Computational protocol: The first report of a Pelecaniformes defensin cluster: Characterization of β-defensin genes in the crested ibis based on BAC libraries

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Protocol publication

[…] According to the order and orientation as well as the clustering results of phylogenetic analysis described below, the crested ibis defensin genes were named following the nomenclature proposed previously. The mRNA was extracted from the liver and spleen, and the full-length sequences of β-defensin cDNA were obtained by GeneRacer™ Kit (Invitrogen) using the primers listed in . All the cDNA sequences were used for gene annotation and deposited in GenBank (accession number: KM272304–KM272316). The defensin genes undetected in the liver and spleen tissues were analyzed with BLAST based on homologies to known chicken genes and the coding regions were predicted by GeneWise.The chicken (chromosome 3: 107,022,000–107,104,000 bp) and zebra finch (chromosome 3: 110,733,400–110,860,340 bp) defensin cluster sequences were both obtained from Ensembl (, while the duck defensin sequences were obtained from NCBI (NW_004678274.1, NW_004683518.1, KB742693.1, and NW_004716371.1). The several gaps in and between these four duck defensin scaffolds were filled by applying long-range PCR (), cloning, and sequencing. We predicted tRNA genes in defensin clusters for four birds by tRNAscan-SE, analyzed GC content with 200 bp windows using Isochore (, and further conducted Pearson's chi-squared test in SPSS 20.0 (SPSS Inc., Chicago, IL, US) to identify any significant differences between each duplicated region and the non-duplicated region. Finally, we drew dot plots using PipMaker to perform syntenic analysis between the crested ibis and other bird defensin clusters. [...] Amino acid sequences were aligned by MUSCLE in MEGA5.0. According to the amino acid sequence alignment, nucleotide sequences were aligned and subsequently used for phylogenetic analysis with chicken, zebra finch, and duck β-defensin genes. Nucleotide sequences encoding the signal and mature peptides were used for phylogenetic reconstruction in MrBayes 3.2.2 with the green lizard AcBD15 (GenBank accession number: FR850158) as the outgroup. Two independent runs of 600,0000 generations with a sample frequency of 100 were performed, and the first 25% of the samples were discarded as “burn-in.” An ML tree was also constructed using the PhyML web service with default settings. The rates of synonymous and non-synonymous substitutions (dS and dN, respectively) were calculated in MEGA5.0. Since the saturated non-synonymous sites in paralogous genes could bias the comparisons, the dN/dS ratios (ω) were calculated pairwise for orthologous genes among the four birds (AvBD14 genes were only compared among the chicken, duck, and crested ibis). The overall dN/dS ratios were calculated separately for signal peptide and mature peptide coding regions. […]

Pipeline specifications

Software tools GeneWise, tRNAscan-SE, PipMaker, MEGA, MrBayes, PhyML,
Databases ACBD
Applications Genome annotation, Phylogenetics, Genome data visualization
Organisms Nipponia nippon