Novel approaches for generating and manipulating diploid strains of Chlamydomonas reinhardtii

Abstract

Genetic study of haploid organisms offers the advantage that mutant phenotypes are directly displayed, but has the disadvantage that strains carrying lethal mutations are not readily maintained. We describe an approach for generating and performing genetic analysis of diploid strains of Chlamydomonas reinhardtii, which is normally haploid. First protocol utilizes self-mating diploid strains that will facilitate the genetic analysis of recessive lethal mutations by offering a convenient way to produce homozygous diploids in a single mating. Second protocol is designed to reduce the chance of contamination and the accumulation of spontaneous mutations for long-term storage of mutant strains. Third protocol for inducing the meiotic program is also included to produce haploid mutant strains following tetraploid genetic analysis. We discuss implication of self-fertile strains for the future of Chlamydomonas research.

Studying Whole-Genome Duplication Using Experimental Evolution of Chlamydomonas

“…DNA should be extracted using a method that produces a high yield, such as traditional cetrimonium bromide (CTAB) and phenol-chloroform extractions or genomic DNA extraction kits, while Qubit (Invitrogen, Carlsbad, CA, USA) can be used for fluorometry. This method was, for example, used by Kariyawasam et al [65], and the quantification of the DNA from our own extractions seems to support the accuracy of this method reasonably well. Nevertheless, we would recommend treating the results with care.…”

TALE homeobox heterodimer GSM1/GSP1 is a molecular switch that prevents unwarranted genetic recombination in Chlamydomonas

“…Vegetative (mitotic) diploids are a critical research tool for analyzing allelic interactions in C. reinhardtii . One way to generate vegetative diploids is to collect cells that have escaped normal zygote development by transferring early‐stage zygotes to a double‐selection growth medium requiring two markers in the haploid parental strains (Ebersold, ; Palombella and Dutcher, ; Kariyawasam et al ., ). Under this selection scheme diploidization requires nuclear fusion, which does not occur in gsm1 ‐null or gsp1 ‐het zygotes.…”

“…Complemented gsp1‐3 C ( gsp1‐3 ; T‐ GSP1 ) was generated from gsp1‐3 mt+ × T‐ GSP1 mt− (Lee et al ., ). Zygotes for tetrad isolation were prepared as previously described (Kariyawasam et al ., ).…”

Grand Challenges in Microalgae Domestication

“…Major breakthroughs could be anticipated if sexual reproduction could be controlled in routine in Chlorella, Dunaliella, Scenedesmus, Klebsormidium, Phaeodactylum etc. We need to keep in mind that in some algal species, the existence of mating types may limit the capacity to develop homozygous diploids requiring the development of self-mating lines (Kariyawasam et al, 2019 ).…”

“…Diatoms have diploid vegetative cells allowing the exploration of this property. The development of autopolyploids from haploid cells has been attempted in pioneering experiments by treatments of Chlamydomonas with molecules blocking cell division, like colchicine (Wetherell and Krauss, 1956 ) or colcemide (Kwak et al, 2017 ; Kariyawasam et al, 2019 ). In diploid Chlamydomonas lines, the lipid yield was improved (Kwak et al, 2017 ).…”