| The second generation of fuel ethanol currently fully commercialized is based primarily on industrial Saccharomyces cerevisiae fermenting lignocellulosic biomass hydrolysates.However,lignocellulose produces complex inhibitors during hydrolysis that adversely affect the growth of Saccharomyces cerevisiae and ethanol production by fermentation,hindering the large-scale implementation of these alternative strategies for lignocellulosic ethanol production.Furfural is one of the main inhibitors,which can induce the accumulation of reactive oxygen species?ROS?,decrease on the key enzyme activities of various metabolic pathways,and eventually lead to programmed cell death.At present,the main methods on the use of reverse genetics?from genetic changes to phenotypic changes?to identify possible genes and transcriptional regulators associated with tolerant inhibitors that respond to metabolic pathways and modify microorganisms?overexpression or knockout?to improve tolerance.However,there are many unknown areas in the systemic mechanism of furfural tolerance,which cannot be completely resolved.At the same time,most of the research object strains belong to experimental strains,and the industrial application of Saccharomyces cerevisiae is more complex.Therefore,in this study,the mechanism of furfural tolerance of Saccharomyces cerevisiae was deeply analyzed from the perspective of molecular ecology and forward genetics?changes in the nature of genes to natural phenotypic changes?,Also,an effective and feasible method for the study of the tolerance of other cellulose inhibitors was provided.In this study,48 strains of industrial Saccharomyces cerevisiae were used as starting strains to determine the phenotypic of furfural tolerance at different concentrations.The maximum phenotypic value was 21 mmol·L-1 and the minimum phenotypic value was 5mmol·L-1.The phenotypic value of most strains was 15 mmol·L-117 mmol·L-1.Due to the degeneration of sporulation ability in industrial Saccharomyces cerevisiae,phenotypic value of 21 mmol·L-1 CICC 31144?diploid?was selected as the high tolerance parent and 11 mmol·L-1 CICC 1373?diploid?as the sensitive strain.Spore production was performed on McClary sporulation media to analyze the sporulation capacity of the two strains.CICC31144 sporulation rate was 41%±0.12%,while CICC 1373 sporulation rate was 55%±0.17%.Rapid identification of haploid mating type using PCR technique,phenotypic screening and HO gene knockout.Finally,a parental P1?CICC 31144-a?with a phenotypic value of 21mmol·L-1 as a high furfural tolerance and a parental P2?CICC 1373-??with a phenotypic value of 11 mmol·L-1 as a low resistance to furfural.A large number of progeny populations were cross-bred with parental strains for genetic mapping.The phenotypic distribution of furfural resistance in 200 F2 populations was analyzed by SPSS 19.0 software,the skewness coefficient was-0.375 and the kurtosis coefficient was 0.238.The coefficient of the two is less than 1,which is approximately a normal distribution,indicating that furfural tolerant traits are continuous quantitative traits suitable for quantitative genetic studies.The existing microsatellite?SSR?loci?190?were genotyped between the two parental haploid strains,and a total of 82 polymorphic loci were screened out with a polymorphism ratio of 43%.By using these polymorphic loci for genotyping of the two extreme F2 populations?39 strains?and genotype difference analysis,the results showed that two microsatellite markers were identified.Among them marker 2P3had significant differences and marker 2P5 had extremely significant differences.Furthermore,72.2%of high tolerant individuals contained the marker 2P5?333 bp?derived from P1,the high tolerant parent;100%of low tolerant individuals contained the marker 2P5?318 bp?derived from P2,the low tolerant parent,indicating that the locus may have a highly significant genetic relationship with the QTL that controls the trait.The genetic map was constructed using Kosambi function,with a total of 79 markers and14 linkage groups with the length of 1499.07 cM.By using the Inclusive Composite Interval Mapping?ICIM?to map the location of furfural tolerant quantitative traits,the LOD curve had significant peaks on chromosomes 2,7,and 16 with phenotypic variation rates of 43.1%,28.65%,and 12.34%,respectively.Among them,qTCHRII is located between 2P5 and 2P6?205476258897?,qTCHRVII is located between 7P17 and 7P14?1043692156030?,and qTCHRXVI is located between 16P16 and 16P17?591668834952?.The phenotypic variation of chromosomes 2 and 7 are more highly,indicating that these interval are the main effect QTL affecting furfural tolerance.This study establishes an appropriate density genetic map for preliminary maping of quantitative traits of furfural resistance,laying the foundation for further accurate maping and selection of favorable alleles?molecular marker assisted breeding MAS?to improve the accuracy and predictability of breeding. |
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