Amphitrema flavum3 no 18S rDNA sequence

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• Amphitrema flavum 3 no 18S rDNA sequence
• Arcella sp. 1 no 18S rDNA sequence
• Assulina muscorum 55 AJ418791
• Assulina seminulum 41 no 18S rDNA sequence
• Bullinularia indica 28 no 18S rDNA sequence
• Cetropyxis aerophila 1 no 18S rDNA sequence
• Centropyxis laevigata 39 no 18S rDNA sequence
• Corythion dubium 18 no 18S rDNA sequence
• Euglypha compressa 50 no 18S rDNA sequence (G)
• Euglypha laevis 1 no 18S rDNA sequence (G)
• Euglypha strigosa 8 no 18S rDNA sequence (G)
• Heleopera sylvatica 2 no 18S rDNA sequence
• Hyalosphenia elegans 55 no 18S rDNA sequence
• Hyalosphenia minuta 2 no 18S rDNA sequence
• Hyalosphenia papilio 56 no 18S rDNA sequence
• Nebela griseola 1 no 18S rDNA sequence
• Nebela miltaris 3 no 18S rDNA sequence
• Nebela tincta 57 no 18S rDNA sequence
• Phryganella acropodia 3 no 18S rDNA sequence

(Mitchell et al. 2000)
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Phylogeny of Euglyphid Testate Amoebae
Order EUGLYPHIDA
MONADOFILOSA
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DNA samples and PCR with EUK-primers
300-303 (triplicates) A 312-315
(triplicates) B intermediate 324-327
(triplicates) C tendency ombrotrophic 336-337
(triplicates) D intact
300 312 324 336 N 300 312 324 336
1 µl DNA (110)
2 µl DNA (110)
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Design of Euglyphida-specific PCR primers for 18S
rRNA gene sequences
Filosea100f Filosea1170r Filosea1630r Filosea170
0r DNA 336 (D) 120, 1µl, 35 cycles
Filosea 100f-1170r Filosea 100f-1630r Filosea
100f-1700r
51 54.2 56.2 57.3
51 54.2 56.2 57.3
51 54.2 56.2 57.3
300 312 324 336 N 300 312 324 336
1 µl DNA
2 µl DNA
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PCR on DNA from different sites 100f-1630r
Samples 312 (B intermediate) 1µl from each
triplicate pooled gt 120 gt 1ml for PCR
(30c) Samples 336 (D intact) 1µl from each
triplicate pooled gt 120 gt 1ml for PCR (30c)
300 312 324 336 N 300 312 324 336
49.9 51 52.4 54.2 56.2 57.9 59.2 61
49.9 51 52.4 54.2 56.2 57.9 59.2 61
312 B
336 D
1 µl DNA
2 µl DNA
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PCR on DNA from different sites 100f-1700r
Samples 336 (D intact) 1µl from each triplicate
pooled gt 120 gt 1ml for PCR (35c)
49.1 49.9 51 52.4 54.2 56.2 57.9
59.2 61
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Cloning and sequence analysis

• Samples 336, 337, 338 (D intact)
• 1µl from each triplicate pooled gt 120 gt 1ml for
  PCR (30c)
• Each pooled DNA amplified (4x) with both primer
  sets
• Separate clone libraries
• First Results
• Primer sets seem to be specific
• Sequence identities of clone sequences (500bp)
• Trinema enchelys 95-98
• Euglypha rotunda 93-98
• Assulina muscorum 94
• Euglypha tuberculata 94-95

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Next steps

• Cloning DNA from all sampling points A-D in
  Chaux-dAbel
• Use sequence data to
• Perform phylogenetic analysis of the
  Euglyphida-communities
• Design probes for Fluorescence in situ
  Hybridization
• Compare sequence data with sequences from
  isolates (?)
• Compare molecular and direct observation approach
• Use sequence data from non-Euglyphida isolates
  for new primers (?)
• Develop a fingerprint screening method