Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.
Part 1: Preparation of strains for gametogenesis
Note: All manipulations should be carried out at room temperature. Higher or lower temperatures will affect the speed of zygote germination and may adversely affect viability.
Important consideration: It is essential that the strains to be mated are growing robustly and are free of contaminating fungi or bacteria. If the strains have not been transferred regularly, passage on rich medium should precede generation of gametes.
Part 2: Mating
Part 3: Tetrad Dissection
Note: Generation of vegetative diploids is not covered here, but essentially involves plating the mating mixture on medium which will not support the growth of either parental strain, but will support the growth of the diploid (e.g. two different auxotropic markers). Plates are kept in the light to avoid the formation of zygotes.
Genetic analysis is simple in Chlamydomonas, but it requires particular techniques to generate gametes and separate tetrad progeny. With healthy strains, the techniques are readily acquired. With some mutant strains, it becomes more of an art, and readers should refer Volume 1 of the recently-published Chlamydomonas Sourcebook for historical notes and practical hints. Additionally, it should be noted that there are many variations on the basic theme described above, ranging from the method for inducing gametogenesis, to the pattern of zygote distribution and dissection. Ultimately, each researcher finds their comfort zone, or adapts to laboratory-specific protocols.
Certain technical details mentioned above, should be reiterated. In particular, the cells must be healthy and without any infection. It is well known that laboratory-adapted strains which have not been crossed for long periods may not mate well, and water quality may also affect gametogenesis.
The authors have nothing to disclose.
This protocol is based on techniques learned in the laboratory of Francis-André Wollman at the Institut de Biologie Physico-Chimique, Paris, France. The authors owe a debt of gratitude to Jacqueline Girard-Bascou, who originally introduced D.B.S. to Chlamydomonas genetic techniques. Chlamydomonas research in the authors’ laboratory is supported by National Science Foundation award MCB-0646350.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
CC-125 | Mating type plus strain | Chlamydomonas Stock Center | ||
CC-124 | Mating type minus strain | Chlamydomonas Stock Center | ||
Tris-Acetate-Phosphate (TAP) | Growth medium | |||
N10 | Growth medium | |||
Difco agar | Reagent | |||
Fisherbrand Stainless-Steel Blades | Tools | Fisher Scientific | 08-916-5B | |
BD Surgical Blade Handles | Tools | Fisher Scientific | 08-914-5 | |
Fisherbrand Metal Scalpels | Tools | Fisher Scientific | 08-920A |