Similar protocols

Protocol publication

[…] sed to amplify CENH3 and its domains, NTT and HFD, from rye cDNA are presented in Supplementary Table . For amplification of HFD CENH3 from Triticum and Aegilops species, we used a set of degenerated primers designed for monocotyledons., RT-PCR products were purified using a Qiagen Purification Kit (Qiagen) and cloned using an InsTAclone PCR Cloning Kit (Thermo Fisher Scientific). Both strands of 12–20 clones of each accession were sequenced using an ABI 3130 × 1 Genetic Analyzer (Applied Biosystems Inc., CA) and an ABI BigDye Kit according to a standard protocol. Similarity searches between the obtained rye CENH3 sequences and their orthologous from other species were carried out using the TBLASTN software in the NCBI database (http://blast.ncbi.nlm.nih.gov/Database/). Multiple alignments of amino acids and coding sequences were performed online using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo). Alignments were further refined manually and used for downstream analysis with the aid of statistical, phylogenetic programs and for visualization (http://www.jalview.org). The deduced protein sequences were examined for potential posttranslational modifications using NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos)., Phylogenetic trees were drawn using MEGA6. Mean pairwise amino acid and nucleotide distances were also calculated using MEGA6 according to the Poisson and T92 + G models. Bootstrap values were calculated from at least 1,000 replications., Sequences were analyzed for deviations from neutrality with the McDonald–Kreitman test using DnaSP. Analysis of the ratios of nonsynonymous (Ka) to synonymous (Ks) substitutions (ω) was performed using DnaSP. The statistical significance of positive selection was calculated by Fisher’s exact test as im […]

Pipeline specifications

Software tools TBLASTN, Clustal Omega, Jalview