Investigation of Genetic Relationships Between Hanseniaspora Species Found in Grape Musts Revealed Interspecific Hybrids With Dynamic Genome Structures Méline Saubin, Hugo Devillers, Lucas Proust, Cathy Brier, Cecile Grondin, Martine Pradal, Jean Luc Legras, Cécile Neuvéglise To cite this version: Méline Saubin, Hugo Devillers, Lucas Proust, Cathy Brier, Cecile Grondin, et al.. Investigation of Genetic Relationships Between Hanseniaspora Species Found in Grape Musts Revealed Interspecific Hybrids With Dynamic Genome Structures. Frontiers in Microbiology, Frontiers Media, 2020, 10, pp.1-21. 10.3389/fmicb.2019.02960. hal-02478447 HAL Id: hal-02478447 https://hal.archives-ouvertes.fr/hal-02478447 Submitted on 13 Feb 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License fmicb-10-02960 December 28, 2019 Time: 15:50 # 1 ORIGINAL RESEARCH published: 15 January 2020 doi: 10.3389/fmicb.2019.02960 Investigation of Genetic Relationships Between Hanseniaspora Species Found in Grape Musts Revealed Interspecific Hybrids With Dynamic Genome Structures Méline Saubin1, Hugo Devillers1, Lucas Proust1, Cathy Brier1, Cécile Grondin2, Martine Pradal3, Jean-Luc Legras3 and Cécile Neuvéglise1* 1 Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 2 Micalis Institute, INRA, Edited by: AgroParisTech, CIRM-Levures, Université Paris-Saclay, Jouy-en-Josas, France, 3 SPO, Univ Montpellier, INRA, Montpellier Matthias Sipiczki, SupAgro, Montpellier, France University of Debrecen, Hungary Reviewed by: Hanseniaspora, a predominant yeast genus of grape musts, includes sister species Lisa Solieri, University of Modena and Reggio recently reported as fast evolving. The aim of this study was to investigate the Emilia, Italy genetic relationships between the four most closely related species, at the population Giuseppe Blaiotta, level. A multi-locus sequence typing strategy based on five markers was applied Università degli Studi di Napoli Federico II, Italy on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, Anthony Borneman, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the The Australian Wine Research Institute, Australia level of intraspecific nucleotide diversity, and differences in heterozygosity between *Correspondence: species indicate different life styles. No clear population structure was detected based Cécile Neuvéglise on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two [email protected] distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids Specialty section: were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization This article was submitted to using flow cytometry, karyotypes and genome sequencing showed different genome Food Microbiology, structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. a section of the journal Frontiers in Microbiology Subculturing of an allotriploid strain revealed chromosome loss equivalent to one Received: 19 September 2019 chromosome set, followed by an auto-diploidization event, whereas another auto- Accepted: 09 December 2019 diploidized tetraploid showed a segmental duplication. Altogether, these results suggest Published: 15 January 2020 that Hanseniaspora genomes are not only fast evolving but also highly dynamic. Citation: Saubin M, Devillers H, Proust L, Keywords: MLST, yeast, biodiversity, evolution, Hanseniaspora uvarum, Hanseniaspora guilliermondii Brier C, Grondin C, Pradal M, Legras J-L and Neuvéglise C (2020) Investigation of Genetic Relationships INTRODUCTION Between Hanseniaspora Species Found in Grape Musts Revealed Interspecific Hybrids With Dynamic Grape must is a complex ecosystem that combines grape and cellar micro-organisms. Many species Genome Structures. are interacting with each other, including yeasts and bacteria (Jolly et al., 2014; Capozzi et al., Front. Microbiol. 10:2960. 2015; Raymond Eder et al., 2017). It is now well recognized that natural microbial populations doi: 10.3389/fmicb.2019.02960 play an important role in winemaking, notably by increasing the complexity of wine aromas Frontiers in Microbiology| www.frontiersin.org 1 January 2020| Volume 10| Article 2960 fmicb-10-02960 December 28, 2019 Time: 15:50 # 2 Saubin et al. Hanseniaspora Interspecific Hybrids (Gamero et al., 2016; Padilla et al., 2016; Bagheri et al., 2018; This method, initially developed for the identification of Escribano et al., 2018). The occurrence of microorganisms clones within populations of pathogenic bacteria (Maiden in grapes is dependent on biotic and abiotic factors such as et al., 1998), is progressively becoming classic for genomic geographic location, soil, grapevine cultivar, viticultural practices, diversity studies in yeasts from different environments, and and climate (Bokulich et al., 2014; Drumonde-Neves et al., many markers have been reported (Bougnoux et al., 2002; 2016). Yeast biodiversity in must is thus generally vintage Ayoub et al., 2006; Odds and Jacobsen, 2008; Jacques et al., dependent (Vigentini et al., 2015). Whereas Saccharomyces 2017; Tittarelli et al., 2018). In this study, MLST analysis cerevisiae, already well known for its importance in diverse performed on Hanseniaspora strains was used to clarify the fermented food production, is the dominant species at the end of complex of the four wine-growing species examined and vinification, the yeast species commonly found on grapes and in allowed detection of inter-specific hybrids, which were then musts at the beginning of spontaneous fermentations are rather investigated by flow cytometry and karyotyping. Finally, genome Saccharomycotina than Basidiomycotina. They mostly belong to sequencing and comparison to parental species genomes genera Hanseniaspora, Lachancea, Metschnikowia, Pichia, and provided more details into the contribution of each of them to Starmerella (Wang et al., 2015; Varela and Borneman, 2017; the genome of the hybrids. Cioch-Skoneczny et al., 2018; Lorenzini and Zapparoli, 2019), and the genus Hanseniaspora is generally the most abundant at the onset of the fermentation. MATERIALS AND METHODS The Saccharomycotina genus Hanseniaspora belongs to the Saccharomycodaceae family. Due to their lemon-shaped cell Hanseniaspora Strains structure, Hanseniaspora species have been called apiculate A total of 120 Hanseniaspora strains were examined in this study, yeasts, together with the closely related species of the genera 35 of them were collected from French grape must in Occitanie Saccharomycodes and Nadsonia. The genus includes 21 described region, France in 2015 and 2016 (Table 1). The other strains species, which can be separated into two lineages based on have been collected from grape musts or from other substrates phylogenetic relationships deduced from D1D2 domains of in distant French regions and in countries mainly from Europe, the 28S rRNA subunit (Boekhout et al., 1994; Ouoba et al., South America and Africa. Yeast cells were cultivated on YPD 2015; Martin et al., 2018) or from the concatenation of medium (yeast extract 10 g/L, peptone 10 g/L, glucose 10 g/L) ◦ taxonomic markers (Cadez et al., 2019). Recently, based on at 28 C. whole genome sequence comparison, Steenwyk et al.(2019) confirmed Hanseniaspora to be composed of two lineages, a fast- DNA Extraction evolving lineage (FEL) and a slow-evolving lineage (SEL), which DNA extractions were carried out on cells grown in complete differ by their evolution rate and the extent of their gene loss. medium to stationary phase with two different methods: Species found on grapes and musts belong to both FEL (mostly extraction with the Masterpure yeast DNA purification kit H. uvarum) and SEL (H. vinae). Several studies report that (Epicentre, France), and with an in-house protocol involving a they play an important role in wine fermentation by producing mechanical and chemical lysis. Briefly, cells were resuspended flavors, modulating the growth and metabolism of Saccharomyces in 200 mL of lysis buffer (Tris 10 mM pH8, EDTA 1 mM, NaCl cerevisiae and affecting wine color (reviewed in Martin et al., 100 mM, Triton 2% and SDS 1%), 200 mL of phenol chloroform 2018). However, population structure and genetic relationships isoamyl alcohol 25:24:1, and 0.3 g of glass beads. This step was between Hanseniaspora species remain unclear. Nine species of followed by 4 min of vortexing and 5 min of centrifugation at FEL are particularly close phylogenetically and some of them 13,000 rpm. Then, the aqueous phase was mixed with ethanol could be difficult to differentiate probably because they diverged and the precipitated DNA pellet washed twice with ethanol quite recently (Cadez et al., 2002). D1D2 regions of the ribosomal 70%, dried and resuspended in 100 mL of TE with 1 mL of subunit, which are generally used for taxonomic classification RNase A at 10 mg/mL. and phylogenetic trees, differ