Creating diploids

Fission yeast is a haplontic species. It grows as a haploid, mates by fusion (conjugation) between a Minus and a Plus strain to generate a diploid zygote. The diploid is generally short lived: immediately, so sometimes after a few cell divisions, the zygote will go into meiosis and generate a 4 spored ascus. This tetrad of spores are the 4 meiotic products that have a highly resistant spore wall which contains starch which we can visualise using iodine.

To obtain diploid strains, cells need to be induced to conjugate, but before continuing to meiosis be moved to a medium that induces continued growth while selecting against haploids. See for more information at the Forsburg webpage.

Complementation

To select against haploids and only allow diploid cells to grow, choose strains that each can complement each other preferably with two closely linked markers or with the marker in the same locus, to avoid recombinants to grow on the medium selection against either of the markers. For example, take a leu1+ and a leu1::hphMX and transfer the cells to an PMG+hygromycin plate. Or a common combination: ade6-M216 and ade6-M210 and grow on medium without adenine; these alleles can complement each other.

• Mix the two strains on a mating plate and incubate at 25oC for 12-20 hours.
• Take a dollop of the cell mix
• Streak out on the plate that selects against the independent haploids but that allows for diploids with the complementing markers to grow.
• incubate the plate at 32-36C (see pombenet)
• when colonies are visible, pick these and transfer to YES potentially with a selective antibiotic

Note that the diploids are unstable and can have gone through meiosis even on rich medium, so best to check if what you have is really a diploid strain. Use a plate with Phloxine B for this.

When moving the diploids to a mating plate the diploids should form azygotic asci.