PCR-based assay for mating type and diploidy in Chlamydomonas

The unicellular green alga Chlamydomonas reinhardtii has become an important genetic model system for studying photosynthesis, cell motility, and organelle biogenesis (1). Chlamydomonas has many of the same advantages as budding yeast for doing genetics, including rapid growth, the ability to maintain both haploids and diploids, tetrad analysis, easy transformation with DNA, and a completely sequenced genome. Because of these advantages, Chlamydomonas is often referred to as “green yeast.”

The primary strength of Chlamydomonas is its powerful genetics. During most genetic procedures, it becomes necessary to determine the mating type of a given strain. Chlamydomonas is a heterothallic organism, with two mating types denoted mt+ and mt−. Mating type is determined by two different alleles of a single MT locus, and unlike budding yeast, there are no additional silent copies in the genome. Mating type is normally assessed using functional mating tests with tester strains (2). These functional mating tests often fail because many mutants of interest (e.g., mutants with defective flagellar motility) are very poor maters. Moreover, mating efficiency can be affected by variation in media or precise growth conditions. Thus there is a need for a rapid method to determine mating type that will work even in mutants with low mating efficiency.

A related problem concerns testing for diploidy. Diploid strains are extremely valuable because they allow complementation tests and the determination of whether a given mutation is recessive or dominant. One selects for diploids by mating cells that contain combinations of closely linked auxotrophic markers such that a diploid, but neither haploid parent strain, can grow on minimal media. However, both spontaneous revertants for either marker, or recombination occurring between the linked markers, can result in haploid cells that still grow on minimal media. It is therefore always critical to verify that a putative diploid strain is actually a diploid. Some workers have reported the analysis of diploids through the use of Southern blot analysis, either with probes within the MT locus (3) or with probes outside the MT locus that detect an restriction fragment-length polymorphism (RFLP) within MT that distinguishes the two alleles (4). However, Southern blot analysis is time-consuming and inconvenient for analyzing a large number of strains.

We have developed a PCR-based method for assaying mating type and for distinguishing haploids from diploids. This method takes advantage of the fact that the mt+ and mt− alleles of the MT locus each contain unique sequences not present in the other allele. The use of PCR allows us to avoid Southern blot analysis steps. A PCR-based method for detecting the mt+ and mt− alleles has been reported (5). However, it was reported that this method falsely classified some known diploid cells as haploid (5), calling the reliability of the method into question. This previously described method required two steps, a resin-based genomic DNA purification followed by a PCR step.

We have developed a new one-step PCR-based approach that circumvents these difficulties. Based on the mt+ and mt− specific allele sequences (6,7) we designed four primer pairs specific to each allele (Table 1). PCR was carried out using the conditions listed below, with DNA isolated from wild type mt+ or mt− cells. As illustrated in Figure 1A, all four mt+ specific primer pairs gave products of the expected size when used in reactions with DNA from mt+ cells. In contrast, none of the mt+ specific primers gave any detectable product with DNA from mt− cells. Likewise, all four mt− specific primer pairs gave products of the expected sizes when used in reactions with DNA from mt-cells and gave no product in reactions with DNA from mt+ cells. This establishes that the primer pairs are specific for the MT region.

Table 1.  PCR Primer Pairs for Chlamydomonas Mating Type Analysis

We then selected one mt+ specific primer pair and one mt− specific primer pair, which give different size products, allowing both primer pairs to be combined in a single PCR. The mt+ allele was detected using the pair of primers MTP2F and MTP2R (Table 1), which were designed to detect a 423-bp piece of the mt+ specific gene FUS1 (Gen-Bank^® accession no. U49864) and which encodes a mating type plus specific glycoprotein (6). The mt− allele was detected using the pair of primers MTM3F and MTM3R (Table 1), which were designed to detect a 689-bp piece of the mt− specific gene MID1 (Gen-Bank accession no. U92071, also known as IMP 11) and which encodes a putative leucine-zipper transcription factor involved in sex determination (7). PCRs using MTM3F and MTM3R with DNA isolated from the strain cf181, an mt− strain carrying a deletion of the MID1 gene, gave no detectable product (data not shown), further confirming the specificity of this primer pair.

PCR analysis of the MT locus was performed as follows. Single colonies of cells from a plate were suspended in 5 µL of water and boiled for 5 min in a PCR tube. Into this same tube, the four primers MTP2F, MTP2R, MTM3F, and MTM3R (38 pmol of each) were added along with nucleotides, polymerase mixture, buffer (both from BD Biosciences Clontech, Palo Alto, CA, USA), and water to a final reaction volume of 25 µL, according to manufacturer's instructions. PCRs were performed using the Advantage^®GC kit (BD Biosciences Clontech). Reactions were run for 30 amplification cycles, each consisting of denaturation at 94°C for 30 s, followed by annealing/elongation at 68°C for 1.5 min. Figure 1B illustrates the application of this assay to wild-type test strains, and confirms that we can distinguish haploid mt+, haploid mt−, and diploid cells. Diploid cells are easily distinguished by the presence of two bands.

In order to check the reliability of diploid detection in our procedure, we isolated 38 independently generated diploid strains (formed by mating arg2 mt+ with arg7 mt− and selecting for arg2/arg7 diploids that can grow on media lacking arginine due to intragenic complementation between the arg2 and arg7 alleles). These diploid lines were subjected to PCR analysis (Figure 2), which indicated that, out of 38 diploid lines, all showed the expected pair of products.

In contrast to previously described mating type assays, PCR-based assays are easily applied to large numbers of strains and to strains that mate poorly. The procedure documented here represents a significant advance over that reported by Werner and Mergenhagen (5), in that our method (i) eliminates the need for a separate DNA isolation step and (ii) detects diploids with 100% reliability.