Computational protocol: Identity of Two Sympatric Species of Orius (Hemiptera: Heteroptera: Anthocoridae)

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Protocol publication

[…] The 18S ribosomal gene internal transcribed spacer 1 (ITS-1) has been used previously to assess the phylogenetic relationship among various Onus species including O. insidiosus and Orius tristicolor (White) from North America (). Genomic DNA sequence data for ITS-1 were derived from specimens of O. insidiosus and O. pumilio collected from our laboratory cultures and from O. tristicolor collected from butterfly bush, Buddleja sp., growing in Wapato, Washington for comparison with previously published sequences from anthocorid species. Genomic DNAs of 50 pooled adult O. insidiosus, O. pumilio, or O. tristicolor were isolated using the Wizard Genomic DNA Isolation System (Promega, www.promega.com) according to the manufacturer's protocol for animal tissues. Direct PCR for the ITS-1 and flanking regions were amplified from the genomic DNAs as template using Taq PCR kit (New England Biolabs, www.neb.com) with the forward primer, 5′?-ACCGCCCGCGCTACTACCGAT -3′?, and reverse primer, 5′?-TGTTCATGTGTCCTGCAGTTCACA-3′? (Integrated DNA Technologies, www.idtdna.com), as identified by Muraji et al. (). The amplification program was 94°° C for 3 min with 40 cycles of 92°° C for 40 s, 58°° C for 40 s, and 72°° C for 40 followed by 72°° C for 4 min. The PCR products were cloned into pGEM-T Easy (Promega) and sequenced on contract using ABI 3130 DNA sequencing at Interdisciplinary Center for Biotechnology Research (University of Florida). A total of six sequences were examined for each species to establish nucleotide identities.The nucleotide sequences for ITS-1 reported from anthocorid species were aligned using ClustalW as a component of Mac Vector 7.2.3 software (MacVector, Inc., www.macvector.com). The phylogenetic and molecular evolutionary analyses were conducted with PAUP** v 4.0b10 () and Mega version 4 (). Alignments were subjected to analysis with PAUP** Maximum Parsimony and trees were established as rooted using Cimex lectularius Linnaeus as the outgroup with a 50% majority rule consensus and a bootstrap of 1000 replicates. The same sequence alignment was subjected to analysis with Mega4 Neighbor-Joining, Minimum Evolution and Maximum Parsimony-Close-Neighbor Interchange, and all phylograms were established as rooted using C. lectularius as the outgroup with a 50% majority rule consensus and a bootstrap of 1000 replicates. […]

Pipeline specifications

Software tools Clustal W, MacVector, MEGA
Application Phylogenetics