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Deliverables D9 Genetic diversity of protistan groups. 1. Develop 18S rRNA gene based detection of selected protistan groups (testate amoebae) 2. Develop and apply 18S rRNA gene based screening tool for testate amoebae. 18S rRNA gene detection. Euglypha rotunda. Euglypha rotunda. - PowerPoint PPT Presentation
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Deliverables D9Genetic diversity of protistan groups
1. Develop 18S rRNA gene based detection ofselected protistan groups (testate amoebae)
2. Develop and apply 18S rRNA gene basedscreening tool for testate amoebae
Phylogeny of Euglyphid Testate Amoebae
0.01
Euglypha rotunda
Euglypha rotunda
Euglypha filifera
Euglypha tuberculata
Euglypha rotunda
Euglypha rotunda
Euglypha acanthophorea
Euglypha filifera
Assulina muscorum
Trachelocorythion pulchellum
Trinema enchelys
Tracheleuglypha dentata
Cyphoderia ampulla
Paulinella chromatophora
Thaumatomonas sp.
Cercomonas caudatus
MO
NA
DO
FILOS
AOrderEUGLYPHIDA
18S rRNA gene detection
5%
Cercomonas longicauda Cyphoderia ampulla
Paulinella chromatophora
>3-H11
>2-G4>4-D3>3-A3
>3-E7>1-E10>1-C9
>2-F2
>4-B11>4-D2>4-E9>4-E3
>2-H1
>4-D7
>2-E2
>3-A8
Assulina muscorum
>4-A2
Tracheleuglypha dentata
>1-D4
>3-B8
>4-F8
>1-B12
>1-A6-OTU1-corr
>4-H8
>1-C7-OTU2-corr
>3-D3
>2-B3
>4-F11
>3-B11
>2-F7
>1-E11
>2-C10
2-A7>2-E1
>1-H8>1-H3
>4-G11>2-E9
>3-A6>2-H10
>1-F4-OTU2-corr>1-B11-OTU2-corr
>4-B8>3-H7
>3-F1>3-C4
>1-C12-OTU2-corr>1-C8-OTU2-corr
Trinema enchelys>3-C10
>1-D10-OTU1-corr>1-E1-OTU1-corr
Trachelocorythion pulchellum >1-F11
>4-G3>4-A1
Euglypha filifera Euglypha acanthophora
>3-D5>1-D8
>4-F10>2-G8Euglypha rotunda 83
Euglypha rotunda 84 >1-D6>1-E7>1-A4>1-E4
Euglypha filifera 86 Euglypha tuberculata
Eyglypha rotunda X92 Euglypha rotunda 82
100
76
89
91
91
96
96
82
97
98
90
89
99
92
99
87
8795
95
100
89
90
99
97
81
Trinema cluster
E.rotunda cluster
Assulina cluster
Trachelocorytion cluster
Chaux-d`AbelABCD
18S rRNA gene detection
5%
Allas JJP-2003
Tracheleuglypha dentata
Contig-1-E1Contig-1-C12
Contig-1-B11
Contig-3-H11Contig-1-A6
Euglypha tuberculata
Contig-2-H1
Adiplophysa
Contig-3-E7
Pspinosa
Contig-3-C4
Thaumatomonas SA
Contig-2-B2
Euglypha filifera
Euglypha ciliata Rygmossen
Contig-1-D6
Contig-4-A1
Trinema lineare
Contig-2-F7Contig-2-E2
Contig-1-B7Contig-1-C9
Trinema enchelys Contig-1-D4
Assulina muscorum Assulina eminulum Holmegaard Mose
Trachelocorythion pulchellum Contig-2-B3
Euglypha strigosa Chaux d'AbelContig-1-B12
Euglypha rotundaGomeraContig-3-A2
Euglypha rotundaTenerifeContig-2-G8
Euglypha rotundaCREuglypha rotundaCCAPEuglypha filiferaCR
Euglypha acanthophora Millstream
Paulinella chromatophora Cyphoderia ampulla
Allas Thaumatomonas SCCAP
Allas SA Thaumatomonas seravini
100
100
97
82
100
100
7884
97
100
96
70
99
100
72
72
80
68
97
93
92
68
89
99
90
99
18S rRNA gene detection
PCR with fluorescentlylabeled primers
Enzymatic digestion
Fragment separation onsequencing gel
DNA extraction
Terminal Restriction Fragment Length Polymorphism(T-RFLP)
Bray-Curtis similarity of T-RFLP profiles from Chaux-d`Abel
12
34
56
78
9
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9 1
336
337
338
303
302
314
326
325
A
B
C
C
D
• Correlation needed with plant cover, restoration stage and direct observation• Profiles available for Schottland (A, C), France Russey (C, D)
18S rRNA gene diversity screening
PROBLEMS• Only few DNA samples amplifiable• No PCR-products at all with WP2 DNA samples
> consequences for T-RFLP screening of most RECIPE sites> how to continue?
• Other important (more important) groups missing!
Further deliverables decided during the RECIPE meetings
• Clone libraries using primers targeting all eukaryotes> not very useful; mostly „non-target“ (non-protistan) sequences
• Clone libraries using primers targeting all ciliates> done for Chaux-d`Abel> is information/additional screening on ciliate community structure usefull for functional microbial diversity analysis/which manuscripts could profit?
Deliverables D9Genetic diversity of protistan groups
3. Develop FISH probes for the visualization,detection and enumeration of selected protistangroups (testate amoebae)
unstained cells stained target cells
-ACTGGCGTAAGTCAG-
-ACTGGCGTAAGTCAG-
TGACCGCATTCAGTC
Hybridization
Fluorescence in situ Hybridization (FISH)
Accessible sites for FISH oligonucleotide probes
Amplified regionapprox. 500 bp
Detection by FISH
...Make it accessible for FISH oligonucleotide probes!!
> Use of unlabelled oligonucleotide “helper” probes
(Fuchs et al., 2000)
Detection by FISH
5%
Cercomonas longicauda Cyphoderia ampulla
Paulinella chromatophora
>3-H11
>2-G4>4-D3>3-A3
>3-E7>1-E10>1-C9
>2-F2
>4-B11>4-D2>4-E9>4-E3
>2-H1
>4-D7
>2-E2
>3-A8
Assulina muscorum
>4-A2
Tracheleuglypha dentata
>1-D4
>3-B8
>4-F8
>1-B12
>1-A6-OTU1-corr
>4-H8
>1-C7-OTU2-corr
>3-D3
>2-B3
>4-F11
>3-B11
>2-F7
>1-E11
>2-C10
2-A7>2-E1
>1-H8>1-H3
>4-G11>2-E9
>3-A6>2-H10
>1-F4-OTU2-corr>1-B11-OTU2-corr
>4-B8>3-H7
>3-F1>3-C4
>1-C12-OTU2-corr>1-C8-OTU2-corr
Trinema enchelys>3-C10
>1-D10-OTU1-corr>1-E1-OTU1-corr
Trachelocorythion pulchellum >1-F11
>4-G3>4-A1
Euglypha filifera Euglypha acanthophora
>3-D5>1-D8
>4-F10>2-G8Euglypha rotunda 83
Euglypha rotunda 84 >1-D6>1-E7>1-A4>1-E4
Euglypha filifera 86 Euglypha tuberculata
Eyglypha rotunda X92 Euglypha rotunda 82
100
76
89
91
91
96
96
82
97
98
90
89
99
92
99
87
8795
95
100
89
90
99
97
81
Trinema cluster
E.rotunda cluster
Assulina cluster
Trachelocorytion cluster
Chaux-d`AbelABCD
(E.strigosa/ciliata)
?
Detection by FISH
Not yet delivered
• Screening of bulk samples• Using picked organisms to test specificity• Find organism behind abundant 18S rRNA gene sequences not
available as cultures• Top to bottom approach for the first screening > if necessary
design of more specific probes with target sites on the complete 18S rRNA gene sequence
• FISH probed for selected sequenced testate amoebae...
• No probes yet for Assulina; potential sites for Tracheleuglypha dentata and Thaumatomonas clade
• Alternative to T-RFLP screening of RECIPE sites?
