Phylogentic utility of secondary structure of ribosomal ITS2 and Cytochrome oxidase subunit-I (COI) in sarcoptes isolates from different hosts
A Valappil, S Angadi
cytochrome, isolates, oxidase subunit i coi, ribosomal internal transcribed spacer 2 its2, sarcoptes, secondary structure
A Valappil, S Angadi. Phylogentic utility of secondary structure of ribosomal ITS2 and Cytochrome oxidase subunit-I (COI) in sarcoptes isolates from different hosts. The Internet Journal of Genomics and Proteomics. 2008 Volume 4 Number 1.
A comparative study of common core secondary structure in the ribosomal internal transcriber 2 (ITS2) and cyotchrome oxidase subunit I (COI) of 11 and 5 isolates respectively were selected from different hosts was carried out. Sarcoptes cause scabies of different nature in different hosts. Multiple sequence alignment and secondary structural analysis of ITS2 and COI was performed to elucidate the phylogenyetic relationships. This study reveals a phylogenetic relationship among the different isolates of
Internal transcribed spacer 2 (ITS2) refers to a piece of non-functional RNA situated between structural ribosomal RNA (rRNA) on a common precursor transcripts. DNA sequence of the two internal transcribed species (ITS1 and ITS2) of the rRNA transcription unit have proven useful in resolving phylogenetic relationships for closely related taxa due to their relatively rapid evolution rate ( Baldwin, 1992; Schlotterer
The transcripts folding structure of the ITS2 provide some signals that guide the ribosomal coding region when they are processed into small, 5.8S and large ribosomal RNA (Van der Sande et al., 1992; Van Nues
The over all literature cited shows that ITS2 and COI are widely used to resolve the phylogeny of acarina and many other invertebrates. Utility of ITS2 in phylogenetic study is limited with regards to
Materials and method
ITS2 sequence of 11 sarcoptes isolates from different hosts (human, dog, silver fox, red fox, raccoon, dromedary, lynx, goat, swine, wombat and rabbit) and COI sequence of five available isolates (human, wombat, canine (dog), rabbit and goat) that are deposited in GenBank were investigated. The accession numbers of ITS2 and COI isolates are:-
Multiple sequence alignment were performed by using CLC free workbench version 4.5.1 (http://www.clcbio.com) with gap open penalty 15 and gap extension penalty 07.This program align nucleotides using a progress alignment algorithm(Feng and Doolittle, 1987).
Secondary structure prediction
The RNA secondary structure for ITS2 and COI were predicted by using RNADRAW online software (Christoffersen
The phylogenetic service of CLC free workbench was used for phylogenetic tree construction (CLC free workbench 4.5.1 http://www.clcbio.com).Unweighted Pair Group Method using Arithmetic average (UPGMA) (Michener and Sokal, 1957; Sneath and Sokal, 1973). Clustering algorithm was used, for interpreting phylogenies bootstrap values are used.
Sequence analysis of ITS2 and COI regions
The length of ITS2 of 11 selected sacrcoptes isolates ranged in size 296bp and 361bp. Eleven dispersed but unambiguously conserved sequence segments encompassing about a third of the ITS2 length have been identified. They were interspersed with variable regions and gaps where size variations accumulate. The characteristics of sequences for each isolates are shown in the table 1. The length variations were observed with maximum length being 361bp in goat isolate and minimum 276bp in human isolate. The G+C content for the 2 regions of rDNA (5.8S and ITS2) of all the isolates ranged from 32% to 40%. For ITS2 regions the sequence identities ranges, with maximum 99% similarity between human isolate and wombat isolates ; 83 % similarity between rabbit isolate and goat isolate and rabbit isolate and swine isolate; 77% similarity between wombat isolate and silver fox isolates and wombat isolate and dromedary isolate; 49% between silver fox isolates and raccoon isolate and dromedary and raccoon; 35 % between raccoon isolate and red fox isolate; 23 % between red fox isolates and lynx isolate and minimum 9% similarity between dog isolate and lynx isolate. Alignment of ITS2 region is shows simple tandem repeats were present at various locations along the ITS2. The sequence similarity is more towards the 5´ end and with dispersed conserverdness in the middle than towards the 3´ end.
The characteristics of COI sequence of each isolates were shown in the table 2. In COI regions the sequence identities ranges, with maximum 98 % similarity between human and wombat isolate; 94 % similarity between rabbit
and human isolates, and between canine and human isolates; where as minimum being the 34 % between rabbit and canine isolates. Alignment of COI is shown in the figure 2. Simple tandem repeats were present at various locations along the COI sequences. The sequence similarity is more at the 5´ end with dispersed conserverdness in the middle towards the 3´.
Secondary structure in ITS2 and COI regions
Secondary structure of ITS2 region was given in the Table 2. The secondary structures of the isolates from different hosts were classified into 5 groups based on the analysis of conserved stem and loops (Fig 3). Class I includes isolates of dog, red fox and class II includes isolates of dromedary and rabbit, class III includes isolates of wombat and human, class IV includes isolates of raccoon and swine and class V includes isolates of goat. Three common motifs having sequence AAAA and GCUUU respectively were conserved in all classes (Figure 5). Apart from the common conserved motifs shared among the species that are categorized into different classes, variable regions also do exists. The observed similarities at the secondary structural level are further reflected at energy level and of ITS2 and regions of various isolates.
The secondary structure features of COI were is given in the table 2. The secondary structures of the isolates were classified into four groups (Fig 4). Class I includes human isolate, class II includes rabbit and isolates, class III includes goat isolate and class IV includes wombat isolate. The common UUU AND AUAU respectively were conserved in all classes (Figure 6).
Sarcoptes isolates are serious pathogen for scabies of differen
The selected 11 sarcoptes isolates from different hosts occur world wide and they cause scabies of various extensions in their hosts. In the present investigation, the
ITS2 and COI sequences reflected the trend observed in the phylogeny. The isolates from more distantly related hosts
the less was the convergence at the ITS2 and COI level. This study reveals, particularly with COI, although sequence variation was found in ITS2 sequence than in COI sequence, this did not correlate well with morphology and host suggesting that all the sequences studied belong with a single polymorphic species. The study also point out that COI sequence is more reliable, than ITS2, for sequence analysis and secondary structure prediction to resolve the phylogeny of sarcoptes.