening, LAB for the acidification of the dough, and both produce a wide variety of compounds (organic acids, alcohols, aldehydes, esters, ketones, bacteriocins) that act in a synergistic way to prevent or eliminate deleterious microbial contaminations, improving the shelf-life of baked goods. Moreover, microbial compounds contribute to unique flavours and textures of the diverse fermented bread, enhancing also their nutritional and nutraceuticalul quality [1]. The production of bioactive metabolites varies significantly among the different LAB and yeast strains occurring in each sourdough [2; 3], as the result of their genetic intraspecific diversity. Therefore, the molecular and functional strain-level characterization of sourdough microbiota is crucial to give value to traditional or origin protected baked end-products, to develop innovative starter cultures and to design functional cereal-based foods. To this aim, molecular tools for the intraspecific characterization of sourdough microbiota are fundamental, though there is no universally applicable typing method to discriminate among isolates from the same ecological niche, harbouring different microbial communities well adapted to the environment and often closely related among them [4]. [PERSON] et al. (2013) revealed the composition of the LAB microbiota of 18 Italian traditional sourdoughs, using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique with P4, P7 and M13 primers [5]. Multiplex RAPD-PCR with two singly (P4 and MV1) and two combined (OPL-05 + RD1 and P1 + RD1) primers was utilized by [PERSON] et al. (2012) for typing _Lactobacillus surfrancisensis_ isolates from traditional Italian sourdoughs [6]. Another technique, which has been found particularly useful for differentiating sourdough LAB at the strain level, is repetitive element sequence-based (rep)-PCR using (GTG)\({}_{5}\) primer. Such a method was applied by [PERSON] et al. (2016) to analyse a total of 246 LAB strains isolated from 15 different samples of Chinese traditional sourdoughs [7]. Many studies have focused on the intraspecific characterization of LAB, while only a few investigated sourdough yeasts. Among DNA-fingerprinting methods, yeast intraspecific heterogeneity has been studied using RAPD- and rep-PCR. The former has been successfully used, with M13 and RF2 primers, to differentiate three distinct biotypes out of 58 _Saccharomyces cerevisiae_ strains from southern Italian sourdoughs [8]. Recently, [PERSON] et al. (2019) revealed a high polymorphism among the _77 S. cerevisiae_ isolates from Tuscan sourdoughs, using inter-delta region analysis [9]. Rep-PCR with (GACA)\({}_{4}\) primer was utilized to reveal the composition of the yeast microbiota of 18 traditional Italian sweet sourdoughs and 19 typical Italian bread sourdoughs, respectively [5; 10]. [PERSON] et al. (2014), using intron splice site dispersion (ISS-PCR) and (GTG)\({}_{5}\) rep-PCR analyses, discriminated different strains within the species _Candida milleri_ (now reclassified as _Kazachstania huminils_) [11]. Considering that a high genetic intraspecific diversity of sourdough microbial communities may provide differential functional features affecting nutritional, organoleptic and technological properties of leavened baked goods, here, for the first time, we investigated the molecular diversity of Protected Designation of Origin (PDO) Tuscan bread sourdough microbiota at the strain level.