Articles

Demonstration of Loss of Heterozygosity by Single-Nucleotide Polymorphism Microarray Analysis and Alterations in Strain Morphology in Candida albicans Strains during Infection

Anja Forche, Georgiana May, P. T. Magee
DOI: 10.1128/EC.4.1.156-165.2005

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Design of allele-specific oligonucleotides and demonstration of the three possible genotypes. The top panel shows the composition of the allele-specific oligonucleotide attached to the microarray slide. The bottom panel depicts the three possible genotypes obtained through competitive hybridization of an experimental and control probe.

  • FIG. 2.
    FIG. 2.

    Cluster diagram showing SNP genotypes (see also Table 3) for strains SC5314, SC5314a, and SC5314α. The AF (see Materials and Methods) (x axis) is plotted against the square root (for scaling purposes only) of the total fluorescent signal (y axis). Each data point in the diagram represents results averaged over 4 (SC5314), 16 (SC5314a), and 14 (Sc5314α) independent experiments. Vertical lines represent the AF cutoff values for genotype determination: homozygous a1 < 0.4; homozygous a2 > 0.6; heterozygous alleles, 0.4 < a1 and a2 < 0.6. Values of 0.4 and 0.6 were scored as ambiguous.

  • FIG. 3.
    FIG. 3.

    Chromosomal location of SNP loci and LOH events of strains 6-4191 and 6-4205 (map modified from Forche et al.) (15). Numbers above chromosomes correspond to SNP order in Table 3. Except for the strains indicated, all strains analyzed exhibited the same SNP genotype as strain SC5314. Two markers on chromosome 7, YKL084 and 2397/2498, are homozygous in CAI-4 and derivative strains.

  • FIG. 4.
    FIG. 4.

    Karyotypes, colony morphology, and filamentation phenotypes of GAL1 recombinant strains. (A) Karyotypes of GAL1 recombinants that exhibit altered chromosomal patterns compared to those seen with the parental strains. Arrows point to chromosomal changes in strains 6-4193, 7-3990, and 6-4205. (B) Colony morphologies of strains 7-3976 and 7-3990 compared to that of strain AF7 on YEPD-uridine plates incubated at 30°C for 2 days. (C) Filamentation phenotypes at a magnification of ×40 for the GAL1 recombinants 6-4205, 7-3976, and 7-3990 compared to the results seen with the parental strains AF6 and AF7. Each set of recombinant and parent strains was streaked onto the same plate for accurate comparison.

Tables

  • TABLE 1.

    Allele-specific oligonucleotides and primer pairs for detection of SNPsa

    MP groupChromosomal location of SNPName of oligonucleotideAllele-specific oligonucleotide (5′-3′)bPrimers for probe generation (5′-3′)c
    MP-I5M1855/2172-a1-c5AmMC6-ttt ttt ttt ttt ttt TGT TGA CGT TGA TGGF: CCT GAA GTA ATG CCA CCA
        1855/2172-a2-t5AmMC6-ttt ttt ttt ttt ttt TGT TGA TGT TGA TGGR: CCA ATG TCT ATA GAA ACT GCCd
    5MSNF1-a1-a5AmMC6-ttt ttt ttt ttt ttt AGA GCC AAT AGT CAAF: CAC CTG AGA GAG AGG TAA C
        SNF1-a2-c5AmMC6-ttt ttt ttt ttt ttt AGA GCC ACT AGT CAAR: TTG TAG GCG GTG TGG GAA TTd
    5M1922/2344-a1-c5AmMC6-ttt ttt ttt ttt ttt TTA TCG GCG TAG TGAF: GCC ATG GCC TTA TGC TAT
        1922/2344-a2-t5AmMC6-ttt ttt ttt ttt ttt TTA TCG GTG TAG TGAR: GTT GCA CCA TTC CCA TGG GAd
    5MPDE1-a1-c5AmMC6-ttt ttt ttt ttt ttt GCA CAA CTA AGG AACF: ACG CAC AAC ACC ATA GCA
        PDE1-a2-t5AmMC6-ttt ttt ttt ttt ttt GCA CAA TTA AGG AACR: TAA TAT GCT AGG TGG GGG TTC CTTd
    5M1969/2162-a1-a5AmMC6-ttt ttt ttt ttt ttt GAA GTG GAG ATG ATAF: ATA CGG CCA CTC GGT ATG A
        1969/2162-a2-t5AmMC6-ttt ttt ttt ttt ttt GAA GTG GTG ATG ATAR: CAG CTA ATG CCC CAC GTA ATd
    5IDPH5-a1-a5AmMC6-ttt ttt ttt ttt ttt TTC TAC AGC AGA GAAF: CAG CCA ACC AGG AGT CAT TG
        DPH5-a2-g5AmMC6-ttt ttt ttt ttt ttt TTC TAC GGC AGA GAAR: ATT GTG GAT GGT CTC GACd
    5I2093/2390-a1-c5AmMC6-ttt ttt ttt ttt ttt GAT GAG TAC GAA TCAF: TCT CTC TTT GGA GTG AGC
        2093/2390-a2-t5AmMC6-ttt ttt ttt ttt ttt GAT GAG TAT GAA TCAR: GAA AGA TGG CTC CTT ATA TTT TGd
    5I1341/2493-a1-a5AmMC6-ttt ttt ttt ttt ttt GCA ATT TAG TAG CCAF: GTC AGC TTC ACC ACA GTT
        1341/2493-a2-g5AmMC6-ttt ttt ttt ttt ttt GCA ATT TGG TAG CCAR: AGT TCC AAC TCC AAA GCCd
    6OB15B20-a1-a(rc)e5AmMC6-ttt ttt ttt ttt ttt GGT TCA GAA TAA TGAF: GGA ATT GGA AAG AAG TCAd
        B15B20-a2-g(rc)5AmMC6-ttt ttt ttt ttt ttt GGT TCA GGA TAA TGAR: GCA TAT AGT CTA CCC AGT G
    7CYDL114-a1-t(rc)5AmMC6-ttt ttt ttt ttt ttt ACC TCC GTT GAG TAGF: CAA CCA CGT TCA GGA CCAd
        YDL114-a2-c(rc)5AmMC6-ttt ttt ttt ttt ttt ACC TCC GCT GAG TAGR: CCA CTC TCT CTC AAG TCA
    7FYKL084-a1-a5AmMC6-ttt ttt ttt ttt ttt TGG CTA TAC ATG TACF: TAA CCA TGA ACC AGA CCG
        YKL084-a2-t5AmMC6-ttt ttt ttt ttt ttt TGG CTA TAC TTG TACR: ACC AGG GTT GAA TTG TCCd
    7F2397/2498-a1-c5AmMC6-ttt ttt ttt ttt ttt ATT CTG GCT TGA AAGF: CTC ACC CCT TCG TCA CCA
        2397/2498-a2-t5AmMC6-ttt ttt ttt ttt ttt ATT CTG GTT TGA AAGR: GGT ATT GCT GGT ATC GGTd
    7FRBP1-a1-a5AmMC6-ttt ttt ttt ttt ttt TGG TTA CAG TAT CACF: ATT AGC ACC ACC TTT ACC
        RBP1-a2-t5AmMC6-ttt ttt ttt ttt ttt TGG TTA CTG TAT CACR: TGT CTG AAG AAC TTC CACd
    7GYJL54/2-a1-a5AmMC6-ttt ttt ttt ttt ttt TGG GAA TAC ACT GAGF: GGA GAC TGT ACC TTT AGT AG
        YJL54/2-a2-c5AmMC6-ttt ttt ttt ttt ttt TGG GAA TCC ACT GAGR: GCT ACT AAA ATA TGC TCA GTGd
    7G6.1345-a1-g(rc)5AmMC6-ttt ttt ttt ttt ttt CTC CAT GGC CAT TAGF: ATC GGC AAT GGA AGT GGGd
        6.1345-a2-a(rc)5AmMC6-ttt ttt ttt ttt ttt CTC CAT GAC CAT TAGR: GTC GTG GGA TTT TCA GCA AG
    7G6.282/2-a1-t(rc)5AmMC6-ttt ttt ttt ttt ttt TAT CTA ATG AAG CCAF: ACC ACC ACA ATT GGC TTC
        6.282/2-a2-c(rc)5AmMC6-ttt ttt ttt ttt ttt TAT CTA ACG AAG CCAR: GAT CAG CCC ATT GTT GATd
    MP-II5MC2F7-a1-t(rc)5AmMC6-ttt ttt ttt ttt ttt CAC CAC ATT TGA AATF: CGT CGT CTG TTA TAG TCC CCT Td
        C2F7-a2-c(rc)5AmMC6-ttt ttt ttt ttt ttt CAC CAC ACT TGA AATR: AGC CGG AAT AGA AAC TTT TGG C
    5MHST3-a1-c5AmMC6-ttt ttt ttt ttt ttt ACA TCA CCC TCA AATF: CCT GGG CAA AAG ACT AAC AG
        HST3-a2-t5AmMC6-ttt ttt ttt ttt ttt ACA TCA CTC TCA AATR: CCA ATG ACT TGA CAC CAG CAd
    5IHEX1-a1-g(rc)5AmMC6-ttt ttt ttt ttt ttt GGA TTT TTG TAC AAA TCF: GCT CAA TCC ACA CGC TTG TGd
        HEX1-a2-a(rc)5AmMC6-ttt ttt ttt ttt ttt GGA TTT TTA TAC AAA TCR: ACC ACA ACT TCC GTT CCC TT
    5I1817/2082-a1-c5AmMC6-ttt ttt ttt ttt ttt AGT TCT CCT CCA TTTF: GAC CAT TAT TTG AGA GCC AC
        1817/2082-a2-t5AmMC6-ttt ttt ttt ttt ttt AGT TCT CTT CCA TTTR: GGT GCA GTA CTT GTT GAT Gd
    5M1899/2008-a1-c5AmMC6-ttt ttt ttt ttt ttt TGT TTT CCG GTT GATF: TAC GGC CAA GTT TTC CTC AC
        1899/2008-a2-t5AmMC6-ttt ttt ttt ttt ttt TGT TTT CTG GTT GATR: GCT CGG GGA TAT AAT TGG CAd
    5M1445/2395-a1-c5AmMC6-ttt ttt ttt ttt ttt TGC CTT GCG TTA TAGF: GGT TTT GGT TTA TCT CCA GGT TTC
        1445/2395-a2-t5AmMC6-ttt ttt ttt ttt ttt TGC CTT GTG TTA TAGR: AGA TAC ATA CCG TGG GAA GCT ATTd
    5I2340/2493-a1-a5AmMC6-ttt ttt ttt ttt ttt TTT GGA TAA ACG AGTF: GCT TGG GGG TTC TGA TAC TT
        2340/2493-a2-g5AmMC6-ttt ttt ttt ttt ttt TTT GGA TGA ACG AGTR: AGC GAC CAT TAC GCA AGG TAd
    7CLEU2-a1-c5AmMC6-ttt ttt ttt ttt ttt CCC AAT TCA ACG GTTF: GGT GGT GGT CAA GAA AGA GG
        LEU2-a2-t5AmMC6-ttt ttt ttt ttt ttt CCC AAT TTA ACG GTTR: GGT ATT GAG ATT TGG CGT CGd
    7GARG4-a1-t(rc)5AmMC6-ttt ttt ttt ttt ttt ACC AAT GTG CCC ATCF: TCA CGG CAA TTC TTG AAC GGGd
        ARG4-a2-a(rc)5AmMC6-ttt ttt ttt ttt ttt ACC AAT GAG CCC ATCR: GCT AAA GCA CCA GAT CCT AAT GGA G

    • ↵ a Allele-specific oligonucleotides (ASOs) are sorted on the basis of their MP-PCR groupings (MP-I and MP-II). The chromosomal location, allele-specific oligonucleotide sequences for both alleles, and primer pairs for amplifcation of SNP markers are given.

    • ↵ b Each allele-specific oligonucleotide contains an amino-linker (5AmMC6), a 15-nucleotide poly-T-spacer (ttt ttt ttt ttt ttt), and 15 nucleotides of specific sequence with the polymorphic base at the middle position.

    • ↵ c F, forward; R, reverse.

    • ↵ d Primer used in PE reaction.

    • ↵ e (rc), reverse complement.

  • TABLE 2.

    Sizes of MP-PCR fragments and SNP positions

    MP groupChromosomal location of SNP locusName of SNP locusSize of MP product (bp)SNP position (from 5′ of forward primer)
    MP-I5M1855/2172206177
    5M SNF1 11754
    5M1922/2344234207
    5M PDE1 215190
    5M1969/2162303254
    5I DPH5 21533
    5I2093/2390175129
    5I1341/2493273174
    6OB15B2028252
    7C YDL114 21538
    7F YKL084 145112
    7F2397/2498245219
    7F RBP1 230157
    7GYJL54/2188167
    7G6.134523027
    7G6.282/228219
    MP-II5MC2F718877
    5M HST3 18274
    5I HEX1 14628
    5I1817/208214954
    5M1899/200819552
    5M1445/2395207135
    5I2340/2493160134
    7C LEU2 205174
    7G ARG4 19692
  • TABLE 3.

    Genotypes of parental strains and GAL1 derivatives

    SNP map locationaSNP locusGenotype for strain (AF)b:
    SC5314SC5314aSC5314αAF6AF77-3791d6-4191e6-4205e
    1C2F7 a1 a1 a1 a1 a1 a1 a1 a1
    21855/2172a1/a2 (0.51) a2 (0.82) a1 (0.30) a1/a2 (0.43)a1/a2 (0.49)a1/a2 (0.51)a1/a2 (0.42)a1/a2 (0.48)
    31899/2008a1/a2 (0.50) a1 (0.26) a2 (0.87) a1/a2 (0.41)a1/a2 (0.47)a1/a2 (0.46)a1/a2 (0.59)a1/a2 (0.50)
    4 SNF1 a1/a2 (0.52) a1 (0.11) a2 (0.74) a1/a2 (0.52)a1/a2 (0.50)a1/a2 (0.44)a1/a2 (0.54)a1/a2 (0.49)
    5 HST3 a1/a2 (0.51) a1 (0.09) a2 (0.96) a1/a2 (0.50)a1/a2 (0.52)a1/a2 (0.49) a2 (0.64) a1/a2 (0.51)
    61445/2395a1/a2 (0.51) a2 (0.65) a1 (0.36) a1/a2 (0.46)a1/a2 (0.49)a1/a2 (0.49)a1/a2 (0.45)a1/a2 (0.49)
    71922/2344a1/a2 (0.49) a1 (0.32) a2 (0.77) a1/a2 (0.50)a1/a2 (0.50)a1/a2 (0.51)a1/a2 (0.59)a1/a2 (0.50)
    8 PDE1 a1/a2 (0.50) a2 (0.93) a1 (0.16) a1/a2 (0.48)a1/a2 (0.50)a1/a2 (0.51) a1 (0.37) a1/a2 (0.51)
    91969/2162a1/a2 (0.50) a1 (0.16) a2 (0.87) a1/a2 (0.56)a1/a2 (0.53)a1/a2 (0.56) a2 (0.64) a1/a2 (0.50)
    10 HEX1 c a1 a1 a1 a1 a1 a1 a1 a1
    11 DPH5 a1/a2 (0.56) a2 (0.74) a1 (0.06) a1/a2 (0.53)a1/a2 (0.53)a1/a2 (0.57) a1 (0.36) a1/a2 (0.56)
    131817/2082a1/a2 (0.50) a1 (0.03) a2 (0.97) a1/a2 (0.46)a1/a2 (0.50)a1/a2 (0.46) a2 (0.61) a1/a2 (0.50)
    141341/2493a1/a2 (0.51) a1 (0.24) a2 (0.92) a1/a2 (0.52)a1/a2 (0.52)a1/a2 (0.46) a2 (0.62) a1/a2 (0.51)
    152340/2493a1/a2 (0.48) a2 (0.96) a1 (0.10) a1/a2 (0.49)a1/a2 (0.52)a1/a2 (0.52) a1 (0.37) a1/a2 (0.53)
    16B15B20a1/a2 (0.48)a1/a2 (0.53)a1/a2 (0.52)a1/a2 (0.46)a1/a2 (0.49)a1/a2 (0.48)a1/a2 (0.48)a1 (0.35)
    17 LEU2 c a1 a1 a1 a1 a1 a1 a1 a1
    18 YDL114 a1/a2 (0.49)a1/a2 (0.49)a1/a2 (0.48)a1/a2 (0.51)a1/a2 (0.51)a1/a2 (0.51)a1/a2 (0.50)a1/a2 (0.48)
    19 YKL084 a1/a2 (0.53)a1/a2 (0.49)a1/a2 (0.49) a1 (0.02) a1 (0.02) a1 (0.02) a1 (0.05) a1 (0.03)
    202397/2498a1/a2 (0.52)a1/a2 (0.50)a1/a2 (0.50) a1 (0.05) a1 (0.05) a1 (0.05) a1 (0.08) a1 (0.06)
    21 RBP1 a1/a2 (0.51)a1/a2 (0.50)a1/a2 (0.49)a1/a2 (0.51)a1/a2 (0.50)a1/a2 (0.50)a1/a2 (0.50)a1/a2 (0.51)
    22YJL54/2a1/a2 (0.53)a1/a2 (0.46)a1/a2 (0.49)a1/a2 (0.43)a1/a2 (0.50)a1/a2 (0.42)a1/a2 (0.48)a1/a2 (0.50)
    236.1345a1/a2 (0.51)a1/a2 (0.52)a1/a2 (0.46)a1/a2 (0.51)a1/a2 (0.50)a1/a2 (0.51)a1/a2 (0.50)a1/a2 (0.45)
    256.282a1/a2 (0.52)a1/a2 (0.51)a1/a2 (0.54)a1/a2 (0.48)a1/a2 (0.51)a1/a2 (0.48)a1/a2 (0.50)a1/a2 (0.46)

    • ↵ a Numbering corresponds to the locations of SNPs on the SNP map.

    • ↵ b AF values were averaged over a minimum of eight spots (two independent experiments) per strain.

    • ↵ c Signal only obtained for homozygous allele; therefore, no calculation of a1/a2 ratios was performed.

    • ↵ d Strain after mouse passage in AF7 background.

    • ↵ e Strain after mouse passage in AF6 background. Representative SNP genotypes for 19 strains with no LOH.

  • TABLE 4.

    Chromosome 5 haplotypes for strain SC5314 at 15 SNP loci

    SNP map locationaSNP locushaplotypeb
    a1da2
    1C2F7tt
    21855/2172tc
    31899/2008ct
    4 SNF1 ac
    5 HST3 ct
    61445/2395tc
    71922/2344ct
    8 PDE1 tc
    91969/2162at
    10 HEX1 gg
    11 DPH5 ga
    122093/2390cct
    131817/2082ct
    141341/2493ag
    152340/2493ga

    • ↵ a Numbering corresponds to the locations of SNPs on the SNP map (Fig. 3).

    • ↵ b Haplotypes were obtained from sorbose-derived strains SC5314a (a1) and SC5314α (a2).

    • ↵ c Haplotypes were confirmed by sequencing (data not shown).

    • ↵ d Numbering of alleles (a1 versus a2) was random.

  • TABLE 5.

    Summary of genotypic and phenotypic results for parental strains and GAL1 derivativesa

    StrainLOHGrowth rate in YEPD broth (change)bColony morphologyFilamentation on serumKaryotype
    7-3791−−−−−
    7-3972−−−−−
    7-3973−−−−−
    7-3974−−−−−
    7-3975−−−−−
    7-3976−+ (down)++−
    7-3977−−−−−
    7-3978−−−−−
    7-3979−−−−−
    7-3980−−−−−
    7-3981−−−−−
    7-3982−−−−−
    7-3983−−−−−
    7-3990−−+++
    7-4118−−−−−
    7-4130−−−−−
    7-4164−−−−−
    6-3552−+ (up)−−−
    6-4191+−−−−
    6-4193−−−−+
    6-4205++ (down)+++

    • ↵ a “+” and “−” indicate change and no difference, respectively, compared to parental strain results.

    • ↵ b up, increase in growth rate compared to parental strain results; down, decrease in growth rate compared to parental strain results.

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KEYWORDS

Candida albicans
Oligonucleotide Array Sequence Analysis

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