## 2 Materials and Methods
### Yeast and Fungi Community Analysis
#### 2.4.3 Yeast Biodiversity Analysis  
* Meta-barcoding and high-throughput sequencing analysis (HTS)  
HTS analysis targeting rDNA 18S (fungal) was applied to all samples. DNA libraries for fungi were prepared according to the following protocol: a 350 base (on average) 18S rDNA gene fragment was amplified from each DNA sample with the universal primers FR1 (Amplicon PCR Reverse Primer **overhang adapter** = 5\({}^{\prime}\)**GTCTCGTGGGCTCGAGAGATGTATAAGAGACACATCCAATTCGGTANT) and FF390 (Amplicon PCR Forward Primer **overhang adapter** = 5\({}^{\prime}\)**TCGTCGGCGAGGTCAGATGTATAAGAGAGACGCG ATAAACGAGACCT (19). This first PCR targeting regions with specific primersincluding universal sequence primers (amplicon PCR primers) was performed in the laboratory. PCR reactions consisted of 2.5 uL of dilute template (DNAs standardized to 5 ng/uL), 5 uL of each Amplicon PCR Primer 1 uM, 12.5 uL 2X KAPA HiFi HotStart Ready Mix (Roche, Basel, Switzerland). Reactions were cycled for 3 min at 95 \({}^{\circ}\)C, then for 35 cycles of 98 \({}^{\circ}\)C for 30 s, 52 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 60 s, then followed by a final extension period of 8 min at 72 \({}^{\circ}\)C.  
The second PCR consisted of attaching indices and Illumina sequencing adapters using the Nextera(r)XT Index Kit (Juno Beach, FL, USA), made by Plateforme Genome-Transcriptome in Bordeaux. Finally, normalized pool libraries for the Illumina paired-end library were prepared, and cluster generation and 2 \(\times\) 250 bp paired-end sequencing (MiSeq Kit NANO v2) were performed on an Illumina MiSeq instrument.  
Data were subsequently imported into the Find Rapidly OTUs with Galaxy Solution (FROGS) Pipeline [41]. The sequences were cleaned as follows. Preprocess: paired-end assembled, with 5\({}^{\prime}\) primer, with 3\({}^{\prime}\) primer, with expected length (<300 and >400 bps), and without N. Then, sequences were dereplicated before being clustered using SWARM [42] with a local clustering threshold with a distance of 3. This single-link method is robust and independent of the sequence at which it is begun. Chimeras were removed with vasearch [43]: chimeras are sequences formed from two or more biological sequences joined together. The resulting sequences were filtered to remove singletons, using Filter phiX (contaminant databank). Taxonomic assignment of operational taxonomic units (OTUs) corresponding to 185 rDNA sequences was performed using silva132 18S [44] as the reference database. Sequences were filtered on BLAST with a percentage of identity (97%) and percentage of coverage (95%). An affiliation postprocess allowed inclusive amplicon ambiguities to be resolved and OTUs aggregated based on alignment metrics. Finally, OTUs corresponding to _Vitis_ sp. were removed.  
* Yeast diversity analysis by MALDI-TOF MS  
Total yeast population was quantified by the plating method on LT medium. The identification of 30 clones per sample was performed at species level following the manufacturer's protocol ([PERSON], Germany) as previously described and using the oenological laboratory specific database.