Mapping And Validating Of Major QTLs Associated With Low-temperature Tolerance In Native Strains Of Saccharomyces Cerevisiae

Low-temperature fermentation has been a hot spot in modern oenology for its capability in increasing the flavor diversity of wine,which contributes to wine quality improvement.The success of low-temperature fermentation largely depends on the fermentation properties of Saccharomyces cerevisiae,especially on the low-temperature tolerance.Therefore,studying the genetic basis of low temperature tolerance of S.cerevisiae is of great significance for the screening,genetic improvement and application of strains with excellent low-temperature resistance,improving wine quality and enhancing the competitiveness of Chinese wine.In this study,we mapped the major QTLs using bulk segregant analysis?BSA?combined with whole genome sequencing technology in the F₂ segregant population of two native S.cerevisiae strains with divergent tolerance capability at low-temperature.The function of candidate genes in QTLs were validated by reciprocal hemizygosity analysis?RHA?,sequence polymorphism analysis and allele replacement.The main results were summarized as follows:1.Low-temperature tolerance of native S.cerevisiae was analyzed by determining the fermentation capacity and growth at low temperatures?16°C,12°C,8°C,4°C?of 68 native S.cerevisiae isolated from five major wine producing regions.The results showed that the maximum specific growth rate at each temperature of S.cerevisiae followed the Normal distribution.With the temperature decreasing,the growth of yeasts was gradually inhibited,the maximum specific growth rate was greatly reduced and the fermentation capacity of yeasts was also decreased.Compared with other strains,Wb-3-40,ZX11 and ZX13 grew better,and they produced gas faster,indicating the tolerance of these strains was stronger.While 31y3,NX9412,112y4 grew poorly,produced gas slower and their fermentation was severely inhibited,indicating that these strains had weak low-temperature tolerance.These strains were used as stating materials for investigating the genetic basis that underlies low-temperature tolerance in S.cerevisiae.2.The HO gene in six diploid strains with extreme phenotypes was knocked out by the Cre-loxP recombination system.Then,they were sporulated and all the segregants were evaluated for their growth at low-temperature.The ZX11 segregant ZX11?6??MAT?,ho??that displayed the highest?_max value?0.0143?in all segregants at 4?,was chosen as the superior parent strain.A NX9412 segregant,NX9412?4??MATa,ho??only grew slightly with the?_max value of 0.0025,was used as the inferior parent strain.Two auxotrophic strains ZX11?6??MAT?,ho?,ura3::KanMX4?and NX9412?4??MATa,ho?,ura3::KanMX4,lys2::URA3?were constructed and their growth at low-temperature did not change significantly.3.Two parent strains were crossed with each other to obtain the F₁ hybrid.F₁ hybrid was then sporulated and the spores were collected,generated the F₂ segregant population.The growth at low-temperature of all F₂ segregants was determined.Afterward,two pools were made by selecting extreme individuals from the F₂ population segregants with the basic statistics of the phenotypic data.The major QTL_S were identified by pooled-segregant whole-genome resequence analysis.The results showed that a total of 500 F₂ segregants were isolated.Among them,21 superior segregants displaying high tolerance to low-temperature were assembled in the‘Tolerant pool'while 23 inferior segregants displaying low tolerance were selected to assemble the‘Sensitive pool'.A total number of14,618 highly credible SNPs between two parental strains was detected.By analyzing the distribution of??SNP-index?of the two pools,two QTLs were identified.They were located on chromosomes IV and XV,respectively.4.Candidate genes in the two QTLs were validated by RHA,sequence polymorphism analysis,site-directed mutagenesis and allele replacement.The results showed that NAT1 and YOR365C are major genes associated with low-temperature tolerance in S.cerevisiae.The NAT1 gene is involved in the N-terminal acetylation of proteins,and the protein function of YOR365C is unknown.The protein domain of YOR365C was predicted by SMART,and it was found to be involved in cell wall biosynthesis.Through sequence polymorphism analysis,we found two nucleotide variants leading missense amino acid change in NAT1coding region in the two parent strains,and one nucleotide variant?T1187C?was absent in the other 28 S.cerevisiae strains of which the complete genome sequence is known.Using site-directed mutagenesis and mutant allele replacement,this variant was verified to be in almost entirely reconstitution of the phenotype of the ZX11?6?nat1 null mutation strain,which indicated that the nucleotide polymorphism at this site must be a QTN controlling the low-temperature tolerance in S.cerevisiae.The QTN caused a corresponding amino acid change?F396S?,improved the low-temperature tolerance in strains.We also found that the tolerance of original sensitive parent strain?NX9412?was improved by the replacement of its both NAT1 alleles by NAT1^ZX11?6?allele.