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Beoordeel zelf slecht matig voldoende goed zeer goed. Functional Genetics of Industrial Yeasts. Paperback, blz. Over de auteur Over dit boek Artikelen en interviews Recensies. Samenvatting Rubriek: Wetenschap en techniek. Serie: Topics in Current Genetics. Lezersrecensies Beoordeel zelf slecht matig voldoende goed zeer goed. Algemene beoordeling slecht matig voldoende goed zeer goed.

Uw recensie. Therefore, we limited our experimental competitions of the inbred diploids versus JAY—GFP to a maximum of 8 daily transfer cycles, in order to insulate the measured GFP ratios from the effect of de novo mutations. These control experiments also showed that integration of the GFP cassette into the JAY genome did not by itself have an effect on growth kinetics.

Additional JAY—GFP versus JAY control co-culture competitions were included every time a new experimental evaluation of the inbred collection was performed 39 replicates , and in no cases a significant deviation in the GFP ratio was observed before or at transfer cycle 8.

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Many of the inbred diploids displayed growth kinetics that were substantially different from JAY, not only slower but also faster. Importantly, all but one of the inbreds, regardless of the neutral, positive or negative relative growth kinetics profiles, followed a steady unidirectional trajectory from the early cycles until the end of co-culture. This result is consistent with their phenotype being a function of their initial genotype, and not due to the random appearance of de novo mutations during the experiments. In addition, there was very little variation between the independent replicates of each inbred co-culture, further disfavoring a potential influence of de novo mutations over the observed phenotypes.

This shows that the cumulative liquid co-culture competition assay was able to reliably and consistently uncover extremely subtle relative differences in growth kinetics. Thus, the co-culture competition assay offered an opportunity to reliably measure minor phenotypic changes that resulted from the different genotype combinations represented in the inbred collection.

Even though we collected data for cycles 0, 2, 5, and 8, we used data only from cycle 5 for the downstream QTL analysis as it offered an optimal quantification of relative growth kinetics. The cumulative nature of this assay meant that by cycle 8 some of the extreme GFP ratios had already started to reach a plateau, which could lead to an underestimation of their full phenotypic differential.

This cumulative trend can be visualized in Figure S6 as the progressive shift in the phenotypic distribution away from center cycles 0 and 2 and toward the low and high extremes over time cycle 8. The specific mean percentage of each inbred in the co-culture at cycle 5 and the phenotypic distribution in the full strain set are shown in Figure 3B. Figure 3 Phenotypic distributions in the inbred strain collection. A Left side: Median error bars in standard error of the median heat tolerance scores for inbred strains, ordered from lowest to highest; Right side: Frequency distribution of the phenotypic values shown to the left.

In both plots the bars corresponding to the JAY phenotype are highlighted in yellow and indicated by a black arrow.

Functional genetics of industrial yeasts; of ancient skills and modern applications | SpringerLink

We next performed a QTL analysis to identify possible relationships between the specific genotypes at JAY HetSNPs and the phenotypic variation in heat stress tolerance and competitive growth among inbred strains. Using a one-dimensional scan of the genome, log10 likelihood ratio LOD scores were determined for each marker for each trait.

The genome-wide LOD scores for each trait are plotted in Figure 4 , and regions that rose above the 4. A two-dimensional scan of the genome was also performed, but no significant pairwise epistatic interactions were detected data not shown. Figure 4 QTL analyses to identify loci potentially underlying heat stress tolerance A and relative growth kinetics B. The color code in the plots indicates the allele contributing the higher phenotype value red: maternal; blue: paternal.

Black circles indicate the positions of centromeres. Plots were generated in R version 3. Although our inbred population size was relatively small, this analysis was sufficient to reveal multiple genomic segments that may make important contributions to the traits of interest. In total, thirteen regions from eight chromosomes showed association to heat tolerance, and seven regions from six chromosomes to competitive growth Table S6.

This narrower list included 47 and 48 candidate genes within the genomic regions that were significantly associated to heat tolerance and competitive growth, respectively. The genes in both lists belonged to diverse functional annotation groups i. We evaluated which quantitative inheritance model better fit the observations from each region Table S7.

Cytoplasmic inheritance

Most regions 16 of 20 were consistent with an additive variance model in which the heterozygote has an intermediate phenotype. We also found four regions with likely dominance, but no cases of overdominance. In order to facilitate a comparison of the relative contributions between regions to each trait, we also calculated relative PVE values normalized to the locus with the highest PVE. The identification and characterization of specific major genes and alleles that contribute to these traits in JAY was beyond the scope of this particular study.

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However, we noted that none of the significant association regions overlapped between the two traits. In addition, there was no overlap between the inbred strains ranked in the upper or lower tiers of heat tolerance and competitive growth Figure S7. This suggested that the two traits are controlled independently of each other, so different combinations of alleles present at different sets of JAY genomic regions contributed in their own way to the phenotypic variation observed for each trait.

In the inbred collection approach described above, each strain had lost roughly half of the heterozygosis present in JAY, thus a large fraction of the genome was affected. We next took an independent and more conservative approach in which fewer heterozygous loci were manipulated at a time. To do so, we adapted a procedure to induce targeted uniparental disomy UPD i.

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Here, we adapted this approach for use in prototrophic diploid strains by integrating a hemizygous copy of the heterologous forward and counter selectable marker AmdS Solis-Escalante et al. We modified the AmdS cassette by removing the transcriptional terminator sequence, thus enabling constitutive transcription to continue past the ORF and extend through the centromeric sequence.

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Insertions of AmdS cassettes adjacent to centromeres of each the M and P homologs of targeted chromosomes were obtained and stably maintained through forward selection for growth in media containing acetamide as the sole nitrogen source Figure 5A. Then, counter selection for loss of the cassette fluoroacetamide resistance was used to isolate candidate clones carrying chromosome loss.

The final phase, and a key part of the strategy, relied on the observation that monosomic diploid S. Another possible mechanism is that UPD may be formed in a single step through meiosis I-like co-segregation of sister chromatids in mitotic cells Andersen and Petes, Figure 5 Construction and phenotypes of UPD strain pairs. A A cassette containing the counter-selectable marker AmdS under the transcriptional control of the TEF1 constitutive promoter and lacking a terminator sequence was integrated immediately upstream of the centromeric regions of each M or P homolog of Chr4, Chr14, and Chr15 insertion of AmdS at a P homolog is shown in this case.

Transcription of AmdS perturbs centromere function and induces targeted chromosome mis-segregation during mitosis. Cells that lost the AmdS marker were selected for in media containing fluoroacetamide. Spontaneous endoduplication of the remaining homolog results in strains containing UPD, in this case represented as the maternal red homolog. Loss of each homolog was validated by RFLP-PCR genotyping analysis at distal markers on both chromosome arms diamonds , and confirmation of endoduplication was obtained by tetrad dissection and spore viability analysis 4 viable spores per tetrad indicate disomy.

Multiple independently-generated UPD strains were isolated and used in phenotypic tests. B Growth profiles under heat stress of UPD strain pairs. The black line represents the JAY control. C Competitive growth profiles of UPD strain pairs. Color scheme is the same as in B. We conducted a proof-of-concept experiment focused on the generation of strain pairs carrying bidirectional UPD for three chromosomes Chr4, Chr14, and Chr15 , chosen on the basis of their overall chromosome size, and number and distribution of HetSNPs Figure S1. It has been proposed that loss of long S. Chr4 was an attractive candidate for this analysis because it is a large chromosome but has a relatively low number of HetSNPs, which are all clustered in a central region.

We integrated the terminator-less AmdS cassette immediately adjacent to the centromeres of each homolog of these three chromosomes. We then screened multiple independently-generated chromosome loss clones by PCR-RFLP genotyping followed by tetrad analysis to identify those that had undergone UPD to become homozygous for each of the three respective targeted chromosomes Figure S8. We tested by tetrad analysis a subset of the fluoroacetamide resistant clones that had LOH at both the left and right centromere-distal HetSNP markers.

All of the clones tested through tetrad analysis were found to be disomic i. All strains remained heterozygous at those loci, except for the specific chromosome homolog targeted for UPD. Our goal was to test whether localized reduction of heterozygosity in these three chromosomes would be sufficient to cause detectable variations in the heat tolerance and competitive growth phenotypes.