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RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions, and are not known to affect host fitness. In this study, we describe a new S. cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in 1 strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Furthermore, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.

The diploid isolate EM93 is the main ancestor to the widely used Saccharomyces cerevisiae haploid laboratory strain, S288C. In this study, we generate a high-resolution overview of the genetic differences between EM93 and S288C. We show that EM93 is heterozygous for >45,000 polymorphisms, including large sequence polymorphisms, such as deletions and a Saccharomyces paradoxus introgression. We also find that many large sequence polymorphisms (LSPs) are associated with Ty-elements and sub-telomeric regions. We identified 2,965 genetic markers, which we then used to genotype 120 EM93 tetrads. In addition to deducing the structures of all EM93 chromosomes, we estimate that the average EM93 meiosis produces 144 detectable recombination events, consisting of 87 crossover and 31 non-crossover gene conversion events. Of the 50 polymorphisms showing the highest levels of non-crossover gene conversions, only three deviated from parity, all of which were near heterozygous LSPs. We find that non-telomeric heterozygous LSPs significantly reduce meiotic recombination in adjacent intervals, while sub-telomeric LSPs have no discernable effect on recombination. We identified 203 recombination hotspots, relatively few of which are hot for both non-crossover gene conversions and crossovers. Strikingly, we find that recombination hotspots show limited conservation. Some novel hotspots are found adjacent to heterozygous LSPs that eliminate other hotspots, suggesting that hotspots may appear and disappear relatively rapidly.

We sequenced the genome of Saccharomyces cerevisiae strain YJM789, which was derived from a yeast isolated from the lung of an AIDS patient with pneumonia. The strain is used for studies of fungal infections and quantitative genetics because of its extensive phenotypic differences to the laboratory reference strain, including growth at high temperature and deadly virulence in mouse models. Here we show that the [almost equal to]12-Mb genome of YJM789 contains [almost equal to]60,000 SNPs and [almost equal to]6,000 indels with respect to the reference S288c genome, leading to protein polymorphisms with a few known cases of phenotypic changes. Several ORFs are found to be unique to YJM789, some of which might have been acquired through horizontal transfer. Localized regions of high polymorphism density are scattered over the genome, in some cases spanning multiple ORFs and in others concentrated within single genes. The sequence of YJM789 contains clues to pathogenicity and spurs the development of more powerful approaches to dissecting the genetic basis of complex hereditary traits.

Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult 1 , 2 , 3 , 4 , 5 . As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes 6 , 7 . This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae . A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases.

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

Whether in natural populations or between two unrelated members of a species, most phenotypic variation is quantitative. To analyze such quantitative traits, one must first map the underlying quantitative trait loci. Next, and far more difficult, one must identify the quantitative trait genes (QTGs), characterize QTG interactions, and identify the phenotypically relevant polymorphisms to determine how QTGs contribute to phenotype. In this work, we analyzed three Saccharomyces cerevisiae high-temperature growth (Htg) QTGs (MKT1, END3, and RHO2). We observed a high level of genetic interactions among QTGs and strain background. Interestingly, while the MKT1 and END3 coding polymorphisms contribute to phenotype, it is the RHO2 3'UTR polymorphisms that are phenotypically relevant. Reciprocal hemizygosity analysis of the Htg QTGs in hybrids between S288c and ten unrelated S. cerevisiae strains reveals that the contributions of the Htg QTGs are not conserved in nine other hybrids, which has implications for QTG identification by marker-trait association. Our findings demonstrate the variety and complexity of QTG contributions to phenotype, the impact of genetic background, and the value of quantitative genetic studies in S. cerevisiae.

We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer. We show extensive 2μ sequence variation in Saccharomyces cerevisiae and in other species of Saccharomyces and 2μ associations with S. cerevisiae populations, clinical origin and genotypes.

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

We characterized previously identified RNA viruses (L-A, L-BC, 20S, and 23S), L-A–dependent M satellites (M1, M2, M28, and Mlus), and M satellite–dependent killer phenotypes in the Saccharomyces cerevisiae 100-genomes genetic resource population. L-BC was present in all strains, albeit in 2 distinct levels, L-BChi and L-BClo; the L-BC level is associated with the L-BC genotype. L-BChi, L-A, 20S, 23S, M1, M2, and Mlus (M28 was absent) were in fewer strains than the similarly inherited 2µ plasmid. Novel L-A–dependent phenotypes were identified. Ten M+ strains exhibited M satellite–dependent killing (K+) of at least 1 of the naturally M0 and cured M0 derivatives of the 100-genomes strains; in these M0 strains, sensitivities to K1+, K2+, and K28+ strains varied. Finally, to complement our M satellite–encoded killer toxin analysis, we assembled the chromosomal KHS1 and KHR1 killer genes and used naturally M0 and cured M0 derivatives of the 100-genomes strains to assess and characterize the chromosomal killer phenotypes.