Fig 1.
Phylogenetic position and morphology of the freshwater Hydra polyps.
(A) Schematic view of the phylogeny of the four groups of Hydra species as reported in references 13–16: H. vulgaris with the sub-groups H. vulgaris-Pallas and H. vulgaris-NA (blue background), H. oligactis, H. braueri, and H. viridissima. (B) Images of polyps illustrating the similar overall appearance between strains and species despite differences in developmental and cellular responses: H. vulgaris-Pallas (Basel-2 strain), H. vulgaris-NA (AEP-2 strain), H. oligactis (Cold-resistant strain, Ho_CR) and H. viridissima (Nicolet-Geneva strain) groups. Note the stalk peduncle (arrow) typical of the H. oligactis species. Scale bars: 2 mm.
Table 1.
Sequences of the primers used in this study.
Fig 2.
Direct genomic DNA amplification from single Hydra polyp.
(A) PCR amplification of β-actin genomic DNA from three 4–6 mm long, non-budding AEP animals dissociated and resuspended each in 50 μl water. Various amounts (from 0 to 15μL) of the resulting macerates were used as PCR template for β-actin amplification to estimate PCR efficiency. (B) Graphic representation of DNA concentration and DNA purity as deduced from OD measurements at 260, 230 and 280 nm wave lengths. Each dot represents either the OD260 value or the 260/280 or 260/230 OD value ratios obtained from a single polyp. For each DNA, the efficiency of PCR amplification is indicated with a color code. Note the lower DNA content in most reg-16 polyps that were fixed in PFA and stored in methanol for months prior to rehydration, maceration and DNA amplification.
Fig 3.
Phylogenetic relationships within the Hydra genus based on the analysis of the Cytochrome Oxydase I (COI) DNA sequences.
The maximum likelihood tree of the COI sequences was built by adding to the dataset of 85 COI sequences available on Genbank [15] the 10 sequences obtained in the present study (written red, indicated with red arrows, see S2 Table for accession numbers). Black dots indicate the robustness of the nodes as deduced from the bootstrap support (at least 750 over 1’000 bootstraps). This tree confirms that the sequences obtained in this study distribute into the expected Hydra groups, Nicolet strain in H. viridissima, Ho_CR and Ho_CS in H. oligactis, Hm-105, sf-1, reg-16, Basel-1 and Basel-2 in H. vulgaris-Pallas, AEP1, AEP2 in the H.vulgaris-NA sub-group.
Fig 4.
Analysis of the polymorphism of the TG-rich microsatellite c25145 sequence (ms-c25145).
(A) Amplification of the ms-c25145 genomic sequences from 7/9 tested strains that represent H. vulgaris-Pallas (Hm-105, Basel1, Basel2 strains), H. vulgaris-NA (AEP1, AEP2 strains) and H. oligactis (Ho_CR, Ho_CS strains). Yellow arrows point to a smear detected in both AEP1 and AEP2, orange arrows point to a faint second band detected in both Ho_CS and Ho_CR. (B) Sequence alignment of the ms-c25145 region. The salmon-pink color box indicates the central TG-rich microsatellite region embedded within highly conserved regions (grey boxes). Primer sequences used for amplification are indicated with black arrows. Numbers in brackets after the strain name indicate the number of independent positive sequencings, numbers at the 3’ end indicate the size of the PCR product and the number of TG-repeats (bold). Red writings indicate transcriptomic (t) or genomic (g) sequences available on the HydrATLAS (HA) server [32, 40, 41], the NHGRI Hydra web portal for the Hydra 2.0 genome (g2.0) [34] and Juliano transcriptomes (Jul) [38] or the Compagen (Co) server [37, 42] (see S2 Table). (C) Graphical representation of the different ms-c25145 amplicons as deduced from sequencing data. Each dot corresponds to a distinct amplicon confirmed by one or several sequencings as indicated by the number of sequenced colonies (see S2 Table). Green, red and yellow color dots correspond to expected sizes, lighter color dots refer to sequences with errors (PCR or sequencing), the grey dot indicates missing data.
Fig 5.
Analysis of the polymorphism of the AT-rich microsatellite region of the Aryl-Hydrocarbon Receptor-Interacting Protein gene (ms-AIP).
(A) Amplification of the ms-AIP genomic sequences in six out of nine tested strains, which represent two distinct H. vulgaris sub-groups, H. vulgaris-Pallas (Basel1, Basel2, Hm-105, reg-16) and H. vulgaris-NA (AEP1, AEP2). White arrows point to a faint band observed only in Hm-105 polyps, yellow arrows indicate a size difference between Basel1 and Basel2, and the orange arrows show a second band detected in AEP2 but not in AEP1. (B) Alignment of the ms-AIP sequences. The color boxes indicate the AT-rich central region (salmon-pink) and an A-rich motif (green) embedded within highly conserved regions (grey). Primer sequences used for amplification are indicated with black arrows. Numbers at the 3’ end indicate the PCR product size and the number of AT-repeats (bold). Red writings indicate transcriptomic (t) or genomic (g) sequences available on HydrATLAS (HA) [32, 40, 41], NHGRI web portal for the Hydra 2.0 genome (g2.0) [34] and Juliano transcriptomes (Jul) [38], or Compagen (Co) server [37, 42] (see S2 Table). (C) Graphical representation of the ms-AIP amplicons as deduced from sequencing data. Dot legend as in Fig 4. (D) Amplification of ms-AIP in five transgenic lines ecto-GFP and endo-GFP produced in uncharacterized AEP [42], AEP1_Wnt3 [49], AEP2_203 and AEP2_293 (QS, unpublished).
Fig 6.
Analysis of the polymorphism of the AT-rich microsatellite detected in the Cyclin-D-Binding Myb-Like Transcription Factor 1 gene (ms-DMTF1).
(A, B) Amplification of the ms-DMTF1 genomic sequence is restricted to the AEP strains, either unmodified (AEP1, AEP2) or transgenic (Q82-293, ecto-GFP, Wnt3::GFP, endo-GFP) lines. (C) Alignment of the ms-DMTF1 sequences. The color boxes indicate the AT-rich central region (salmon-pink) embedded within highly conserved regions (grey). Primer sequences used for amplification are indicated with black arrows. Numbers in brackets after the strain name indicate the number of independent positive sequencings, numbers at the 3’ end indicate the size of the PCR product and the number of AT-repeats (bold). Red writings indicate transcriptomic (t) or genomic (g) sequences available on HydrATLAS (HA) server [32, 40, 41], NHGRI web portal for the Hydra 2.0 genome (g2.0) [34] and Juliano transcriptomes (Jul) [38], or Compagen (Co) server [37, 42] (see S2 Table). (D) Graphical representation of the size of the ms-DMTF1 amplicons as deduced from sequencing data. Red color dots correspond to expected sizes, the grey dot indicates missing data.
Fig 7.
Summary scheme showing the value of each microsatellite for efficient discrimination between Hydra species and Hydra strains.