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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
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Parasites & Vectors

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