Over the last decades genetic studies using the budding yeast Saccharomyces cerevisiae
have led to discovery of a variety of cellular signaling components as well as many other fundamental cellular processes. One of the advantages of yeast genetics is that it is straightforward to isolate desired mutant strains and identify the underlying mutations. In principle, such genetic approaches can be applied only in the haploid backgrounds because it is difficult to isolate recessive mutations in diploids due to complementation of the phenotype by the second copy of the gene. This becomes an issue when mutant strains defective in diploid-specific developments such as meiosis, sporulation, spore germination, bipolar budding pattern, and pseudohyphal development need to be isolated. Although a yeast homozygous knockout library of the S288C background is available 
, this genetic background has lost some of these specific phenotypes and hence those are commonly studied in other strain backgrounds. Therefore, a method for efficient construction of homozygous double mutants is required.
The yeast sexual cell types are designated a
, which are conferred by the MATa
alleles of the M
), respectively 
. In general, homozygous diploid mutant strains (i.e. MATa/α xxxΔ/xxxΔ
) are constructed by crossing strains of the opposite mating-type, which need to be constructed individually. When the two haploids have different prototrophic or antibiotic resistance markers, the diploids can be easily selected on plates lacking both nutrients or containing both antibiotics because auxotrophy or antibiotic sensitivity are complemented by each genotype (). The HO endonuclease, which mediates mating-type switch, can be used to obtain diploids via mating of MATa
cells within colonies 
. Alternatively, zygotes (dumbbell-shaped cells) can be isolated by micromanipulation during conjugation of two cells. However, these methods are unsuitable for large-scale analysis. Thus, there has been no easy way to construct and select diploid strains from single haploids at high throughput so far.
Strategy for construction of homozygous diploid strains.
Decreasing gene dosage by RNAi (restored by introducing Dicer and Argonaute from S. castellii
or by haplo-insufficiency (heterozygous mutant) 
may be useful for studying diploid-specific developments. Since these methods do not completely abolish gene function and consequently might give false negative or positive results, an efficient method to create homozygous deletion mutants is desired. In this study, we present an efficient method for construction of diploid strains using a galactose-inducible mating-type switch gene (PGAL1-HO
) and a counter selection marker gene (PSTE18-URA3
We applied our method to the study of yeast morphological developments. In the S. cerevisiae
Σ1278b background, diploid cells develop pseudohyphae (filamentous growth) under nitrogen starvation. Since filamentous growth is essential for virulence of yeast pathogens such as Candida albicans 
, discovery of positive regulators for filamentous growth using S. cerevisiae
as a model organism can contribute to understanding common conserved mechanisms. The high-osmolarity glycerol (HOG) response MAPK pathway, which plays a central role in osmoadaptation 
, negatively regulates filamentous growth and deletion of the HOG1
MAPK gene leads to hyper-filamentous phenotype even under nutrient-rich conditions 
. In order to identify positive regulators essential for filamentous growth, we performed large-scale construction of homozygous double mutants in theΣ1278b hog1Δ/hog1Δ
background. The screen identified 49 suppressor mutations, showing that our method is useful for genetic study.