This is the first report on identification and characterization of an isoamylase gene from the rye genome. Hexaploid spring wheat (Triticum aestivum L.) cv. Chinese Spring and diploid spring rye (Secale cereale L.) cv. Rogo

were grown under controlled environmental conditions (24 °C day, 20 °C night with a 16 h photoperiod of 240 μmol m− 2 s− 1) in the same growth cabinet. Various plant materials (stem, leaf, root, seed) were sampled, flash frozen in liquid nitrogen, and stored at − 80 °C until used. Genomic DNA was extracted from young leaf tissue at Zadoks growth Stage 22 [20] using a DNeasy Plant Mini Kit (Cat. No. 69104, Qiagen Inc., click here Mississauga, ON, Canada). Total RNA was isolated from immature seeds (12 days post anthesis, DPA) according to a phenol/SDS protocol [21]. RNA was further purified using the RNeasy Plant Min Kit (Cat. No. 74904, Qiagen Inc., Mississauga, ON, Canada). Primers for cloning the rye isoamylase gene were designed according to the conserved regions of Aegilops tauschii isoamylase gene sequence (GenBank accession no. AF548379) [22], wheat iso1 mRNA sequence (GenBank accession no. AJ301647) [23] and barley isoamylase mRNA sequence (GenBank accession no. AF490375) [14]. Ten pairs of primers were designed to amplify the overlapping genomic DNA sequences that correspond I-BET-762 in vivo to the rye isoamylase gene. Furthermore, three pairs of primers

were developed to amplify the overlapping cDNA sequences. Typically, 25 μL of PCR mixture contained 20 pmol primers, 30 ng of genomic DNA or 5 μg of cDNA, 1 × buffer, 1 × Q-solution and 1.25 U of Qiagen HotStar HiFidelity Polymerase (Cat. No. 202605, Qiagen Inc., Mississauga, ON, Canada). Reverse transcription (RT)-PCR was performed using total RNA as the template with Superscript III Reverse Transcriptase (Cat. No. 18080-093, Invitrogen, Burlington, ON, Canada). Primer sequences and PCR conditions are listed in Table 1. Amplified isoamylase DNA fragments were cloned into the PCR4-TOPO vector (Cat. No. K4575-02, Invitrogen, Burlington, ON, Canada) and at least three independent clones for

each fragment were sequenced in both directions by the DNA Sequencing Service Centre, University of Calgary (Calgary, Glutathione peroxidase Canada). Rye isoamylase sequences and the corresponding protein were blasted with the NCBI BLASTN tool (http://blast.ncbi.nlm.nih.gov) and aligned with previously reported isoamylase sequences using DNAMAN software v5.0 (Lynnon Biosoft, U.S.A.). The putative encoding regions of transit peptides and mature proteins of isoamylase genes from different plant genomes were predicted using the ChloroP 1.1 server (http://www.cbs.dtu.dk/services/ChloroP/). Total RNAs were isolated from rye leaves, stems, roots and rye seeds at different developmental stages (9, 15, 24 and 33 DPA) with an RNA Extraction Kit (Cat No. 74904, Qiagen Inc., Mississauga, ON, Canada).