The C. albicans
genome is quite plastic, exhibiting a broad range of mitotic recombination events (Lephart et al., 2005
; Lephart and Magee, 2006
; Forche et al., 2008
; Legrand et al., 2008
)in addition to aneuploidies due to chromosome loss (Janbon et al., 1998
) or acquisition of whole chromosomes or chromosome segments (Selmecki et al., 2005
). To distinguish these different mechanisms, the minimal number of markers required is 4 per chromosome, with two markers per chromosome arm. A total of 152 SNP markers previously used on our SNP microarray ((Forche et al., 2005
; Forche et al., 2008
; Legrand et al., 2008
), ) were screened for the presence of restriction enzyme sites that digest SNPs within the SNP marker sequence. These SNP markers are on average 200 bp in length and often include several SNPs (Forche et al., 1999
; Forche et al., 2004
; Forche et al., 2005
). Of the 152 SNP markers analyzed, 134 (88 %) included one or more SNPs that were recognized by a restriction enzyme (RE) (, Tables S2
). Of these, 112 (74 %) included unique RE sites (those digested only once within the marker sequence) ().
Figure 1 Whole genome map of SNP-RFLP markers. Homolog ‘a’ is colored in black, homolog ‘b’ is colored in grey, Major repeat sequences are indicated as black filled boxes except for Chr3 (RB2 only; hashed box). The centromere is (more ...)
Summary of SNP-RFLP makers sorted by chromosome
We selected a set of 32 markers (8 chromosomes, 4 per chromosome) from the 112 SNP-RFLP markers with unique restriction enzymes recognition sites (). PCR amplification of these markers, followed by digestion with the relevant RE results in 3 distinguishable fragments: one fragment from the uncut allele and two smaller fragments from the allele that carries the polymorphism recognized by the RE. Strain SC5314, which is heterozygous for all SNP markers, was tested for the correct restriction pattern (data not shown). Thirty one SNP markers gave the expected 3 restriction fragments (3 fragments; one fragment for uncut allele and 2 fragments for the cut allele), and restriction digest of marker 1765/2519 (Chr3) yielded the expected 4 restriction fragments ().
Figure 2 Use of the diagnostic SNP-RFLP marker set confirms heterozygosity of all 32 markers in strain SC5314. The chromosome is indicated below each set of gel panels. For each digest the uncut (u) and cut (c) PCR products are shown. Order of the SNP markers (more ...)
In a previous study, haplotypes for each of the eight pairs of chromosomes for strain SC5314 were assigned based on SNP microarray analysis of strains with existing and induced aneuploidies (Legrand et al., 2008
). For 9 out of 32 markers the SNPs analyzed by microarray and by SNP-RFLP were identical () and thus the assignment of haplotype identity was straight-forward. For marker F12n4 on Chr1 no SNP-RFLP haplotype could be assigned (see note below ). Based on the assumption that original trace sequences for strain SC5314 were approximately 400 bp in length, and based on the fact that array SNPs and RFLP SNPs within a specific marker were on average no further apart than approximately 80 bp, each SNP (nucleotide) analyzed by SNP-RFLP was matched up with the appropriate homolog ( a or b) containing the SNP (nucleotide) present on the SNP array (Legrand et al., 2008
). Based on the restriction digest results (cut or uncut) haplotypes were assigned for the remaining 21 SNP-RFLP markers ().
By screening strains of interest, we asked if the SNP-RFLP markers would detect known genotype changes including LOH (the appearance of only a single uncut band or the two cut bands) at a single locus, LOH of a chromosome arm or LOH at all loci across an entire chromosome. In addition, we asked if trisomy of a chromosome arm or of an entire chromosome could be detected as a change in the relative ratio of the uncut band to the two cut bands (Legrand et al., 2008
; Selmecki et al., 2008
As a proof-of-principle, we performed SNP-RFLP analysis of strains known to have genotypic alterations when compared to their parental genotype profiles (Table S1
, ). For example, strain YJB10019 (derived from SC5314) was known from a previous study (Forche et al., 2008
) to have undergone changes on multiple chromosomes including a single LOH event on Chr1, a chromosome arm LOH event on Chr2, and a whole Chr LOH on ChrR. Along with strain SC5314, YJB10019 was subjected to SNP-RFLP analysis of markers from chromosomes R, 1, and 2. shows the results of the restriction digests. Strain SC5314 was heterozygous for all 4 markers on ChrR, Chr1, and Chr2, respectively (). In strain YJB10019, a single LOH event on Chr1 was detected at the telomere-proximal 1799/2450 marker (). As expected, the Chr2 arm event spanned both markers on Chr2L (2051/2483 and 1414/2481) (). The most extensive LOH event was observed for ChrR, where all 4 SNP markers were homozygous. This indicates that LOH occurred across most parts or the entire chromosome. Whole chromosome LOH most likely arises via one or more chromosome non-disjunction events
Detection of LOH and trisomy by SNP-RFLP analysis
Aneuploidy, especially trisomy is frequently observed in drug resistant C. albicans
isolates (Selmecki et al., 2006
; Selmecki et al., 2008
). Array Comparative Genome Hybridization (aCGH) (Selmecki et al., 2005
) is the most comprehensive method for detecting aneuploidies. However, aCGH can be expensive and requires microarray technology that may not be readily available to all labs. Because trisomy results in skewed allelic ratios (Legrand et al., 2008
), we asked if it is possible to detect skewed allele copy number by SNP-RFLP. For this experiment we chose two sets of strains that had included chromosomes with segmental or whole chromosome aneuploidies.
We used a well documented set of strains derived, by transformation, from CAF-2 (diploid). These strains had become trisomic for Chr2 (CAI4-F2 and CAI4-F3) (Selmecki et al., 2005
). The CAI4-F3 strain also became trisomic for Chr1 (Chen et al., 2004
; Selmecki et al., 2005
). SNP-RFLP analysis was carried out for 4 markers of Chr1 and Chr2 for the parental strain CAF-2 and for the two versions of CAI4. Strain CAF-2 was heterozygous for all 4 markers on Chr1 and Chr2 with relative band intensities appropriate for 1:1 amounts of the two different alleles ( left images). In contrast, in strain CAI4-F3, allele ratio bias was evident for 4 markers on Chr2 ( right image) and for 1 marker on Chr1 ( right image). Furthermore, as expected, markers on Chr2, but not on Chr1 exhibited skewed allele ratios for strain CAI4-F2 ( middle images). This demonstrates that SNP-RFLP analysis generates data consistent with the idea that these chromosomes are trisomic in the strain. When multiple markers on a single chromosome exhibit a skewed allelic ratio, it provides more confidence in the idea that the chromosome may be aneuploid. Of course, aCGH would be necessary to confirm this hypothesis.
We next compared strains FH1 and FH6 (White, 1997a
; White, 1997b
, Selmecki et al., 2008
). From aCGH, it was known that FH6 carries i(5L), a segmental aneuploidy in which there are two extra copies of the left arm of chromosome 5, and SNP array analysis had found that markers on Chr5L were present in a 2:1 ratio (Selmecki et al., 2008
). It is important to note that FH1 and FH6 are clinical isolates and likely possess a different SNP distribution across their genome. Thus, although we analyzed all 4 diagnostic SNP-RFLP markers from Ch5, only 2 of the Chr5 markers were heterozygous for both FH1 and FH6 (). Similarly, when markers are not heterozygous, it is difficult to infer changes in ploidy with high confidence. For example, while a skewed allelic ratio (2:1) for the first marker (10080A) on Chr5L can be detected for strain FH6 (), and this is consistent with trisomy of Chr5L detected in this strain (Selmecki et al., 2008
), no skewed allelic ration (2:1) can be detected for strain FH6 for the second marker (SNF1–4) on Chr5L. Marker 2222A on Chr5R was homozygous in both strains and thus is not informative with regard to alterations in the genome ().
In conclusion, we describe here a practical, inexpensive and simple approach to determining if chromosomes underwent loss of heterozygosity using a diagnostic set of SNP-RFLP markers to detect LOH events in the Candida albicans
genome. This method provides the ability to distinguish short range events that generate LOH of a single SNP, LOH events that involve multiple SNPs on a chromosome arm as well as LOH of SNPs spanning entire chromosomes. Combined with our HapMap (Legrand et al., 2008
), haplotype exchanges can also be detected. Furthermore, this SNP-RFLP approach can be used as a preliminary test for trisomy of chromosomes that remain heterozygous. Trisomy is more difficult to detect and thus the absence of a skewed ratio of RE fragment alleles is not definitive. However, the presence of a skewed ratio at multiple markers on a chromosome provides more confidence in the interpretation of a potential trisomic chromosome.
A major advantage is that this diagnostic set of SNP-RFLP markers provides a rapid and accurate method to detect genomic changes on all 8 chromosomes after strain manipulations such as transformation, which can lead to increased levels of aneuploidy and/or LOH (Bouchonville et al., in preparation). The use of 2 SNP markers per chromosome arm reveals distinct mechanisms of LOH. Importantly, this method relies upon simple techniques available in all molecular biology labs, enabling the generation of data that is easily compared between research groups. In this regard, it should be considered a simple, rapid and accessible alternative to SNP microarrays and MLST for testing strain integrity.