The Genetics of Non-conventional Wine Yeasts: Current Knowledge and Future Challenges

Saccharomyces cerevisiae is by far the most widely used yeast in oenology. However, during the last decade, several other yeasts species has been purposed for winemaking as they could positively impact wine quality. Some of these non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, Lachancea thermotolerans, etc.) are now proposed as starters culture for winemakers in mixed fermentation with S. cerevisiae, and several others are the subject of various studies (Hanseniaspora uvarum, Starmerella bacillaris, etc.). Along with their biotechnological use, the knowledge of these non-conventional yeasts greatly increased these last 10 years. The aim of this review is to describe the last updates and the current state-of-art of the genetics of non-conventional yeasts (including S. uvarum, T. delbrueckii, S. bacillaris, etc.). We describe how genomics and genetics tools provide new data into the population structure and biodiversity of non-conventional yeasts in winemaking environments. Future challenges will lie on the development of selection programs and/or genetic improvement of these non-conventional species. We discuss how genetics, genomics and the advances in next-generation sequencing will help the wine industry to develop the biotechnological use of non-conventional yeasts to improve the quality and differentiation of wines.


INTRODUCTION
In oenology, alcoholic fermentation is generally performed by Saccharomyces cerevisiae yeast, the "conventional" wine yeast. Currently, the winemakers have the choice between hundreds of S. cerevisiae starters that have been selected for various characteristics including their ability to complete alcoholic fermentation in oenological conditions, their low release of off-flavor compounds, their positive impact on wine aromas, etc., (Pretorius, 2000;Marullo and Dubourdieu, 2010). The growing demand for more diversified wines or for specific characteristics (low ethanol content, etc.) has led to the exploration of new species for winemaking. These non-conventional yeasts may contribute to the wine's flavor and taste by producing a broad range of secondary metabolites and extracellular enzymes (Hong and Park, 2013;Ciani et al., 2014;Wang et al., 2015). Some species could be interesting for alcohol level reduction in wine (Masneuf-Pomarede et al., 2010;Bely et al., 2013) or for greater fermentative ability in harsh conditions due to enhanced fructophily (Sutterlin, 2010;Magyar and Tóth, 2011). It has to be noted that, as only some Saccharomyces species (i.e., S. cerevisiae, S. uvarum, and some interspecific hybrids) are able to consume all the sugar contained in grape must, non-Saccharomyces yeasts must be used in co-or sequential-fermentation with a Saccharomyces spp. able to secure AF completion (Jolly et al., 2006;Bely et al., 2013).
The wine industry currently proposes starters of a few nonconventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, Lachancea thermotolerans, etc.), while several other species (Hanseniaspora uvarum, Starmerella bacillaris, etc.) are the subject of various studies to assess both positive contribution (Table 1) and negative impact (if any) on wine quality Maturano et al., 2015). These non-conventional yeasts are widely distributed amongst the Saccharomycetales (Figure 1). In order to evaluate the oenological potential of a given species, several strains are usually compared for phenotypes of interest like fermentation ability (Renault et al., 2009) or glycerol production (Magyar and Tóth, 2011). However, in most cases, neither the relationships between the tested strains are described, nor the genetic structuration of the species is known. This lack of genetic knowledge is clearly detrimental, since we are not able to determine whether the phenotypic diversity described is representative of the species or not. The recent advances in next-generation sequencing (NGS) have triggered the development of genomic and genetic tools for some of these non-conventional yeasts, but the field is still in its infancy. The objective of this paper is thus to review the current state-of-art of the genetics of non-conventional wine yeasts and to discuss the future prospects and challenges from an oenological viewpoint.

BASIC GENETIC KNOWLEDGE OF WINE YEASTS
As a model organism, the genomic outline of S. cerevisiae is well-known: its genome size is around 12 Mb organized in 16 chromosomes, with a mitochondrial genome of 85 Kb ( Table 1). The genome sequences of several hundreds of strains of various origins are available, and much more sequences are produced easily using NGS technology and subsequently assembled even by lab with moderate bioinformatics skills. The population genomics of S. uvarum has been improved recently with the sequencing of more than 50 strains of various origins (Almeida et al., 2014). The type strain CBS7001 T has a genome size of 11.5 Mb and 16 chromosomes (Cliften et al., 2003). By contrast, such basic knowledge (genome size, chromosome number, etc.) is available only for a small number of nonconventional wine species: T. delbrueckii has a genome of 9-11 Mb distributed on eight chromosomes; L. thermotolerans has a 10.4 Mb genome with eight chromosomes. Other wine yeast species usually have genome size ranging from 8 to 12 Mb, with chromosomes number unknown yet (P. kluyveri, M. pulcherrima, etc.). Moreover, there is still a lack of reference genome sequence for several non-conventional wine yeasts of interest like S. bacillaris, P. fermentans, etc., (Table 1). Disparities exist also for the mitochondrial genome, with full sequences available for some species like L. thermotolerans or H. uvarum, and partial sequences for other species (C. stellata, P. membranifaciens, etc.). Thus, although the genomic data of non-conventional wine yeast greatly increased this last decade, there is still a lot of work to achieve in this field.

THE LIFE-CYCLE OF WINE YEASTS
The life cycle of Saccharomyces wine species is well-known: both S. cerevisiae and S. uvarum are diploid species that divide asexually by mitosis. They are able to enter meiosis and form asci containing generally four haploid spores (tetrads). While haploid cells can undergo mitosis, the haploid level is generally transient and crosses between haploid spores of opposite mating types are readily observed, leading to diploid zygote formation. Moreover, haploid cells are usually able to switch mating type at mitosis (homothallism). The physical proximity between mother and daughter haploid cells of opposite mating type usually results in high level of inbreeding (Ruderfer et al., 2006;Cubillos et al., 2009;Warringer et al., 2011). Variations in this breeding system were described for S. cerevisiae like near-dioecy or higher level of outcrossing, but seemed quite rare and associated with environmental specificities (Knop, 2006;Al Safadi et al., 2010;Murphy and Zeyl, 2010).
By comparison, the precise life-cycle of most non-Saccharomyces yeasts is unknown yet. Sporulation was observed for most non-conventional yeast, albeit forming non-tetrad asci in many cases (T. delbrueckii, D. hansenii, H. vinae, etc., Table 1). No evidence of sporulation ability was recorded to date for Starmerella/Candida species. Data regarding the occurrence of sexual reproduction is usually scarce for most non-Saccharomyces yeasts, so classical genetic manipulations are impossible to date. To circumvent this limitation, both intra and inter specific hybridizations by protoplast fusion can be achieved as demonstrated in the past (Ball, 1984;Pina et al., 1986).
The basic ploidy level is also usually unresolved ( Table 1): T. delbrueckii has been considered as a haploid species for a long time, but the detection of several strains harboring several loci with two alleles (26.4% of strains showing heterozygosity), its ability to sporulate and the presence of mating type genes is more congruent with a diploid status (Albertin et al., 2014a). Conversely, for S. bacillaris, the proportion of heterozygous strains was almost null (0.01%). This, combined with its inability to sporulate, is more consistent with an hypothesis of an haploid status  but has still to be formally demonstrated. Finally, despite its fully sequenced genome, the ploidy status of L. thermotolerans is controversial: haploid or diploid depending on the authors (Souciet et al., 2009;Freel et al., 2014). In conclusion, the biological lifecycle of many non-Saccharomyces yeasts remains to be elucidated.

ECOLOGY OF WINE YEAST
Most wine yeasts can colonize several ecological niches, including wine-related environments like grape, must, winery equipment and premise (Table 1). Moreover, many of them can be isolated from other human-associated processes (brewery, bakery, dairy,

2000)
Controversial: haploid (Freel et al., 2014) or diploid (Souciet et al., 2009) One to four spores per ascus (Barnett et al., 2000).   (Kimura, 1980), phylogenetic tree was built using Neighbor joining clustering method and bootstrapping (1000 replicates) was used to assess the robustness of the nodes by means of R package ape (Paradis et al., 2004). Schizosaccharomyces pombe was used as outgroup species. bioethanol, distillery, etc.) and also from wild substrates (soil, insect, plant, etc.). Isolation from clinical specimens is rarely described yet possible (yeasts being opportunistic microorganisms), and most wine yeasts are Generally Recognized As Safe (GRAS). Dissemination and transfer between the different ecological reservoirs could be performed through insects (Parle and Di Menna, 1966;Stefanini et al., 2012;Palanca et al., 2013), but also through human activities like material exchanges, etc., (Goddard et al., 2010). Indeed, although most wine yeasts are described as ubiquitous from an ecological viewpoint, some species have a restricted substrate range. This is the case of H. guillermondii and Starmerella species for example, which are very rarely isolated from non-wine-related substrates . Thus, the study of most wine yeast should consider not only wine strains but also isolates from other technological processes and substrates in order to assess their biodiversity.

ADAPTATION TO WINEMAKING ENVIRONMENTS AND EVOLUTIONARY MECHANISMS
Wine environments are particularly harsh and inconstant: winemaking is a seasonal practice, so that yeasts present at the surface of grape berries at harvest suddenly have to survive in grape must containing high sugar concentrations, usually with sulfur dioxide content. Moreover, from an ecological viewpoint, the ensuing alcoholic fermentation is a rapidly fluctuating ecosystem: within a few days, grape must is depleted of nitrogen nutrients, while ethanol concentration and temperature increase steadily thanks to Saccharomyces spp. metabolism, thus conferring a fitness advantage for Saccharomyces spp. over the other wine yeasts (Goddard, 2008;Salvadó et al., 2011). In addition, the range of temperature can be quite high, with either short-term variations (daily variations) or long-term evolution (seasonal variations). As a result, within wine yeast species, some strains show specific wine-adaptation  like sulphite resistance (Divol et al., 2012), ethanol tolerance (García-Ríos et al., 2014), low pH adaptation (Pretorius, 2000), temperature adaptation (Naumov et al., 2000), etc. The underlying adaptive mechanisms vary greatly from one species to another: in S. cerevisiae, molecular approaches identified allelic variations as molecular causes of adaptation to the winemaking process (Aa et al., 2006;Marullo et al., 2007;Ambroset et al., 2011;Salinas et al., 2012;Jara et al., 2014). At the chromosome level, translocations were shown to be responsible for adaptation to sulfite (Zimmer et al., 2014). Polyploidy and hybridization are also major evolutionary processes that probably triggered adaptation to wine environments (Borneman et al., 2012;Erny et al., 2012) and are currently explored for biotechnological application (Timberlake et al., 2011;Plech et al., 2014;Blein-Nicolas et al., 2015;. Large genomic introgressions were evidenced in S. uvarum strains associated with human-driven fermentations, suggesting a link between introgressions and domestication (Almeida et al., 2014). Various horizontal gene transfers were also evidenced for wine S. cerevisiae strains (Novo et al., 2009), and were shown to favor adaptation to the nitrogen-limited wine fermentation environment (Marsit et al., 2015). Other evolutionary mechanisms were described (Dujon et al., 2004;Barrio et al., 2006;Scannell et al., 2007), and it is highly probable that further investigations will allow the identification of additional adaptation processes in wine yeasts. In particular, it could be interesting to focus on transposon families and their possible implication in environmental adaptation (Zeyl, 2004;Sarilar et al., 2015), to explore the impact of mitochondrial genome variation regarding adaptation to wine environments and practices (Picazo et al., 2015;Wu et al., 2015) or to describe the landscape of gene duplication and prion involvement in fitness issues (Landry et al., 2006;Jarosz et al., 2014). However, to date, most of these data were obtained from Saccharomyces species and could now be obtained from non-Saccharomyces of interest.

POPULATION GENETICS OF YEAST SPECIES ASSOCIATED WITH WINEMAKING
Within a given species, the colonization of different ecosystems can led to the evolutionary differentiation of the subpopulations, in relationship with their adaptation to environmental specificities. This is the case of S. cerevisiae species that shows genetic subgroups of wild and domestic strains associated with human activities like wine, bread, beer, sake, etc., (Fay and Benavides, 2005;Liti et al., 2009;Sicard and Legras, 2011;Almeida et al., 2015), that probably originated through multiple domestication events (Schacherer et al., 2009). In a recent study, Almeida et al. (2014) showed that S. uvarum was also divided in genetic subgroups, one of domestic strains used in both winemaking and cidermaking and associated with the northern hemisphere, while others subgroups were composed of wild isolates from South America and Australasia. The current hypothesis is that a Patagonian "wild" sub-population gave rise to the domestic subpopulation through a recent bottleneck (Almeida et al., 2014). Another wine species was recently described as domesticated: T. delbrueckii is also divided in genetic subgroups of wild and domestic strains (Albertin et al., 2014a). Moreover, the wine/grape-related group showed an increase ability to ferment sugar in oenological condition, confirming the occurrence of phenotypic domestication . By contrast, no hint of domestication was recorded to date for S. bacillaris and H. uvarum whose genetic diversity is shaped by geographical localization and/or time variation Albertin et al., 2016).
In addition to population genetic clustering, microsatellites allow measuring the genetic diversity of a given species in specific conditions. In S. cerevisiae, the genetic diversity varied greatly, from 0 (fully clonal populations) to 1 (fully diversified population, Table 1). The precise impact of S. cerevisiae diversity (or absence of diversity) on wine quality is still debated/studied (Egli et al., 1998;Howell et al., 2006;King et al., 2008) and the direct link between microbial diversity and wine complexity should be considered with caution. S. uvarum and T. delbrueckii showed also a large range of diversity (0.

FUTURE CHALLENGES
Definite progresses in the genetics of non-conventional yeasts were made in the last decade. However, there is still a great lack of data compared to the conventional wine yeast S. cerevisiae. Such knowledge is nowadays within reach thanks to the NGS revolution (Solieri et al., 2013). NGS allows the development of genome-assisted approaches like whole genome sequencing and resequencing, transcriptome profiling, ChIP-sequencing to identify DNA-structure, etc., (Solieri et al., 2013). De novo sequencing is greatly needed as some wine species still lack of nuclear and mitochondrial reference genomes (S. bacillaris, P. fermentans, M. pulcherrima, etc.). However, de novo assembly is sometimes difficult to conduct due to high heterozygosity level or sequence repeat, and led to draft genome with high number of contigs or scaffolds. For example, H. uvarum DSM 2768 genome displays 335 contigs, P. kudriavzevii M12 has 621 scaffolds, and P. anomala NRRL Y-366 shows 1932 scaffolds. Thus, the first aim of non-conventional wine yeast studies should be the completion of robust genomic sequences. Then, additional genome sequencing could be performed: genome re-sequencing using NGS captures individual genotypes and allows population genetics and ecologic studies within species. Such comparative genomics approaches were successfully applied to S. cerevisiae (Liti et al., 2009) and S. uvarum (Almeida et al., 2014), and could now address non-Saccharomyces yeasts of technological interest. In addition to intraspecific genomics, comparative genomics between yeast species is particularly useful to understand genome evolution . The identification of specific metabolic pathways, gene duplications or functions between species may increase our appreciation of adaptation's mechanisms and their biotechnological interest (Blein-Nicolas et al., 2015). It has to be noted that several species genetically close to wine yeasts show no peculiar affinity with winemaking environment (Figure 1). This is the case of S. paradoxus: despite being the most closely related species to S. cerevisiae, S. paradoxus is essentially associated with wild environments and particularly trees (Sniegowski et al., 2002;Johnson et al., 2004). Comparative genomics of wine vs. non-wine yeast species could thus increase our knowledge of the common genomic requirement for grape/wine colonization, if any. Finally, NGS technologies have greatly improved genome-assisted approaches aiming at detecting genetic variants associated with phenotypes in S. cerevisiae (Ehrenreich et al., 2010). In particular, QTLseq or genome-wide association studies (GWAS) could now be applied to non-conventional yeasts depending on whether classical breeding is possible (QTL-seq) or not (GWAS). These fields are blank pages waiting to be filled in the next future of oenology microbial research. The use of mixed-cultures, combining both non-conventional yeasts and one Saccharomyces species able to complete AF, is increasing in winemaking. Thus, another challenge lies in understanding yeast-yeast interactions and their underlying mechanisms (Ciani et al., 2010;Ciani and Comitini, 2015). Indeed, several types of yeast-yeast interactions have been described in enological conditions: competition for nutriments, release of toxic compounds (Fleet, 2003), and even "quorumsensing" like mechanisms Renault et al., 2013). Understanding these complex interactions is of first importance as the combination of some yeast strains seems condemned to failure: for example, cell-cell contact was recently shown to be involved in the death of strains of T. delbrueckii and L. thermotolerans during mixed-culture alcoholic fermentation with S. cerevisiae Kemsawasd et al., 2015). In some cases, yeast death was associated with the release of metabolites or killer toxin (Pérez-Nevado et al., 2006;Albergaria et al., 2010;Branco et al., 2015;Ramírez et al., 2015). The precise impact of such interactions regarding wine quality and aromas is still unclear (Ciani et al., 2006), but will have to be considered to control and optimize complex mixed oenological fermentation.
Finally, in addition to NGS-assisted approaches and interactions studies, another prospect in the field of nonconventional wine yeast lies in classical genetic approaches: indeed, one of the limits of the previously detailed approaches is their low ability in elucidating the basic life-cycle of wine yeasts, particularly regarding the occurrence and control of sexual reproduction. Still, classical breeding is one of the key issues for genetic improvement of industrial strains of S. cerevisiae (Pretorius, 2000;Giudici et al., 2005;Marullo et al., 2006; and represents a technological barrier that must be overcome for actual improvement of non-Saccharomyces wine yeasts. There is an important need for traditional sporulation assays, spore microdissection attempts, subsequent segregant analyses, breeding assays, etc. In addition, genetic transformation of non-conventional wine yeasts would be a welcomed tool for subsequent functional studies (Pacheco et al., 2009;Roberts and Oliver, 2011). These classical approaches are time-consuming and necessitate traditional yeast-manipulation know-how, sometimes viewed as old-fashioned and therefore neglected. However, these old approaches are essential for our future understanding of the genetics of non-conventional wine yeast, and are complementary to the more en vogue NGS-assisted approaches.

FUNDING
This work was funded by the European Union's Seventh Framework Programme (FP7/2007(FP7/ -2013 under grant agreement n • 315065 WILDWINE project: "Multi-strain indigenous Yeast and Bacterial starters for "Wild-ferment" wine production."