RESEARCH ARTICLE E/cient and rapid identi¢cation of Candida albicans allelic status using SNP-RFLP Anja Forche, Musetta Steinbach & Judith Berman Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA Correspondence: Present address: Anja Forche, Department of Biology, Bowdoin College, 234 Druckenmiller Hall, 6500 College Station, Brunswick, ME 04011, USA. Tel.: 11 207 725 3365; fax: 11 207 725 3405; e-mail: aforche@bowdoin.edu Received 26 March 2009; revised 22 May 2009; accepted 5 June 2009. Final version published online 20 July 2009. DOI:10.1111/j.1567-1364.2009.00542.x Editor: Richard Calderone Keywords Candida albicans; SNP-RFLP; LOH; aneuploidy; genome rearrangement. Abstract Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life-threatening deep-tissue infections. Recent studies provide strong evidence that C. albicans possesses an arsenal of genetic mechanisms promoting genome plasticity and that it uses these mechanisms under conditions of nutritional or antifungal drug stress. Two microarray-based methods, single nucleotide polymorphism (SNP) and comparative genome hybridization arrays, have been developed to study genome changes in C. albicans. However, array technologies can be relatively expensive and are not available to every laboratory. In addition, they often generate more data than needed to analyze specific genomic loci or regions. Here, we have developed a set of SNP-restriction fragment length polymorphism (RFLP) (or PCR-RFLP) markers, two per chromosome arm, for C. albicans. These markers can be used to rapidly and accurately detect large-scale changes in the C. albicans genome including loss of heterozygosity (LOH) at single loci, across chromosome arms or across whole chromosomes. Furthermore, skewed SNP-RFLP allelic ratios are indicative of trisomy at heterozygous loci. While less comprehensive than array-based approaches, we propose SNP-RFLP as an inexpensive, rapid, and reliable method to screen strains of interest for possible genome changes. Introduction Candida albicans is the most prevalent opportunistic fungal pathogen in the clinical setting, causing a wide spectrum of diseases ranging from superficial mucosal lesions to life- threatening deep-tissue infections. Although host immunity is a major contributor to the development and progression of disease (Ashman et al., 2004; Tuite et al., 2004; Ashman, 2008), genome plasticity of the eukaryotic pathogen is an important factor in the adaptation of this fungus to its many niches in the warm-blooded host (Fradin et al., 2003; Hube, 2004; Forche et al., 2009). Candida albicans is a diploid yeast and, although a parasexual cycle has been defined (Bennett & Johnson, 2003, 2005), no meiosis has been observed and the range of diversity observed in clinical strains is consis- tent with a predominantly clonal and asexual life cycle (Pujol et al., 1993; Gr¨ aser et al., 1996; Forche et al., 1999). Thus, classical segregation analyses (such as those applied to Saccharomyces cerevisiae) are not used to study and detect genetic and genomic changes. The diploid assembly of the C. albicans SC5314 genome sequence (Jones et al., 2004; Van het Hoog et al., 2007) revealed a high level of natural heterozygosity, with c. 55 000 single nucleotide polymorph- isms (SNPs) (c. 3% of the 16-Mb genome). In many C. albicans isolates, segmental or whole-chromo- some aneuploidies arise in response to environmental stresses (Janbon et al., 1998; Perepnikhatka et al., 1999; Selmecki et al., 2006; Rustchenko, 2007). For example, gain and loss of nutrient assimilation is associated with chromo- somal alterations (Rustchenko et al., 1994), and the growth on specific carbon sources can result in the loss of a specific chromosome (Janbon et al., 1998). A large proportion of C. albicans strains that acquire resistance to fluconazole, the most commonly used antifungal drug, form a segmental aneuploidy, isochromosome 5L [i(5L)], in which the left arm of Chr5 [i(5L)] is duplicated. Candida albicans that acquire i(5L) become highly resistant to the antifungal drug flucona- zole due to extra copies of two genes on Chr5L: ERG11, FEMS Yeast Res 9 (2009) 1061–1069 c  2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved YEAST RESEARCH Downloaded from https://academic.oup.com/femsyr/article-abstract/9/7/1061/512645 by guest on 20 May 2020