Mating type determination

There are a variety of methods to define the mating type of a pombe strain. The main ways are: doing test crosses or performing a PCR for mat1.

Mating type switching in pombe

Mating types in fungi define if a haploid cell can fuse with another haploid cell. The gene(s) at the mating type locus regulate a number of downstream genes that indicate cell identity (i.e. being Plus or Minus for pombe) and are required to induce mating (see Marlini et al. 2013 for a good review). The genes differentially expressed are:

• mating type pheromones (map2 for P-factor and mfm1, mfm2, mfm3 for M-factor)
• Pheromone receptors (map2 and mam3)
• Sexual agglutination proteins (map3 and mam4)
• The M-factor transporter (mam1; no transporter is required for P-factor)
• Pheromone protease for degradation of opposite pheromone (sxa2)

In most fungi, mating type is a trait that is genetically defined (by the 'allele' at the mating-type locus) and is under Mendelian inheritance. But not in fission yeast or in most budding yeasts. During asexual growth, a cell goes through a mitotic division resulting in two daughter cells with different mating types. If the cell was Plus, the genes at the mat1 locus will become replaced by a copy of the Minus genes, and if it was Minus, the Plus genes are introduced. As a template for this change, two non-expressed copies of each mating type (P at mat2 and M at mat3) are present in the genome; but it is only the genes at the active locus that matter. So, determining a mating type is here not possible, as each group of cells will always consist of half P and half M cells.
There are many non-switching strains out there too (these are called heterothallic) and for those we can determine the mating type.

PCR for the active mat1 allele

The mating type is defined by the mating type allele at the active locus mat1. To find the mating type at the mat1 locus we simply do two PCRs, both using primer 46 to the left (centromere proximal) of mat1 as forward (mat1l-K-1-F, 5'-TGGGATGAGTGCTTGCTTTG-3') and with as a reverse

• primer 66 (Mat-minus-R, 5'-CCACATCTCTCCAACCAGCT-3') yielding a 980bp fragement
• primer 68 (Mat-plus-R, 5'-CGGTAGTCATCGGTCTTCCA-3') yielding a 1120bp fragment

Do a colony PCR at 60°C annealing or use a standard Taq PCR on clean DNA (58°C).

Iodine staining to test for homothallism (based on Bresch et al 1968)

A homothallic strain (i.e. a switcher) can mate within a single cell colony, because after switching the two daughter cells are of opposite mating type and can thus fuse and continue with meiosis. After meiosis the newly formed haploid produces will form 4 spores in one ascus. These spores contain starch that will be used during germination and we can use this to detect spores at the macroscopic level by staining for starch using Iodine vapour.

1. On a plate to induce mating PMG or EMM-N
   • Patch the strain to be tested
   • Plate out a population or mix in a low dilution to obtain single cell colonies
2. Incubate the plate for 2 to 7 days at 25°C (longer incubation gives clearer result)
3. In the fumehood, pour into an empty petridish some iodine beads and place the to be tested plate over it (upside down). Make sure the beads don't get onto your plate.
4. Wait for 1 to 2 minutes; the longer you wait the stronger the effect, but the more likely the toxic Iodine fumes will have killed your cells.
5. If a patch or colony does not stay completely yellow, it is homothallic, or was a mix of cells of two opposite mating types. Not yellow for the standard labstrain means that the colony turns black when stained long enough, but for many natural isolates and mutants, this can be anything from purple at the edges, faintly black/purple, black with yellow spots etc.

Test crosses with iodine staining

To test the mating type, simply set up a set of crosses with strains of known mating types.

1. Take a dollop of cells and make three patches on a mating plate
   • Add to the first patch some cells from a known Plus and mix
   • Add to the last patch some cells from a known Minus and mix
   • Leave the one in the middle alone
2. You can easily test 21 strains on a single 9cm plate.
3. Also make a control for the two references you use:
   • grow each alone
   • mix the two together
4. Incubate for 3 days at 25°C
5. Stain the cells using iodine and note which cross worked
   • Only the cross with P --> the focal strain was Minus
   • Only the cross with M --> the focal strain was Plus
   • All three --> the focal strain was homothallic
   • None --> the focal is sterile (Better hope you did not forget to add the control cross, because now is a good moment to check if your plate was correct or you used the wrong reference, or something else might have happened).