Computational protocol: Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

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[…] Sequences for all PCR and sequencing primers are provided as supporting information (). All of these primers were designed to be microsporidian-specific in order to avoid amplification of host DNA. A fragment of rDNA was amplified from each N. bombycis, N. granulosis and V. cheracis isolate using the primers HG4F and 5SR. The rDNA fragment was approximately 550 bp in length and contained the intergenic spacer (IGS) separating the 16S and 5S ribosomal RNA subunits. A second rDNA fragment, approximately 830 bp in length and containing the internal transcribed spacer (ITS) separating the 16S and 18S subunits was amplified from the single V. cheracis isolate, the Scottish N. granulosis isolate and the N. bombycis isolates from China and England using the primers ILSUF and 530R. Fragments of rDNA were also amplified from N. apis and V. necatrix isolates using the primers HG4F and HG4R. The resulting rDNA fragments were approximately 800 bp in length and contained the internal transcribed spacer (ITS) separating the 16S and 18S ribosomal RNA genes.A fragment of the largest subunit of the housekeeping gene RNA polymerase II (RPB1) was amplified from each N. granulosis and N. bombycis isolate and from the isolates of N. apis, V. cheracis, V. necatrix, V. disparis and N. lymantriae. In each case, the general microsporidian primers RPB1F, RPB1R, AF1, AF3 and GR1 were used to obtain preliminary sequence data. This was then used as a template to design species-specific primers. A fragment of the housekeeping gene elongation factor-1 alpha (EF-1α) was also amplified from each N. bombycis isolate with the primers EF1αF and EF1αR while the gene for spore surface antigen protein 30.4 (Sap 30.4) was amplified from the three N. bombycis isolates using the primers SAPF and SAPR. Southern blot analysis indicates that RPB1 occurs as a single copy in Vairimorpha necatrix . Sequence similarity searches of the genomes of N. ceranae (the only Nosema genome currently available) and E. cuniculi (the only fully assembled microsporidian genome) were performed for RPB1, EF1-α and Sap 30.4 using BLASTn and tBLASTx, implemented on the NCBI website with an alignment score cut-off of 200. These detected a single copy of RPB1 and EF1-α in each genome. No sequences similar to Sap 30.4 were detected in either genome, suggesting that this gene has evolved recently in the lineage containing N. bombycis.All PCR products were sequenced directly in both directions using BigDye® terminators on an ABI 3100 high throughput sequencer. Chromatograms of RPB1, EF-1α and Sap 30.4 sequences were studied carefully by eye and double-peaks indicating single nucleotide polymorphisms within a isolate were noted. This could not be accomplished for rDNA sequences because multiple indels resulted in overlapping sequences. PCR products were cloned using TOPO TA according to the manufacturer’s instructions. Plasmid DNA was purified using Qiagen’s QIAprep® Spin DNA purification kit and sequenced with primers T7 and T3. For products greater than 1 kb in length, internal primers were designed to allow full sequencing. Extensive overlap between fragments amplified by general and species-specific primer pairs ensured that most regions of DNA were amplified and sequenced at least twice, independently. This increased the likelihood of detecting PCR and sequencing artefacts. [...] Sequences were aligned using Clustal W and adjusted manually. Fasta files of sequence alignments are provided as supporting information (). In the cases of the rDNA spacer regions (ITS and IGS), multiple, overlapping indels rendered it impossible to align sequences with confidence in highly variable regions. Such hypervariable regions were therefore excluded from the alignments. In order to avoid cloning artifacts, differences in sequence between cloned DNA fragments were accepted as genuine polymorphisms only if they corresponded to double peaks obtained through direct sequencing. The numbers of cloned rDNA and protein-coding sequences used in the analyses are provided in .Changes in nucleotide diversity along each rDNA sequence alignment were calculated as the average heterozygosity per site (π) and the average number of nucleotide differences per site (θW) within a sliding window of 50 base pairs with an increment of 25 base pairs, implemented in Proseq 3.2 . Haplotype networks were constructed for the cloned rDNA sequences obtained from N. bombycis, N. granulosis and V. cheracis, and for the EF-1α sequences obtained from N. bombycis. Connection distances between haplotypes were calculated using Arlequin and the network was visualised using a force-directed algorithm, implemented in Hapstar .Where several different sequences were cloned from the same gene within the same host species, recombination events were detected using RDP4 . This program allows a sequence alignment to be analysed simultaneously with the RDP , BootScan , GeneConv , MaxChi , Chimaera , SiScan , 3Seq and LARD methods to provide a single multiple comparison (MC) Bonferroni-corrected p-value for each recombination event. […]

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