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Table 2 Genotyping by PCR-RFLP and number of polymorphisms at six genetic loci detected by PCR sequencing

From: Identification and genetic characterization of Toxoplasma gondii in free-ranging bristle-spined porcupine (Chaetomys subspinosus), a threatened arboreal mammal from the Brazilian Atlantic Forest

  No. of polymorphisms detected by sequencing  
Isolate Genotype PCR-RFLP Indel Ts Tv Total Sequence with the highest-scoring segment pairs in ToxoDB Identity (%); Expected value
Marker SAG1 (225 bp) – Chromosome VIII Coding function: Surface antigen gene
TgCsBr01 I 0 0 0 0 (0.0 %) TgUgCh83 (EF534734.1) 100; 4e-113
TgCsBr02 I 0 0 0 0 (0.0 %) TgUgCh83 (EF534734.1) 100; 4e-113
TgCsBr03 I 0 0 0 0 (0.0 %) TgUgCh83 (EF534734.1) 100; 4e-113
Marker SAG2 (385 bp) – Chromosome VIII Coding function: Surface antigen gene
TgCsBr01 I 1 2 3 6 (1.5 %) TgCkNg1 (EU650330.1) 99; 0.0
TgCsBr02 I 1 2 3 6 (1.5 %) TgCkNg1 (EU650330.1) 99; 0.0
TgCsBr03 I 7 4 3 14 (3.6 %) TgCkNg1 (EU650330.1) 97; 0.0
Marker SAG3 (115 bp) – Chromosome XII Coding function: Surface antigen gene
TgCsBr01 III 0 0 0 0 (0.0 %) Tg strain CTG (JX218227.1) 100; 3e-52
TgCsBr02 III 1 1 1 3 (2.6 %) Tg strain CTG (JX218227.1) 99:2e-49
TgCsBr03 III 2 0 0 2 (1.7 %) Tg strain CTG (JX218227.1) 98:5e-44
Marker c22-8 (485 bp) – Chromosome Ib Coding function: unknown “conserved hypothetical protein”
TgCsBr01 I 47 64 72 183 (37.7 %) TgCatBr5 (EU258488.1) 90; 1e-94
TgCsBr02 III 2 3 0 5 (1.0 %) Tg PTG (EU258476.1) 100; 0.0
TgCsBr03 III 3 1 1 5 (1.0 %) Tg PTG (EU258476.1) 98; 0.0
Marker PK1 (660 bp) – Chromosome VI Coding function: Protein serine/threonine kinase gene
TgCsBr01 III 5 0 1 6 (0.9 %) TgCkNg1 (EU650328.1) 99; 0.0
TgCsBr02 I 5 0 1 6 (0.9 %) TgCkNg1 (EU650328.1) 99; 0.0
TgCsBr03 I 0 0 1 1 (0.1 %) TgCkNg1 (EU650328.1) 99; 0.0
Marker Apicoa (461 bp) – Apicoplast chromosome
TgCsBr01 III 1 0 2 3 (0.6 %) T. gondii Apicoplast, comp. genome (U87145.2) 99; 0.0
TgCsBr02 III 42 27 52 121 (26.2 %) T. gondii Apicoplast, comp. genome (U87145.2) 95; 5e-30
TgCsBr03 III 1 0 0 1 (0.2 %) T. gondii Apicoplast, comp. genome (U87145.2) 99; 0.0
Total of polymorphisms at six different genetic loci detected by PCR sequencing of T. gondii isolatesb
Total of polymorphisms (%)
Isolate Genotype PCR-RFLP Indel Ts Tv Total Tajima’s relative rate testc Tajima’s D neutrality testd
TgCsBr01 Atypical 54 66 78 198 (8.5 %) u = 137  
TgCsBr02 Atypical 51 33 57 141 (6.0 %) u = 2  
TgCsBr03 Atypical 13 5 5 23 (1.0 %) u = 4  
average between samples 39.3 34.6 46.6 120.6 (5.2 %) P = 0.00000 D = 0.372232
  1. aThe sequences were aligned with the T. gondii apicoplast complete genome
  2. bThe number of insertions and deletions (Indel), transitions (Ts) and transversions (Tv) were calculated comparing the sequence of each isolate with the pattern obtained from GT1, ME49, VEG, TgCATBr5, TgCATBr9, FOU, RUB, VAND, p89, MAS, TgPgBr06, TgPgBr07, TgPgBr08, TgPgBr09, TgPgBr10, TgPgBr11, TgPgBr12, TgPgBr13, TgPgBr14, TgPgBr15, TgPgBr16, 54, 124 and 127 reference strains. The size of each amplicon means the number of base pairs that matched in all samples after the multiple alignment
  3. cThe equality of evolutionary rates between the sequences TgCsBr01, TgCsBr02 and TgCsBr03. “u” means unique differences in each sequence. All positions containing gaps and missing data were eliminated. There were a total of 1604 positions with 1461 identical sites in all three sequences and 0 divergent sites between all three sequences. A P-value less than 0.05 is often used to reject the null hypothesis of equal rates between lineages
  4. dThe analysis involved 27 multi-locus nucleotide sequences (GT1, ME49, VEG, TgCATBr5, TgCATBr9, FOU, RUB, VAND, p89, MAS, TgPgBr06, TgPgBr07, TgPgBr08, TgPgBr09, TgPgBr10, TgPgBr11, TgPgBr12, TgPgBr13, TgPgBr14, TgPgBr15, TgPgBr16, 54, 124, 127, TgCsBr01, TgCsBr02, TgCsBr03). All positions containing gaps and missing data were eliminated. There were a total of 1870 bases aligned with 388 segregating sites. A negative Tajima’s D indicates an excess of low-frequency polymorphisms. Evolutionary analyses were conducted in MEGA6