Computational protocol: Chromosomal assignment of centromere specific histone CENH3 genes in rye (Secale cereale L.) and their phylogeny

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Protocol publication

[…] RTPCR products were purified using a Qiagen Purification Kit (Qiagen) and cloned using an InsTAclone PCR Cloning Kit (Thermo Fisher Scientific). Both strands of each of 15–20 clones of each parental variety and addition line 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 CENH3 sequences and their orthologs in other species were carried out using the TBLASTN software () in the NCBI database ( [...] Amino acids alignments were performed online using Clustal Omega () ( and used for downstream analysis and visualization () ( The phylogenetic tree based on amino acid sequences was constructed using the Neighbor Joining algorithm in MEGA6 (). Bootstrap values were calculated from 1000 replicates. […]

Pipeline specifications

Software tools TBLASTN, Clustal Omega, Jalview, MEGA
Applications Phylogenetics, Amino acid sequence alignment
Organisms Secale cereale, Triticum aestivum, Hordeum vulgare