Genetic Diversity Analysis Of Oenococcus Oeni Strains Isolated From Changli And Yinchuan Regions And Screening Of Promising Strains

Oenococcus oeni(O.oeni)is the predominant species during MLF,and one of the tailors for the final organoleptic properties of wine.The studies on genetic diversity and screening promising indigenous strain of O.oeni have been mostly carried out by developed wine-making regions abroad,such as France,Spain,Italy,Australia,Argentina and Chile,while the research on different wine-making regions of China is still scarce.In this study,fluorescent amplified fragment length polymorphism(AFLP)and multilocus sequence typing(MLST)methods were used simultaneously,to investigate the genetic diversity and population structure of O.oeni strains isolated from different wine-making regions in China.Then,the genetic typing abilities of the two methods were evaluated.The more efficient method was then applied for further genetic analysis of O.oeni strains isolated from Changli and Yinchuan.Subsequently,a three-step screening system for promising indigenous O.oeni strains was established: firstly,the specific-PCR technology was used to detect the functional genes for a rapid and accurate preliminary screening;secondly,the ability of stress resistance was evaluated in different stress conditions of model wine;finally,the wine-making characters were tested.The experimental results were as follows:(1)Fluorescent AFLP and MLST methods were used to investigate the genetic diversity and population structure of 38 O.oeni strains isolated from different wine-making regions in China.The results indicated that AFLP was markedly more efficient than MLST for typing O.oeni strains.AFLP distinguished 37 DNA patterns compared to 7 sequence types identified using MLST,corresponding to discriminatory indices of 99.9% and 60.2%,respectively.The AFLP results revealed a high level of genetic diversity among the O.oeni strains from different regions of China,since two subpopulations were observed.Phylogenetic analysis of the O.oeni strains using the MLST method also identified two major phylogroups,which were differentiated into two distinct clonal complexes by minimum spanning tree analysis.Neither intragenic nor intergenic recombination by SplitsTree verified the existence of the clonal population structure of the O.oeni strains.These results can provide theoretical basis and methodological support for the genetic diversity analysis of indigenous O.oeni.(2)O.oeni strains isolated from 5 different wineries in Changli wine-making region were identified by Species-specific PCR,and their genetic diversity were analysed by fluorescent AFLP technique.AFLP distinguished 221 AFLP-genotypes among 222 identified O.oeni strains,and the similarity coefficient among the strains was 74~98%,revealing a high level of genetic diversity.Based on the unweighted pair-group method with Arithmetic means(UPGMA),two major phylogroups A and B were deciphered at 81.7% similarity level.And more importantly,the strains from the same winery formed the unique clusters,indicating the close genetic relationships with their origins.(3)O.oeni strains isolated from Cabernet Sauvignon wine and Cabernet Gernischt wine in Yinchuan wine-making region were also identified by Species-specific PCR,and their genetic diversity were analysed by fluorescent AFLP technique.Fluorescent AFLP distinguished 206 AFLP-genotypes among 207 identified O.oeni strains,and the similarity coefficient among the strains was 63 ~ 97%,also revealing a high level of genetic heterogeneity.Based on UPGMA,three clear phylogroups A,B and C were deciphered at 79.88% similarity level.Especially,the phylogroups A and B were the major phylogroups with 81.7% similarity level,and interestingly,the strains in the two phylogroups were from different varieties of wine.These results indicated the abundant resources and the obvious differences in population structure of O.oeni strains in different varieties of wine grape.(4)Based on specific-PCR technology,the nine functional genes bgl,estA,gshR,arcABC,hdc,odc and tdc were detected from 170 indigenous O.oeni strains isolated from Changli and Yinchuan,for a rapid and accurate preliminary screening.Among the 170 indigenous O.oeni strains,bgl,estA,gshR(relating to the aromatic compound production and strain stress resistance)and arcA(relating to the synthesis of ethyl carbamate compound)were all recognized;the odc and tdc(relating to the synthesis of putrescine and tyramine)were all none detected;13 indigenous strains with arcB and arcC(relating to the synthesis of ethyl carbamate compound)deletion were found;50 indigenous strains with hdc(relating to the synthesis of histamine),odc and tdc deletion were verified,and,among these 50 strains,1 strain with arcB and arcC gene deletion possessed high risk of the synthesis of high-level ethyl carbamate.So,through the preliminary screening,49 indigenous strains with potential promising fermentation characteristics were obtained.(5)Compared with the strain SD-2a,the ability of stress resistance of the strains from preliminary screening was evaluated in different stress conditions of model wine.9-10,7-04,9-51,9-13,9-50,8-17 and 3-31 showed a strong stress-resistance ability by significance analysis(P<0.05).Then,their MLF performance was evaluated by inoculating in Chinese Marselan wine.3-31 and 7-04,like SD-2a,showed strong L-malic acid metabolism ability.The wine fermented by the 7 strains showed obvious different aroma styles by GC-MS and principal component analysis.Especially,the 3-31 strain was validated as a special promising starter culture for the application of Marselan MLF fermentation,giving the wine a typical,complex and rich style.