Known Genetic and Physical Interactors of the Htg QTGs Are Not Htg QTGs Themselves
One potential strategy to identify novel QTGs is to test genes that interact genetically (e.g., synthetic lethality) or physically (e.g., in complexes) with known QTGs. No genetic interactions have been reported for mkt1
]. However, end3
has been shown to be synthetically lethal with apm3
], and pan1
] mutations and has a synthetic slow-growth phenotype with rvs161Δ
]. End3p also physically interacts with Pan1p [18
] and, in addition, Mkt1p physically interacts with Pbp1p [20
]. Therefore, we performed RHA in YJM145/S288c hybrids to determine if these genes, the gene products of which interact genetically or physically with the gene products of known Htg QTGs, are Htg QTGs themselves. However, RHA showed that none of these interacting genes make allele-specific contributions to the Htg phenotype; that is, APM3, ARP2, PAN1, RVS161, RVS167,
are not Htg QTGs.
Deletion Analysis of Htg QTGs
To determine the deletion phenotypes of Htg QTGs in the S288c and YJM145 backgrounds, we constructed single (qtg1Δ
) and double (qtg1Δ
) diploid deletion strains and tested their Htg phenotypes. Consistent with previous work [21
Δ S288c and YJM145 background strains were temperature-sensitive at 37 °C. Therefore, End3p is required for growth at even modestly elevated temperatures in both backgrounds.
In the S288c background at 39 °C, the highest temperature tested in this Htg− background, rho2Δ strains grew better than wild-type strains. However, in the Htg+ YJM145 background, rho2Δ strains grew less than wild type at 40 °C and 41 °C (). In the S288c background, mkt1Δ and wild-type strains grew equivalently at 39 °C. However, in the YJM145 background, mkt1Δ strains grew less than wild type at 40 °C, while at 41 °C mkt1Δ strains did not grow. Therefore, while end3Δ mutants in both parental backgrounds were unable to grow at 37 °C, the rho2Δ and mkt1Δ phenotypes were different in the two genetic backgrounds, which suggests that phenotype screening of null mutants may have limited predictive value as a QTG identification strategy.
Phenotypes of Htg QTG Deletion Strains in the Parental Backgrounds
In both the S288c and YJM145 backgrounds, we constructed double mutants to test for synthetic interactions between individual Htg QTGs. No synthetic lethal interactions were noted for any pair of deletions or for deletion of all three Htg QTGs in either background. All end3Δ-containing strains (end3Δ mkt1Δ, end3Δ rho2Δ, and end3Δ mkt1Δ rho2Δ) grew similarly to end3Δ strains in both backgrounds. However, in contrast to rho2Δ and mkt1Δ strains, mkt1Δ rho2Δ strains in the S288c background grew less than wild type at 39 °C, and thus exhibit a synthetic Htg− phenotype (). When tested in the YJM145 background at 40 °C, however, the growth defect of mkt1Δ rho2Δ strains was the sum of the mkt1Δ and rho2Δ growth defects, consistent with Mkt1-145p and Rho2-145p contributing additively to Htg ().
Genetic Interactions between Htg QTG Alleles
As the first step in assessing the genetic interactions between Htg QTG alleles, we determined the Htg phenotypes of a heteroallelic YJM145/S288c hybrid versus deletion heterozygotes; for example, END3–145
, and END3–145
. Consistent with the Htg+
alleles of MKT1, END3,
being dominant, deletion heterozygotes containing only the Htg+
QTG alleles had Htg phenotypes similar to a heteroallelic hybrid containing both Htg+
alleles (). As predicted from our previous work (Table S1
], deletion heterozygotes containing only an Htg−
allele had a highly significant growth disadvantage at high temperature relative to a heteroallelic hybrid.
Growth Difference at 41 °C between a Heteroallellic Hybrid and Hybrids Heterozygous for Deletions of One or Two Htg QTGs
Having determined the Htg phenotypes of a heteroallelic hybrid versus all single-deletion heterozygotes, we tested for interactions between Htg QTG alleles by determining the relative Htg phenotypes of a heteroallelic hybrid versus double-deletion heterozygotes. In competitions of a heteroallelic hybrid versus hybrids with the Htg− alleles of RHO2 and END3 deleted (RHO2–145/Δ Δ/END3–288), there was no difference between the hybrids. In contrast, in competitions of the heteroallelic hybrid versus hybrids with the Htg+ alleles of RHO2 and END3 deleted (Δ/RHO2–288 END3–145/Δ), the deletion heterozygotes were Htg− relative to the heteroallelic hybrid. However, the approximately two-fold growth differential in these competitions was much less than that observed when single Htg+ alleles of RHO2 and END3 were deleted (), suggesting a complex genetic interaction between alleles of RHO2 and END3.
In competitions of a heteroallelic hybrid versus hybrids with Htg+ alleles of MKT1 and END3 deleted (Δ/MKT1–288 END3–145/Δ), the deletion heterozygotes had Htg phenotypes significantly less than the heteroallelic hybrid. The Htg phenotypic differential in these competitions was approximately equal to the sum of the differences observed in the single-deletion heterozygote analyses (), indicating MKT1–145 and END3–288 additivity. Similarly, in competitions of a heteroallelic hybrid versus hybrids with Htg+ alleles of MKT1 and RHO2 deleted (Δ/MKT1–288 Δ/RHO2–288), the deletion heterozygotes had Htg phenotypes significantly less than the heteroallelic hybrid, consistent with the earlier observed MKT1–145 and RHO2–145 additivity.
Expression Analysis of the Htg QTGs
To further characterize the Htg QTGs and gain insight into the mechanism of the quantitative trait and the location(s) of the phenotypically relevant polymorphism(s), we carefully examined the steady state mRNA levels of the three Htg QTGs as a function of both temperature and allele. Since isogenic reciprocal hemizygotes contain only one allele of a QTG, in a comparison between reciprocally hemizygous strains, any allele-specific differences in steady state mRNA levels must be due to cis-acting polymorphisms. Therefore, we performed Northern analysis of two pairs of reciprocal hemizygotes for each QTG. Temperature (25 °C versus 37 °C) had no effect on RHO2 and END3 steady state mRNA levels. However, MKT1 steady state mRNA levels increased in an allele-nonspecific manner at 37 °C (37 °C/25 °C = 2.0). No allele-specific fold differences larger than two-fold were observed in steady state mRNA at either temperature for any of the genes.
Homologous Allele Replacement of the Htg QTGs
As a first test of the hypothesis that the phenotypically relevant differences between Htg+ and Htg− QTG alleles were contained within the genes, and most likely from expression results within the coding regions, we ectopically integrated alleles of RHO2 and alleles of MKT1 (separately) at the HO locus. However, we found that the resulting strains had insert-specific, Htg QTG allele-nonspecific, dominant-negative growth defects (unpublished data).
Since these ectopic integrations were problematic, we then constructed isogenic homologous allele replacement strains that differed (see Table S2
) solely with respect to the S288c versus YJM145 nonsynonymous coding region polymorphisms and one 5′ and one 3′ UTR polymorphism in MKT1
(a−104g D30G K453R a+54t), or nonsynonymous coding polymorphisms in END3
(S258N D268N) or RHO2
(F91C) (see Table S3
). We then compared the Htg phenotypes of isogenic strains that differed solely in these MKT1, END3,
For isogenic S288c, YJM145 and YJM145/S288c hybrid background strains with different RHO2
alleles, there were no statistically significant differences in Htg phenotype (). Therefore, the single nonsynonymous RHO2
coding polymorphism (C91F) is phenotypically neutral. Since there are no RHO2
5′UTR polymorphisms, this suggested that RHO2
3′UTR polymorphisms contribute to phenotype (see Table S3
Growth Difference between Coding Allele Exchange Strains for Htg Genes in the S288c, YJM145, and YJM145/S288c Hybrid Backgrounds
To test the RHO2 3′UTR hypothesis, we replaced the 3′UTR of RHO2–288 with the 3′UTR of RHO2–145; that is, we constructed RHO2–288 3′UTR-145 strains. We then performed Htg competitions between heteroallelic hybrids (RHO2–145 3′UTR-145/RHO2–288 3′UTR-288) versus hybrids with the S288c-derived RHO2 3′UTR (Δ/RHO2–288 3′UTR-288) versus hybrids with the YJM145-derived RHO2 3′UTR (Δ/RHO2–288 3′UTR-145). Consistent with our RHA results, the Δ/RHO2–288 3′UTR-145 strains grew as well as the heteroallelic hybrids, while the growth difference between Δ/RHO2–288 3′UTR-288 strains and the heteroallelic hybrid was similar to the growth difference between Δ/RHO2–288 3′UTR-288 and Δ/RHO2–288 3′UTR-145 strains (). Therefore, the RHO2 3′UTR is primarily and possibly solely responsible for the allele-specific contribution of RHO2 to the Htg phenotype.
Growth Difference at 41 °C between YJM145/S288c Hybrids Containing YJM145- versus S288c-Derived RHO2 3′UTRs
For both isogenic S288c and YJM145 background strains with different END3
coding alleles, there were no significant differences in Htg phenotype (). However, consistent with our previous results [1
], Htg+ END3–288
allele-containing YJM145/S288c hybrid background strains had highly significant Htg advantages over Htg− END3–145
In the S288c background, there was no significant difference in growth between strains with different MKT1
alleles ( and S2
). However, in the YJM145 background, Htg+ MKT1–145
allele-containing strains had highly significant Htg advantages over Htg− MKT1–288
allele-containing strains. Similarly, in the YJM145/S288c hybrid background, Htg+ MKT1–145
allele-containing strains had highly significant Htg advantages over Htg− MKT1–288
allele-containing strains. Therefore, these END3
coding (S258N D268N) and MKT1
coding/noncoding (a−104g D30G K453R a+54t) polymorphisms are responsible for much of the END3
allele-specific contributions to the Htg phenotype, and their effects are highly influenced by genetic background.
Identification of the MKT1 and END3 Quantitative Trait Nucleotide
To determine which of the coding single-nucleotide polymorphisms (SNPs) in the two major Htg QTGs, MKT1 and END3, are relevant quantitative trait nucleotides (QTNs) for the Htg phenotype, we used site-directed mutagenesis to replace the two coding SNPs in MKT1 and END3 individually. The isogenic strains were created such that they differed solely in the coding SNP under investigation. RHA competitions were done to determine the effect of each SNP replacement in each gene on the Htg phenotype.
For MKT1, RHA analysis of SNP replacement strains showed that the A1358G SNP (where the S288c base-nucleotide numbers from the start-YJM145 base), resulting in a conservative amino acid change from lysine (K) in S288c to arginine (R) in YJM145 (K453R), had no effect on Htg. However, the A89G SNP, resulting in a nonconservative amino acid change from aspartic acid (D) in S288c to glycine (G) in YJM145 (D30G), affected Htg with 89G (30G)-containing strains growing better at high temperature ().
Growth Difference at 41 °C between YJM145/S288c Hybrids Containing YJM145- versus S288c-Derived MKT1 and END3 Coding Polymorphisms
For END3, RHA analysis of SNP replacement strains showed that the C802T SNP, resulting in an amino acid change from aspartic acid (D) in S288c to asparagine (N) in YJM145 (D268N), had no effect on Htg. However, the C773T SNP, resulting in a nonconservative amino acid change from serine (S) in S288c to asparagine (N) in YJM145 (S258N), affected Htg with 773C (258S)-containing strains growing better at high temperature ().
Phenotypic Heterogeneity among the Htg QTGs in Diverse Genetic Backgrounds
Marker-trait association studies assess the association between a sequence variant and a trait. Marker-trait association, which may be affected by genetic background [2
] and is one of the applications of the data coming from the HapMap project, aims to aid QTG discovery [23
]. As a first step in determining the impact of genetic background on phenotype and on marker-trait association, we determined the Htg phenotype of the S288c background and of ten additional unrelated genetic backgrounds. The Htg phenotypes of the 11 unrelated genetic background strains varied over a greater than 104
-fold range, demonstrating a high degree of Htg phenotypic heterogeneity, and there was no association between the specific MKT1, RHO2,
polymorphisms shown to be relevant in YJM145/S288c and the Htg phenotype ().
Htg Phenotypes and RHA of Htg Genes in Hybrids between S288c and Other S. cerevisiae Genetic Backgrounds
To further assess the impact of genetic background on phenotype and on marker-trait association, we crossed an S288c background strain with strains from each of the ten unrelated Htg+ and Htg− genetic backgrounds and determined the Htg phenotypes of the resulting hybrids. All hybrids, except YJM627/S288c, showed positive heterosis (); that is, the Htg phenotype of the hybrids was greater than either parent, consistent with a general benefit of heterozygosity at many loci. Therefore, although the S288c genome constitutes one-half of the genome content of all hybrids, the genetic composition of the entire genome affects the Htg phenotype. Again, there was no association between the specific MKT1, RHO2, and END3 polymorphisms shown to be relevant in YJM145/S288c and Htg phenotype.
As a final step in determining the impact of genetic background on phenotype and on marker-trait association, we performed RHA of the Htg QTGs in the ten hybrid genetic backgrounds () [1
]. RHA of MKT1, END3,
in hybrids between S288c and the ten unrelated genetic backgrounds further demonstrated the complex impact of genetic background on QTG contributions to phenotype. For example, in MKT1,
all of the non-S288c backgrounds have the same coding polymorphism (30G) that was found to be Htg+
in the YJM145/S288c hybrid. However, in the hybrids between S288c (30D) and these other genetic backgrounds, the 30G-containing MKT1
alleles present in all of the hybrids could be phenotypically Htg+
, neutral, or Htg−
and, conversely, the MKT1–288
(30D) allele present in all of the hybrids could be Htg−
, neutral, or Htg+
. There are similar cases for the END3
coding polymorphism 258S. Our analysis shows therefore that QTGs identified in one background do not necessarily make the same contribution to phenotype in other genetic backgrounds, and that QTL architecture (that is, the identity and arrangement of QTGs) is therefore not conserved and instead varies with genetic background.