Abi bigdye kit

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^{39 (link)} 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^{40 (link)} (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^{41 (link)} (http://www.jalview.org). The deduced protein sequences were examined for potential posttranslational modifications using NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos).

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 (Altschul et al. 1990 (link)) in the NCBI database (http://blast.ncbi.nlm.nih.gov/Database/).