Antonie van Leeuwenhoek 73: 229–236, 1998. 229 c 1998 Kluwer Academic Publishers. Printed in the Netherlands. Complementation analysis with new genetic markers in Phaffia rhodozyma P. Retamales, R. Le´ on, C. Mart´ ınez, G. Hermosilla, G. Pincheira & V. Cifuentes Laboratorio de Gen´ etica, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile ( author for correspondence: e-mail: vcifuent@abello.dic.uchile.cl) Received 10 October 1997; accepted in final form 20 January 1998 Key words: auxotrophs, mutagenesis, nystatin enrichment, protoplasts fusion, Phaffia rhodozyma Abstract Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N -nitro-N-nitrosoguanidine(NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade and met in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385. Introduction Phaffia rhodozyma, a carotenoid-producing yeast with properties of a basidiomycetous origin (Miller et al., 1976), has been isolated from exudates of deciduous trees in cold regions of Alaska, Japan and Russia (Phaff & Starmer, 1989). This yeast is a basidiomycete that forms holobasidia with terminal basidiospores (Gol- ubev, 1995). P. rhodozyma has the capacity to ferment glucose and to synthesize carotenoids, with astaxanthin as its principal pigment (Miller et al., 1976; Johnson & Lewis, 1979; Andrewes et al., 1976). This nat- ural pigment is useful as a food additive in salmon aquaculture (Johnson et al., 1977; Torrissen et al., 1989; Sanderson & Jolly, 1994). The production of carotenoids in P. rhodozyma may be enhanced by clas- sical mutagenic treatments (An et al., 1989; Lewis et al., 1990). However, the more effective development of astaxanthin overproducing strains may be related to a better genetic knowledge of this yeast. Classi- cal genetic analysis of P. rhodozyma has been diffi- cult because the perfect state of its reproductive cycle has been described, only recently, by Golubev (1995). Furthermore, some genetical aspects of P. rhodozyma, such as the characterization of chromosome composi- tion by CHEF analysis (Nagy et al., 1994; Adrio et al., 1995; Cifuentes et al., 1997), isolation of auxotrophic mutants (Palagyi et al., 1995; Adrio et al., 1993) and complementation analysis by protoplast fusion (Chun et al., 1992; Girard et al., 1994) have also recently been communicated. Studies using isoenzymes, restriction fragment length polymorphism and random amplified polymorphic DNA (Varga et al., 1995) and phyloge- netic analysis of its actin gene (Wery et al., 1996) have improved our understanding of its phylogenetic and genome organization. Moreover, several methods for genetic transformation of P. rhodozyma have been developed. One of these methods uses plasmid DNA for the transformation of protoplasts with the bacte- rial kanamycin resistance gene (Adrio et al., 1995). An other method allows a high copy number integra- tion in ribosomal DNA by using the polyethyleng- lycol/lithium acetate technique (Wery et al., 1997). An other procedure uses linear DNA of a wild-type strain by an electroporation technique (Rubinstein et al., 1996). In our laboratory, astaxanthin mutants were