The genetic architecture underlying a quantitative trait is determined by the number of genes that contribute to their additive and interactive effects on variation of the trait phenotype.36
Although many genomes have been fully sequenced and the number of genes with annotated function increases rapidly in the era of functional genomics, it still remains a great challenge to reconstruct the architecture with a full insight of the genetic parameters. Budding yeast, the single-cell eukaryote, but with probably the richest structural and functional information of the genome, provides an ideal model for dissecting the molecular basis of genetic variation in typical quantitative traits such as high-temperature growth12
and sporulation efficiency37
and in genetic control of transcriptome abundance.38
The present study extends these successful examples by dissecting multiple-threshold phenotypic variation of yeast cell aggregation into a genic level.
Several genes have been reported previously in the literature to control clumping phenotype variation in budding yeast. Of these, the most prominent are the FLO gene family responsible for many cellular adhesion phenotypes, including flocculation.10
In sharp contrast, the present study reveals the two novel genes, AMN1
, in addition to previously identified two genes, FLO1
, that are significant determinants for the phenotype. Success of the present study in resolving the mapped QTLs into the underlying QTL genes can be attributed to that the major QTLs have been mapped at the high resolution by which direct gene targeting becomes possible. We noted that the simulation study by Li et al. predicted a much larger confidence interval for mapping categorical traits from a simulated backcross population with 200 individuals genotyped at genetic markers of every 10 cM. When compared with the simulation study, the present analysis has employed a substantially larger sample size (292 segregants) and higher density of markers in the QTL regions. These together with the fact that the budding yeast has a highly recombiogenic and compact genome paved the basis for the mapping resolution we have achieved.
The combined effect of these QTL genes explains 46% of the overall phenotypic variation, leaving the remaining 54% of the variation to be residual. The residual variation can be explained as two parts as well as their interaction, the genetic component whose underlying genetic effects are too small to be detected by the experiment, and the component contributed by environmental modification to the quantitative trait. In addition, we demonstrated that the QTL genes exhibited clear epistatic effects on the aggregation trait. These show that the underlying mechanism of yeast cell aggregation is more complicated than expected in the previous literature.
Of these QTL genes, AMN1
plays the leading role in regulating cell aggregation. We provide strong and direct experimental evidence that supports this is through the genes modulating the mitotic exit network in S. cerevisiae
We have identified the AMN1
as a major QTL gene of cell aggregation through a genome-wide QTL scan in a natural population and provided here the direct experimental evidence for the V368D substitution as the causative genetic variant. Our results also strongly indicate the putative domain of AMN1
, and the functional significance of V368D, which has been recently predicted as a superfamily being highly conserved (e-value of 6.38e-96) across species.39
On the other hand, the RGA1
gene, a member of the GTPase-activating family, is first identified in the present study as a major gene affecting cellular clumping in budding yeast. This gene is required for yeast cells to switch from the apical to isotropic growth phase.40
The present study creates an opportunity to explore the joint effect of this cell aggregation QTL gene, a budding pattern controller, and another QTL gene AMN1
, also a mitotic exit regulator. Genetic modification to these genes through gene knockouts disperses the well-known grape-like strings of cells (Fig. ).
In addition to the above two major genetic factors, FLO1 and FLO8 were also detected as the QTL genes through the present forward genetic analysis. Highly expressed FLO1, a cell surface adhesion regulator, has been widely recognized as a biomarker for cell flocculation in many candidate-based analysis. However, we present here the role of the gene, as merely one of the building elements in the genetic architecture underlying the complex phenotype, and show that FLO1 is highly background dependent in determining the phenotype of its carrier. Unlike what has been widely accepted in the literature of yeast cell flocculation, expressed FLO1 does not necessarily confer a clumping phenotype to its carriers. We demonstrate that the FLO1 allele from a non-clumpy parental strain, in the presence of the other three QTL genes from a clumpy parental strain, creates the genotype for the clumping phenotype that substantially surpasses performance of both the parents, i.e. transgressive segregation of the QTL genes. Taking all these together, the QTL genes unveiled in the present study have been shown to be involved in multiple pathways that govern the cellular development (RGA1), the behaviour of daughter cells (AMN1), and the cell-surface adherence property (FLO1 and FLO8). In contrast to the studies in which biological functions and their phenotypic significance of these QTL genes were examined either independently or jointly, the present study unveils the QTL genes as major determinants of the clumping phenotype in a systematic way. This clearly indicates the power and appropriateness of the forward genetic approach employed in the present study in uncovering the genetic basis of an aggregate polygenic trait such as cellular clumping that involves multiple constituent components.
The evolutionary significance of cellular clumping has recently been highlighted in the single-cell species because the clumping phenotype can protect inner individuals in cell clumps from biotic or/and abiotic stresses such as alcohol, anti-microbials, and physical attacks. The FLO1
gene is recognized as a ‘green beard gene’ because its induced expression in a non-flocculent laboratory strain, S288C, can restore its cells to flocculate, and the cellular flocs, thus, generated provide a cooperative shield to protect from stresses in the living environment.1
Our data demonstrate that expression of FLO1
may not be the biomarker for a clumping phenotype, although an unexpressed FLO1
allele from the non-clumpy strain, which is isogenic to S228C, may enhance clumping of its offspring strains carrying other major clumping genes. In the polygenic system uncovered here, AMN1
is found to be a more important genetic determinant than FLO1
in controlling expression of the clumping phenotype, and this endogenously expressed gene may make a more appropriate target for investigating the molecular mechanism underlying the ‘green beard’ effect.