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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Publications at this Location » Publication #401608

Research Project: New Bioproducts for Advanced Biorefineries

Location: Bioenergy Research

Title: A PCR based method for rapid identification of mating type in Rhodosporidium toruloides yeast

item LOPES, DAIANE - Orise Fellow
item Dien, Bruce
item SINGH, VIJAY - University Of Illinois
item JAGTAP, SUJIT - University Of Illinois
item RAO, CHRISTOPHER - University Of Illinois
item Hector, Ronald - Ron

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/25/2023
Publication Date: 5/1/2023
Citation: Lopes, D., Dien, B.S., Singh, V., Jagtap, S., Rao, C., Hector, R.E. 2023. A PCR based method for rapid identification of mating type in Rhodosporidium toruloides yeast [abstract]. Symposium on Biomaterials, Fuels, and Chemicals . Poster No. M13/RM3-A.

Interpretive Summary:

Technical Abstract: Rhodosporidium toruloides (RT) is an oleaginous yeast species with increasing industrial interest because of its robust growth characteristics and high lipid yields. Polyploid yeast are favored in general for industrial use because mating gives the yeast hybrid vigor and allows for increased population diversity. Increased diversity is generated by direct mating and selection of the most adapted progeny or by the fusion of protoplasts between yeasts with desired characteristics. Generating higher ploidy RT yeast requires knowledge of the yeasts’ mating types and ploidies. Herein, a convenient and quick protocol is described to determine mating type (A1 or A2). The method is based on colony direct PCR using a standard PCR reagent. Conserved regions of the pheromone receptor Ste3 (Ste3.A1 and Ste3.A2) from DNA sequences of RT published in GenBank were used to design the forward and reverse primers (4 pairs of primers). The method works well with either pre-boiled cell supernatant or purified DNA. Assay results were validated using a set of 14 RT yeast, which included haploids and diploids. The amplification products were around 400 bp for MAT-A1 and 620 bp for MAT-A2. This allows for testing for the presence of both mating types in a single multiplex reaction. Assay results were validated by visual confirmation of mating type by microscopic examination using A1 and A2 RT tester strains. We are also currently developing a ploidy assay based on use of a relatively low-cost image analyzer designed for use with yeast cultures. This work can help in the rapid screening of mating types in strains of the RT species, aiming at the development of strains better adapted for growth in industrial sized bioreactors.