# Yeast Diversity during Spontaneous Fermentations and
## 3 Results and Discussion
### Genetic Diversity of _S. cerevisiae_ by Interdelta Polymorphism Fingerprinting  
Since _S. cerevisiae_ represented the most abundant species in spontaneous fermentation, the 290 indigenous _S. cerevisiae_ isolates were subsequently analysed by Interdelta profiles to evaluate their genetic diversity. A total number of 33 distinct genotypes were obtained, and were coded NX1 to NX33 (Figure S1 and Figure 3A). Strains representing NX1 to NX9 genotypes had been isolated from the same vineyard from earlier vintages [36], indicating these indigenous _S. cerevisiae_ strains might have successfully colonised the vineyard over vintages. Besides vintage scales, the genetic diversity of indigenous _S. cerevisiae_ in vineyards can also be affected by the wide application of commercial starter cultures, which can be transferred from the winery either by insect vectors, or from liquid and solid winery deposits. Consequently, their subsequent sexual reproductive cycles would lead to the dispersal of commercial strains in the vineyard. Likewise, several studies have reported that  
Figure 2: The distribution and dynamics of yeast during spontaneous fermentation. (**A**): Colony numbers of yeast species in spontaneous fermentations with different grape cultivars. (**B**): Relative abundance of yeast species during spontaneous fermentation with/without SO\({}_{2}\) addition. The relative abundance of yeast species during SO\({}_{2}\)-free spontaneous fermentation with Semillon (**C**) and Cinsault (**E**). The relative abundance of yeast species during spontaneous fermentation with Semillon (**D**), Cinsault (**F**) treated with SO\({}_{2}\). Must, before fermentation; Middle, the middle stage of fermentation; End, the end stage of fermentation. a few _S. cerevisiae_ strains isolated from spontaneous fermentations shared the same genetic pattern with commercial yeast strains commonly used in the corresponding viticultural region [51; 52]. However, our results show that none of the Interdelta profiles of the 290 isolates were genetically related to that of Lalvin RC212, a commercial _S. cerevisiae_ strain which has been extensively used in Yuma Winery (Figure S1). Comparison has further been made to the published Interdelta profiles of other commercial strains (EC1118 and Levuline SEWA) commonly used by Chinese winemakers [16]. Again, no colonisation of these commercial yeasts in the vineyard has been found (Figure S1). Taken together, our research outcome suggests that natural biodiversity of _S. cerevisiae_ in Yuma vineyard may not be impacted by commercial starters, at least for the current single vintage. Future studies targeting indigenous _S. cerevisiae_ population over consecutive years in Yuma vineyard are essential to elucidate the influence of commercial cultures. Distribution of the _S. cerevisiae_ genotype was strongly associated with grape variety, which was further confirmed by ANOSIM analysis (R = 0.5934, \(p\) = 0.0001). The most diverse genotypes were observed in fermentations with Merlot (9 types), followed by Semillon (8 types), and Cabernet Gemischer (7 types) (Table S4), indicating the adaptation of specific _S. cerevisiae_ genotypes to the unique microhabitats formed by different grape cultivars. Generally, white wine fermentations harboured 11 distinct genotypes whilst the red wine fermentations contained 25 different genotypes (Table 1). NX10, NX11, NX14, NX15, NX17, NX18, NX19, and NX21 were only specific to white wine fermentations, while 22 genotypes were only found in red wine fermentations (Table 2). Particular attention should be paid to NX1, which was the most frequently encountered genotype in a wide range of spontaneous fermentations (except Cabernet Sauvignon, Cabernet Gemischer and Pinot Noir). Aside from NX1, albeit less frequent, NX13 and NX16 were also observed in both white and red wine fermentations. Given that the distance  
Figure 3: Interdelta fingerprinting of the indigenous _S. cerevisiae_ clustering of isolates from Ningxia region.