| Saccharomyces cerevisiae is widely used in the food, wine, pharmaceutical and many other industries. But the accumulation of organic acids can inhibit the growth of yeast and reduce the intensity of fermentation. In recent years, studies on the mechanism of acetic acid tolerance in yeast were gradually rise, however, the molecular mechanism of acetic acid tolerance in yeast is still unclear. In this paper, the simple sequence repeats(SSRs) which were polymorphic between the two parental genome were used as molecular genetic markers to construct genetic linkage map and detect the major quantitative trait loci(QTLs) of acetic acid tolerance in yeast. The main contents are as follows:(1) Selecting of extreme acetic acid tolerant strainsFifty strains were collected for acetic acid tolerant phenotype identification, then 25 SSR loci were selected to screen the whole genome, the results showed the expected heterozygosity of each locus was ranged from 0.708 to 0.972, polymorphism information content of each locus was ranged from 0.660 to 0.959, which indicated the SSR loci have high polymorphism and the population with high genetic diversity in this experiment. In addition, five loci were related to acetic acid tolerance. These results showed that SSR markers can be used to analysis the acetic acid tolerance of yeast. Two strains(CICC 1374 and CICC 31144) with different phenotypes were screened by indentification after sporulation, two haploids were obtained and named YHA and YLA, respetively. YHA can against the acetic acid concentration of 140 mmol·L-1, and YLA can against the acetic acid concentration of 30 mmol·L-1. HO gene of YHA and YLA were knocked out to obtain a stable mating type.(2) Single marker analysis related to acetic acid tolerant traitOne hundred and sixty segregants were obtained by crossing two yeasts(YHA and YLA) with different phenotypes, 78 markers were polymorphic between the two parental strains screened form 220 SSR markers, the polymorphic ration was 35.5%, among the 78 polymorphic marker loci, of which 63 markers segregated in Mendelian ratio while 15 markers segregated in distortion as indicated by Chi-square test. Half diallel cross experiments showed that the generalized heritability of the acetic acid tolerant trait is as high as 94%, indicating the acetic acid tolerant trait was controlled by major genes. In order to seek the markers associated with acetic acid tolerance of yeast, the polymorphic markers were used to screen the whole genome of 40 strains. As a result, marker 14P2 and 15P2 had significant correlation with acid tolerance(p<0.01), 70.6% of high tolerant individuals contained the 14P2(344 bp) derived from YHA, the high tolerant strain; 91.3% of low tolerant individuals contained the 14P2(331 bp) derived from YLA, the low tolerant strain, the microsatellite marker 14P2 has the obvious tendency of heredity correlative with genes for acetic acid tolerance.(3) Genetic linkage map and quantitative trait loci(QTL) mappingOne hundred and sixty segregants were backcross to parental strains aboved. Phenotypic analysis showed a normal distribution in the population and the phenotypic distribution was meet the continuoud distribution of complex trait. The single marker analysis was used to detect markers associated with acetic acid tolerance by WinQTLCart 2.5, the results showed 12 SSR loci had correlation with acid tolerance(p<0.05), marker 2P14, 4P13, 9P8 and 16P16 had significant correlation with acid tolerance(p<0.01), which can explained ranged from 18.3% to 26.7% of the phenotypic variations. Marker 4P13, 9P8 and 16P16 explained over 20% of the phenotypic variation, indicating that the three SSR loci may linked gene which associated with acetic acid tolerance, and also showed that acetic acid tolerant trait was controlled by multiple genes. The 78 polymorphic SSR loci were used to construct genetic linkage map, it coverd a total length of 2,530.2 cM, and there was 5.6 loci(average value) per group, the maximum distance covered by a single linkage group was 461.1 cM while the minimum was 8.7 cM, the average distance between markers was 39.5 cM. Composite interval mapping(CIM) was used to detect QTL associated with acetic acid tolerance. As a result, 3 QTLs were detected, QTL-1 was at 55 cM between marker 9P4 and marker 9P8, QTL-2 was at 157 cM between marker 12P4 and marker 12P6, QTL-3 was at 85.9 cM between marker 16P12 and marker 16P3, explained 59.1%, 65.9% and 38.8% of the total phenotypic variation, respectively. Of which QTL-1 and QTL-3 explained over 50% of the total phenotypic variation, they were major QTLs which associated with acetic acid tolerance.The genetic linkage map constructed in this work was lowdensity map, which can be used for QTL primary mapping of acetic acid tolerant trait, and it can provides the foundation for further fine mapping and molecular assisted selection. |
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