- Quantitative trait loci analysis can be used to identify those genetic differences between two strains of the same species that are responsible for the differences in a given phenotype. - Whole genome sequencing of this subpopulation and of the unsorted pool of segregants implicated several loci that are involved in lipid accumulation. - Three of the identified genes, PIG1 , PHO23 and RML2 , were investigated in more detail. - Deletions of these genes and the exchange of the alleles between the two parental strains confirmed that the encoded proteins contribute to neutral lipid storage in S. - Backcrossing of one of the segregants with the parental strains for seven generations revealed additional regions in the genomes of both strains with potential causative genes for the high lipid accumulation phenotype.. - Full list of author information is available at the end of the article. - Most of the loci that were enriched in the segregants with high NL content had no known connec- tion to the biochemical pathways contributing to lipid synthesis, storage or degradation. - After crossing the two parental strains, sporulation of the hybrid and germin- ation of their haploid progeny, we picked 2288 single colonies into 96 well microtiter plates to analyze the dis- tribution of the NL content in the F1 generation. - More than 40% of the progeny showed a heterosis effect:. - 22.9% of the strains had a lower FI than the BY4741 par- ental strain, and 18.6% of the segregants showed stron- ger fluorescence than the AWRI1631 parental strain.. - Furthermore, whole genome sequen- cing of the pools of segregants in these two subpopula- tions was performed to obtain a better resolution of the SNVs in the subpopulation of segregants with high NL content compared to the average population. - The mean of the coverage for the average pool was 738-fold, and for the high NL content pool it was 1407-fold. - method, and for which WGS analysis confirmed non- silent SNVs with the highest bias for one of the parental alleles in the corresponding loci of the subpopulation with high NL content. - In the case of RML2, however, the enrichment of the BY4741 allele in this population could be the conse- quence of linkage with the CAN1 locus, which was one of the selection markers, and we therefore could not conclude which allele is beneficial in terms of NL content.. - Based on the results of the X-QTL study, three genes were selected for further analysis and quantification of their contribution to NL storage. - RML2 was included be- cause the SNPscanner analysis proposed the existence of a minor QTL in the vicinity of the CAN1 locus. - 1 TAG and SE content of the two yeast strains AWRI1631 and BY4741. - 0.001 for the comparisons of the two strains in both exponential and stationary phase and for both compounds, TAG and SE.. - The frequencies of alleles that are beneficial for high NL content are shown for a subset of 43 out of the 60 segregants with the highest FI. - Black points: difference between the populations: higher abundance of the red than of the gray signal indicates that this region is enriched for AWRI1631 sequences in the population with high NL content. - Shading denotes the parental origin of the genomic region in the F7 generation of the backcrossing experiment selecting for high NL content (BY4741 – blue. - However, neither RML2 nor any of the other QTLs listed in Table 1 have been implicated in NL storage so far. - Using SNV- specific PCR, we genotyped a subset of 43 out of the 60 strains with the highest NL content among the 2288 sin- gle segregants, according to the Nile Red-based assay.. - 2a), suggesting that these three genes indeed play a role in lipid storage metabolism and that the RML2 allele of the BY4741 strain is beneficial for NL accumu- lation. - Pho23p was characterized as a component of the Rpd3L histone deacetylase complex [26]. - To answer the question whether the opposite effect of the deletion of PHO23 in the two strain backgrounds is specific for this gene or a result of a reduction or loss of function of Rpd3L, we analyzed several mutants that were deleted for other components of the Rpd3L complex. - It should be noted that two out of the four tested genes, SDS3 and. - Hence, the different effect of the complex on lipid storage might be the result of several proteins with slightly altered functionality.. - In the case of RML2, which was characterized as a component of the mitochondrial large ribosomal sub- unit, we randomly selected four other proteins of this complex, Mrpl3p, Mrp7p, Mrpl8p and Mrpl49. - Lipid analysis of the respective knock-out strains showed that none of these mutants accumulated TAG in similar amounts as the strain deleted for RML2 (Supplemental Fig. - In all four mutants the NL content was slightly higher than in the wild-type, but we assume that this change is a consequence of the growth defect of these strains, due to the loss of functional mitochondria. - Furthermore, we constructed double and triple dele- tion mutants of the three genes, to investigate possible genetic interactions. - None of these strains showed a growth defect, except for the slightly slower growth of the mutants with a deletion of RML2. - To confirm the importance of the allelic variations between the two strains, we substituted the three protein coding regions of the genes in both genetic backgrounds with the alleles from the other parental strain. - as expected from the enrichment of the respective AWRI1631 alleles in the subpopulation with. - TAG and SE content of the AWRI1631 (panel a) and the BY4741 (panel b) strains, deleted for PIG1 , PHO23 or RML2 , and combinations thereof. - Importantly, the effect of the substitution showed a rather high variation, indicating that the quantitative contribution of Pig1p A-. - to NL content depends on other factors that were present in only part of the segregants.. - TAG and SE content of the AWRI1631 (panel a) and the BY4741 (panel b) mutant strains. - To assess the potential combined effects of the alleles, we first constructed the double substituted strain AWRI1631 with PIG1 BY4741 PHO23 BY4741 alleles, result- ing in a strain with less efficient alleles for NL accumula- tion in the genetic background of the parental strain with higher NL content. - Sur- prisingly, the additional substitution of RML2 in this strain resulted in a strong increase of the TAG content, although RML2 substitution alone did not show any ef- fect in AWRI1631. - Thus, presence of the RML2 BY4741 al- lele in the AWRI1631 genetic background masked the phenotypic effect of the substitution of the PIG1 and PHO23 alleles, indicating a genetic interaction. - Also, comparison of the single- and double-swapped strains in the AWRI1631 background showed that the contribu- tion of the PIG1 and PHO23 alleles to NL content is non-additive. - On the other hand, the TAG level of the BY4741 PIG1 AWRI1631 PHO23 AWRI1631 strain was not dif- ferent from the wild-type BY4741 strain, despite the fact that these two alleles are apparently beneficial for high NL content. - The strain with the highest NL content (107 mg/g CDW ac- cording to TLC analysis, corresponding to 263% of the AWRI1631 parental strain) from the F1 generation, which also had reproducibly high levels of NL according to the Nile Red method, was crossed with either of the parental strains to obtain the F2 generation for each lineage. - The distributions of the 192 segregants of the F7 generation from both lineages, compared to the 2288 segregants of the F1 generation, are shown in Fig. - The average FI of the F7 generation of the BY lineage was similar to the average FI of the segregants in the F1. - generation, whereas in the AWRI lineage the average FI of the F7 generation was 36% higher than in the F1 gen- eration. - The average FI of the back-crossed populations was higher than in the corresponding parental strains in both lineages (Fig. - 5), but this difference was more pro- nounced in the F7 generation of the BY lineage than in the AWRI lineage. - To identify these remaining regions of the other parent strain, the segregants with the highest NL content from every generation of both lineages, which were also used for the next backcrossing to the lineage’s parental strain, had their genomes sequenced. - S7 shows the genomes of the corresponding strains from F2 to F7. - This selection and backcrossing procedure re- sulted in strains with between 3.2 and 6.5% of the gen- ome from the parental strain of the other lineage (Supplemental Fig. - These values are significantly higher than the value of 0.8% that would have been ex- pected in the 7th generation without any selection, indi- cating at least partial causality of the retained regions for the selected trait. - In the case of the BY lineage, regions from the AWRI1631 parental strain included the whole QTL on chromosome XII, containing PIG1 (Fig. - Swh1p is a member of the family of oxysterol-binding proteins.. - The results from the present study sug- gest that Swh1p is also involved in the regulation of the NL content of S. - It should be noted that we found during the analyses of whole-genome sequences a VIII-t-XVI translocation in the genome of the AWRI1631 parental strain, which has not been previously described in this strain, but is known to be present in a number of wine strains [29].. - the founder of both backcrossing lineages, which however had also the complete chromosome XVI and thus effective duplica- tion of the translocated region (Supplemental Fig. - that of the AWRI1631 strain in the selected strain in the F5 generation (data not shown). - 5 Neutral lipid content of the BY and AWRI lineages after backcrossing. - The figure shows the frequency distribution of the fluorescence intensity of Nile Red, which correlates with the NL content, of the F1 generation (upper panel), the F7 generation of the BY lineage (middle panel) and the F7 generation of the AWRI lineage (lower panel). - The FI of the segregants was normalized to the value of the parental strain BY4741 and according to the log 2 value of the normalized intensity the segregants were distributed into 100 intervals. - the average class of the F1 generation. - the average class of the F7 generation of the BY lineage. - the average class of the F7 generation of the AWRI lineage. - we deleted MKT1 in both strain backgrounds and found no changes in the NL content of the mutants (data not shown), thus confirming that in the case of NL storage MKT1 is not causal.. - This means that some of the genes of BY4741 are more ef- ficient with regard to high NL accumulation than the alleles from the AWRI1631 strain background. - It is noteworthy that many of the genes. - The substitution of PHO23 with the allele from the other background indicates that structure and function of the whole complex and its effect on NL storage depends on several factors because the BY4741 strain bearing the AWRI1631 allele of PHO23 was not altered in its NL content, whereas the NL content of the analogous AWRI1631 mutant was the same as in the strain deleted for PHO23 (Fig. - Another example for the dependence of the effect of an allele on the genetic background is the replacement of PIG1 from AWRI1631 with the allele from BY4741 (Supplemental Fig. - The average decrease of the NL content was 6%, but the variation between individual segregants was remarkably high, ranging from + 3.5 to − 15.1%. - The high variability of the effect of this substitu- tion in segregants derived from the same parents suggests that the function of Pig1p depends on interac- tions with several other proteins that are encoded by different alleles in the two parents.. - We constructed mutants bearing substitutions of the alleles of PIG1, PHO23 and RML2 with the alleles of the other reference strain in both backgrounds and in all possible combinations. - These efforts were based on the assumption that a switch of the alleles between the strains should cause a change in the NL content of the mutant according to the results in the QTL study. - The most likely explanation for this behavior is that the positive effect of the native proteins on NL accumulation in AWRI1631 depends on additional factors. - Although the deletion of PIG1 and PHO23 indicated a positive genetic interaction for these two genes, this result cannot explain the results of the substitution experiments. - As it is the case for all major metabolic pathways, most of the enzymes catalyzing product formation in lipid me- tabolism are known. - The medium for phenotyping (MM-P) was buffered with 20 mM (2- (N-morpholino)ethanesulfonic acid) (MES) and supple- mented with 2 g/L of the following amino acid mixture:. - 100 μL of the exponentially growing pre-culture were transferred to 100 mL MM-L/S in a shake flask. - Over-representation of one of the strain-specific single nucleotide variations (SNVs) in the subpopulation was detected with the algorithm SNPscanner, which assigns so-called prediction signals to variants between the compared genomes [11, 23], based on which over-representation of a variant in the studied subpopulation of segregants from two parental strains can be established.. - To obtain a truly single-nucleotide resolution of the frequencies of SNVs in the subpopulation of segregants with extremely high NL content compared to the aver- age population, WGS of these two subpopulations was performed. - After demultiplexing the data, FastQC was used to assess the quality of the sequences.. - Mapping of the reads to the reference S. - 400 bp downstream of the stop codon) in the recipient strain were first replaced by a loxP-flanked KanMX4 cassette [48], which was then excised through Cre recombinase treatment. - In the donor strain, the KanMX4 cassette was integrated downstream of the ter- minator and the region consisting of the ORF and the marker was amplified by PCR and integrated at the same locus in the recipient strain. - One of the primer pairs was designed to introduce the desired recognition sequence at the 5′-end of the gRNA. - After 24 h, 20 μL of the cultures were inoculated into 180 μL fresh media and incubated for 72 h. - Frequency distributions of the. - Segregants were geno- typed for the presence of the specific alleles using the SNV-specific PCR, as described in [34].. - Quality control of the reads, trimming, mapping and variant calling in haploid mode were performed for the genome of each segregant as described in the section „Identification and quantification of QTLs with whole-genome sequencing. - Mock reference genomes of the parental strains, constructed as described above, were used as ref- erences for SNP calling of all backcrossed strains. - Segregants with a duplicated part of chromosome XVI were identified based on the higher sequencing depth of the corresponding chromosome part and in these segregants the duplicated part of the chromosome was excluded from the visualization. - The CDW of the cultures was determined according to [35]. - In the figures presenting results from lipid analyses, the means of the TAG and SE con- tents and their standard deviations are shown. - The funding agencies had no role in study design, data collection and analysis, and preparation of the manuscript.. - Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A. - Overlapping functions of the yeast oxysterol-binding protein homologues. - Polygenic analysis and targeted improvement of the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae . - Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth
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