Additional file 1 of Extensive non-redundancy in a recently duplicated developmental gene family
Creators: name: E. A. Baker
name: S. P. R. Gilbert
name: S. M. Shimeld
name: Woollard, A.
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Fig. 1. Multiple sequence alignment of all the Wart domain proteins mined from the predicted proteomes of various nematode species (Species abbreviations: Tnat, Trichinella nativa; Tmur, Trichinella murelli; TspT6, Trichinella sp. T6; TspT8, Trichinella sp. T8; TspT9, Trichinella sp. T9; Tpap, Trichinella papuae; Tpat, Trichinella patagoniensis; Tnel, Trichinella nelsoni; Tpseudo, Trichinella pseudospiralis; Trchrs_su, Trichuris suis; Ts, Trichinella spiralis; Bm, Brugia malayi; As, Ascaris suum; Tc, Toxocara canis; Sr, Strongyloides ratti; Pp, Pristionchus pacificus; Cbre, Caenorhabditis brenneri; Cbri, C. briggsae; Cr, C. remanei; Ce, C. elegans.). Alignment was constructed using SeaView software version 4.6.2 and the CLUSTAL Omega programme (default parameters) was used to locally improve the alignment, which was further refined by eye. Fig. 2. IQ-TREE maximum likelihood molecular phylogenetic analysis of the Wart domain sequences mined from selected nematode genomes (Species abbreviations: Tnat, Trichinella nativa; Tmur, Trichinella murelli; TspT6, Trichinella sp. T6; TspT8, Trichinella sp. T8; TspT9, Trichinella sp. T9; Tpap, Trichinella papuae; Tpat, Trichinella patagoniensis; Tnel, Trichinella nelsoni; Tpseudo, Trichinella pseudospiralis; Trchrs_su, Trichuris suis; Ts, Trichinella spiralis; Bm, Brugia malayi; As, Ascaris suum; Tc, Toxocara canis; Sr, Strongyloides ratti; Pp, Pristionchus pacificus; Cbre, Caenorhabditis brenneri; Cbri, C. briggsae; Cr, C. remanei; Ce, C. elegans.). The node labels are ultrafast bootstrap support values. The tree was generated in FigTree. Table 1. (1.1) Table lists the accession numbers of the additional (degenerate) Wart domain containing sequences mined from the nematode species in this investigation. Where legitimate Wart domain containing genes are included, these have been named by our investigation according to our findings, i.e. the previously unannotated four wrt-4 paralogues in P. pacificus and the wrt-x orthologues and putative Hh homologues in Clade I species. (1.2) Table lists the species used in this investigation and the NCBI BioProject accession number of the edition of their genome used. Fig. 3. Alignment of ‘wrt-4′ paralogues in P. pacificus using Clustal Omega (automatic parameters). Only III_PDM72491.1 (Pp_WRT-4iii, chromosome X) is predicted to encode a Hog/Hint motif, i.e. an intein-mediated protein splicing domain (GO0016539) and a protein autoprocessing domain (GO0016540) as annotated by InterProScan 5 [61]. Table 2. Table details the genes directly adjacent to each Warthog family orthologue in a particular nematode species. Purple font indicates that the adjacent gene is not orthologous to the adjacent gene beside the orthologous Warthog in C. elegans. Green font indicates that the adjacent gene is orthologous to the adjacent gene beside the orthologous Warthog in C. elegans. ‘-’ indicates that there was a ‘gene desert’ adjacent to the Warthog locus or that the contig finished and so syntenic relationships could not be established. ‘Gene deserts’ are arbitrarily defined here as regions on a chromosome that do not feature any open reading frames for over 3.5 kb. All genes are represented with their WormBase/NCBI accession numbers. Fig. 4. (A) wrt-7 RNAi knockdown performed on different genotypes. From the left, empty vector control RNAi performed on wildtype, wrt-4(tm1911),wrt-2(ok2810), wrt-8(ok1585);wrt-2(ok2810), wrt-8(ok1585);wrt-4(tm1911), wrt-8(ok1585);wrt-4(tm1911);wrt-2(ok2810) and rrf-3(pk1426) animals; and wrt-7 RNAi performed on wildtype, wrt-4(tm1911);wrt-2(ok2810), wrt-8(ok1585);wrt-2(ok2810), wrt-8(ok1585);wrt-4(tm1911), wrt-8(ok1585);wrt-4(tm1911);wrt-2(ok2810) and rrf-3(pk1426) animals. (B) Wrt-2/4/8 knockdowns on WT and wrt-7(ok3271) animals. From the left, empty vector control RNAi performed on wildtype and wrt-7(ok3271) mutants; wrt-2 RNAi performed on wildtype and wrt-7(ok3271) mutants; wrt-4 RNAi performed on wildtype and wrt-7(ok3271) mutants; and wrt-8 RNAi performed on wildtype and wrt-7(ok3271) mutants. Black bars show mean + SEM. All comparisons are not significant. Table 3. Table details the naturally occurring variations found in wild isolates of C. elegans in the open reading frame of wrt-7 as retrieved from the CeNDR database (Cook et al. 2016). The precise mutational change (and corresponding amino acid change) is listed according to the reference (Bristol N2) and the corresponding change and the effect on the predicted protein is given in the eighth and ninth columns, respectively. Fig. 5. wrt-3(ok2810) animals show severe morphological defects in the middle body despite being outcrossed six times. wrt-3 RNAi animals also display the same middle body defects. The gonad arms appear both abnormally shaped and fragmented, often miniaturised. Large vacuoles also distort the middle body anatomy. The vulvae (arrowheads) also protrude. The severity of the morphological defects meant that we were unable to observe the left–right asymmetry of the middle body in wrt-3(ok2810) mutant animals. Scale bar = 50 μm. Fig. 6. (A) % penetrance of LR defects recorded on specific Wrt family RNAi knockdown on wrt-8(tm1585);wrt-4(tm1911);wrt-2(ok2810) triple mutants (Empty Vector Control, n = 20; wrt-1, n = 23; wrt-5, n = 20; wrt-6, n = 19; wrt-9, n = 25; wrt-10, n = 20). wrt-3 knockdown was not performed due to the severe middle body morphological defects it gives rise to (SD8) (B) % penetrance of Muv defects recorded on specific Wrt family RNAi knockdown on wrt-5(ok670);wrt-3(ok2608) double mutants. (Empty Vector Control, n = 19; wrt-1, n = 19; wrt-2, n = 21; wrt-4, n = 24; wrt-6, n = 22; wrt-7, n = 26; wrt-8, n = 20; wrt-9, n = 29; wrt-10, n = 20) (C) Body size (48 h post L4) of wrt-3(ok2608);wrt-9(ok2732) double mutants upon knockdown of specific Wrt family members. Black bars show mean + SEM. Black asterisks show *P ≤ 0.05, all other comparisons are not statistically significant, i.e. P &gt; 0.05. Fig. 7. (A) Gene tree of Warthog family in selected nematode species as shown in Fig. 6 of the manuscript. (B) wrt-x gene tree (grey). (C) wrt-1 gene tree (bright red). (D) wrt-2 gene tree (bright orange). (E) wrt-3 gene tree (bright green). (F) wrt-4 gene tree (dark orange). (G) wrt-5 gene tree (dark green). (H) wrt-6 gene tree (purple). (I) wrt-7 gene tree (yellow). (J) wrt-8 gene tree (sunset orange). (K) wrt-9 gene tree (dark red). (L) wrt-10 gene tree (blue). Table 4. Table details the strains used in this investigation. All strains were derived from Bristol N2 worms. Warthog mutants were outcrossed with CB4088 a minimum of four times (and in the case of RB2125 and VC2083, six times) prior to use, i.e. phenotypic characterisation and the performance of genetic crosses. All genotypes in cross progeny were followed throughout by PCR except strains crossed into P57657.