Materials
The genetic material included 36 of the 65 barley varieties released in Australia since the 1950s and 5 advanced breeding lines (Additional file 1). All the materials are two-rowed spring type. A sub-set of varieties—Hindmarsh, Gairdner, Skiff, Onslow, Clipper, Franklin, Moondyne and Chevalier—were selected to investigate the sequence variation of Lox1 gene based on their LOX activity (see Lipoxygenase Assay section below).
Field trials
The field plot was planted in a randomised complete block design in plots 1 × 3 m2. Plants were grown at three sites in Western Australia with two replicates at each site. Harvested grains from each site were evaluated for their physical quality, and grains from the best site were used for micro-malting analysis.
Micro-malting analysis
Barley samples were cleaned and sieved over a 2.2 mm screen prior to micro-malting in a Joe White Systems micro-malting unit without additives. A standard malting schedule was used by steeping at 19°C for 7 h wet, 8 h air rest, 3 h wet, 4 h air rest, 1 h wet. Germination took 96 h (48 h at 18°C followed by 48 h at 16°C), then moisture adjusted to 46% for 24 h. Kilning was 2 h at 45°C, 3 h at 50°C, 4 h at 55°C, 3 h at 60°C, 3 h at 65°C, 3 h at 70°C, 2 h at 75°C, and 4 h at 80°C. Malt rootlets were removed using a custom-made rootlet-removing machine (Fraser Fabrications P/L, Malaga, Western Australia).
Lipoxygenase assay
We used the Joe White Malting revised version of the Malt Lipoxygenase (LOX) (original assay from Baxter [6]). All processes were completed on ice unless otherwise indicated.
Preparation of substrate solution (2.5 linoleic acid)
Five milliliters of 0.05 M borate buffer (pH 9.0) was added to a volumetric flask (10 mL) followed by 0.25 mL Tween20, 0.25 mL linoleic acid and 0.65 mL 1 M NaOH. The contents were shaken gently in an ultrasonic bath with ice water until the solution became clear, then distilled water was added to 10 mL.
Enzyme extraction from finished malt
Finished malts were milled in a Retsch ZM200 centrifugal mill (Retsch GmBH, Germany) with a 1.0 mm screen; 5 g milled malt was transferred to 100 mL flask. 50 mL of acetate buffer (pH 5.0) containing 0.1 M NaCl was added and kept in ice water bath for 15 min with occasional shaking. The resulting solution was transferred to a 1.5 mL eppendorf tube and centrifuged for 5 min at 10 000 rpm. The supernatant was subsequently transferred to a new eppendorf tube and stored on ice.
Enzyme assay
The temperature of the cell holder and phosphate buffer (0.1 M, pH 6.8) was equilibrated to 25°C by water circulation. 100 μL enzyme extract and 2850 μL phosphate buffer (0.1 M, pH 6.8) was added to 50 μL substrate solution, mixed, returned to the cell holder and absorbance recorded at 1 min and 4 min at 234 nm. Blank absorbance was measured using 50 μL substrate solution and 2950 μL phosphate buffer at 1 min.
Calculation and expression of results
One unit of LOX activity represents an increase in absorbance at 234 nm per minute, per gram malt on dry basis, as calculated using the following formula:
where Abs (4 min) is absorbance at 234 nm at 4 min of reaction, Abs (1 min) is absorbance at 234 nm at 1 min reaction, M is moisture content of malt sample, and B is volume (μL) of enzyme extract of malt used.
DNA extraction
Genomic DNA was extracted from the leaves of two-week-old seedlings for each variety following the phenol chloroform standard protocol. DNA samples were quantified using the Nanodrop and adjusted to a final concentration of about 25 ng/μL for PCR analysis.
Primer design and PCR amplification for isolation of Lox1 promoter and gene
The full-length Lox1 promoter sequence has been isolated from Himalaya [15] (NCBI database, U83904). Full-length cDNAs of the Lox1 gene have been isolated from barley varieties Triumph [3] (NCBI database, L35931) and Haruna Nijo (NCBI database, AK252639). Based on the sequences from the NCBI database (http://www.ncbi.nlm.nih.gov), PCR primers were designed for sequencing with 100–200 bp overlaps between adjacent fragments (Additional file 2).
PCR reactions were carried out in a final volume of 50 μL containing about 100 ng of genomic DNA, 10× PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 400 nmol of each primer, and one unit of Taq DNA polymerase. The reaction was initially denatured at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, annealing for 45 s and 72°C for 1 min. The PCR was terminated at 72°C for 7 min.
Transformation, cloning and sequencing
The PCR-amplified DNA was excised separately from agarose gels and extracted using QIAEX II Gel Extraction (QIAGEN). The DNA sequence was ligated into pGEM®-T Easy Vectors (Promega) according to manufacturer recommendations. Competent E. coli cells, strain JM109, were transformed and the clones screened by blue/white colony selection. Plasmid DNA from the colony was isolated by the alkali lysis method [16]. Recombinant DNA was screened for appropriate insert size by digestion with EcoRI restriction enzyme. The pGEM-T Easy cloned products were sequenced with primers M13F and M13R. Sequencing reactions were performed on PCR products according to manufacturer recommendations, using Big-Dye labeling sequencing reaction mixture. Sequences were read on an Applied Biosystems 3730 DNA sequencer (SABC, Murdoch University, Western Australia).
Data analysis
Sequences were edited to remove the vector sequence and extra restriction sites. The promoter region and intron–exon structure of the barley LOX-1 coding region were deduced by comparing the nucleotide sequence of the barley published Lox1 cDNA sequence (NCBI database L35931 and AK252639). The amino acid sequence of Lox1 was deduced using Translate Tools in the ExPaSy web server (http://au.expasy.org/tools/dna.html). Multiple alignments of sequences were used with Clustal X (v 1.82) and performed using GeneDoc (v2.5). Phylogenetic reconstructions were done using the MEGA4.0 package. Distance matrices were constructed using the Kimura two-parameter model and trees constructed using the neighbour-joining algorithm. The dataset was re-sampled 1000 times using the bootstrap method. Phylogenetic analyses are presented for the Lox1 DNA sequences.