Navy bean (cv. Seahawk, cv. Norstar, and cv. Vista) and white kidney bean (cv. Beluga, cv. Silver Cloud, and cv. Lassen) cultivars representative of the two market classes were selected for this study to represent the Middle American and Andean COD, respectively. Seeds from each of the market classes used in this study were obtained from field grown plants at the Colorado State University Agricultural Research, Development and Education Center (ARDEC), Fort Collins, CO. The bean seed used for analysis within each omics approach was of the same physiological age but differed across omics platforms. Seeds for transcriptomics analyses were collected from plants at two and three weeks after anthesis (plant flowering), immediately frozen in liquid nitrogen and stored at -80°C. Seeds for proteomics and metabolomics were harvested at full maturity and air dried and stored at room temperature (RT, 22 ± 2°C) until use.
RNA was extracted from navy and white kidney bean seeds from field grown plants in 2007. Seeds were collected in the field and immediately frozen as described above. Two cultivars, Silver Cloud (white kidney) and Norstar (navy) were selected for spotted array gene expression analysis. After storage, frozen seeds were separated from pods and RNA was isolated according to the TRIzol reagent protocol (Life Technologies, Gaithersburg, MD). The RNA sample was purified with the RNeasy Mini Kit (Qiagen, Valencia, CA) according to Affymetrix (Santa Clara, CA) instructions. RNA concentrations and purity were determined using an ND-1000 NanoDrop spectrophotometer (Thermo Scientific NanoDrop, Wilmington, DE). RNA integrity was evaluated by the Experion Bioanalyzer Automated Electrophoresis System (Bio-Rad Laboratories, Hercules, CA).
Since a commercial Phaseolus vulgaris microarray was not available, a soybean (Glycine max L. Merr.) array was selected because it was the closest phylogenetic relative among available arrays . Although Glycine max and Phaseolus vulgaris differ in chromosome number and genome size (the soybean genome is twice as large as common dry bean), linkage mapping of DNA markers found an average conserved block length of 13.9 cM between the two genomes indicating high conversation and preservation [29, 30]. Twenty-five μg of RNA was used to prepare complimentary DNA for spotted 2-color microarray analysis. Complimentary DNA (cDNA) was synthesized using the Superscript III kit (Invitrogen, Carlsbad, CA) and hybridized to a microarray developed for soybean  using the Genisphere Array 50 kit (Genisphere, Hatfield, PA). Briefly, RNA (29.5 μL) was mixed with dye-appropriate RT primer (1.5 μL) and heated to 80°C for 10 min, and transferred to ice for 2 min. Superase-in RNase inhibitor (1 μL) was added before 18 μL of reaction mix composed of 5× Superscript III first strand buffer (10 μL), 10 mM dNTP mix (2 μL), 0.1 M dithiotreitol (4 μL) and Superscript III enzyme (4 μL). After incubation at RT for 5 min, tubes were placed in an iQ iCycler (Bio-Rad) for 2 h at 50°C. The reaction was stopped with 0.5 M NaOH/50 mM EDTA (7 μL) followed by incubation at 65°C for 10 min and neutralized with 1 M Tris-HCl (10 μL). cDNA from the navy and white kidney bean samples were combined and purified using Geneclean Turbo kit (Qbiogene, Carlsbad, CA). The purified cDNA was stored at -20°C until use. Arrays were cross-linked by exposure to 65 mJ of UV irradiation and blocked by incubation in 250 mL prehybridization solution [5× SSC (18% Sodium Chloride, 9% Sodium Citrate) buffer, 0.1% SDS and 0.01% BSA solution] at 42°C for 1-2 h. The arrays were dried by centrifugation at RT for 5 min and placed into hybridization chambers and incubated at 42°C for 15 min. Formamide-based buffer (2×, 50 μL) and LNA dT blocker (2 μL) were added to the cDNA sample and heated for 10 min at 80°C. A cover slip was placed on the array and the cDNA mix was introduced between the cover slip and the array surface. The solution was distributed evenly on the array by capillary action. Water (15 μL) was added to the wells of the array chambers followed by incubation for 16 h at 42°C. The array was vigorously washed 3 times for 2 min, first with 2× SSC and 0.2% SDS (250 mL) followed by 2× SSC (250 mL) and then with 0.2× SSC (250 mL). The arrays were centrifuged for 3 min (1000 × g) to dry and warmed at 42°C for 15 min. A mix of 2× formamide-based buffer (42 μL), water (35 μL) and 3.5 μL each of Cy3 and Cy5 was prepared in the dark and incubated at 80°C for 10 min. The hybridization mix was then injected onto the soybean cDNA microarray. Arrays were incubated at 42°C for 3 h. After the second hybridization step, the arrays were washed and dried as described above and immediately scanned with a Genepix 4000B scanner (Molecular Devices, Sunnyvale, CA).
Scanned images were analyzed using GenePix Pro 6.0 software (Molecular Devices) where spot features were aligned for annotation. Poorly hybridized spots were discarded from analysis using the software parameters and the intensities of each dye were quantified. Fluorescence intensities were normalized and exported for statistical analyses.
Protein isolation and two-dimensional gel electrophoresis (2DGE)
2DGE was performed with bean extracts according to previously published methods . Approximately 15 mg of bean seed samples from each of the six cultivars listed above were suspended in sample lysis buffer, containing 7 M urea, 2 M thiourea, 40 mM DTT, 2% CHAPS, 1% Pharmalyte pH 3-10 (GE Healthcare, Piscataway, NJ), and trace amount of bromophenol blue (BPB). Insoluble debris was removed after centrifugation for 30 min (1000 × g, 15°C). The supernatant was recovered and used for analysis. Protein concentration was quantified using the Bradford assay.
Bean seed protein (200 μg) was applied to reswelled, immobilized pH gradient (IPG) strips (pH 4-7L, 24 cm; GE Healthcare). The isoelectrofocusing conditions were as follows: 10V to 300 V for 3 h and at 5000 V for a total of 95 kVh at 20°C. After isoelectrofocusing, the strips were equilibrated in a buffer containing 30% glycerol, 6 M urea, 2% SDS, 10 mg/mL dithiothreitol (DTT) and 0.05 M Tris-HCl (pH 6.8) for 15 min and then for an additional 15 min in equilibration buffer in which 42.5 mg/mL iodoacetamide replaced the DTT. The strips were positioned at the top of 13-16% gradient polyacrylamide gels with 0.5% agarose containing Laemmli sample buffer . SDS-PAGE was performed in Laemmli electrophoresis buffer at 150 V at RT. Proteins were stained with alkaline ammoniacal silver staining  scanned with a GS-800 Calibrated Densitometer (Bio-Rad) and analyzed using PDQuest v7.1.1 software (Bio-Rad).
Analysis of gels
Samples were analyzed using 2DGE to obtain quantitative protein profiles within the molecular weight range of 25 to 150 kDa. Each bean cultivar was run in duplicate. Protein spots were automatically detected using PDQuest v7.1.1 software. All spots were also manually confirmed. Images of duplicate gels were superimposed and a master gel generated for each of the six cultivars. Proteins were quantified using spot densitometry.
Comparisons of the 2DGE protein patterns generated an inclusion list for only those proteins that differed significantly between the two COD (greater than 2-fold change). Protein patterns were also compared between cultivars within each COD. Although out of the scope of this work, these spots can be used in the future for protein identification using MALDI TOF MS.
Metabolite extraction and analysis
It is imperative that the majority of the proteins within the dry bean samples are removed prior to metabolomic fingerprinting in order to avoid confounded results from possible protein fragments. The dried bean seeds were boiled to denature proteins and freeze dried for storage. The proteins were then precipitated during the metabolite extraction process using cold ethanol. The boiling procedure is as follows: 0.5 kg of bean was soaked in distilled water for 3 h at RT. After 3 h the beans were drained, rinsed thoroughly with deionized water, and blanched for 5 min at 93°C. Beans were boiled for 60 min in a 1.5% KCl solution using a pressure cooker. Finally, beans were freeze dried (Genesis SQ25LL, Virtis Company, Gardiner, NY) to powder form and stored at -80°C until use.
Metabolites were extracted using cold ethanol (65%, -20°C). Approximately 2.5 g of freeze dried bean powder was added to ethanol (65%, 50 mL) and vortexed to ensure complete mixing. The mixture was sonicated at RT for 2 h and centrifuged (1000 × g, 10 min) to separate the insoluble material from the ethanol extract. The extract was decanted into a clean conical tube and stored at -20°C until analysis up to 1 mo.
Ultra Performance Liquid chromatography-Mass Spectrometry (UPLC-MS)
Sample separation was performed using an Acquity UPLC under the control of MassLynx software (Waters, Millford, MA, USA). The sample set was randomized and held in an 8°C sample manager during the analysis. For each chromatographic run, a 1 μL sample injection was loaded to a 1.0 × 100 mm Waters Acquity UPLC BEH C18 column with 1.7 μm particle size held at 40°C. Separation was performed by reverse phase chromatography at a flow rate of 0.15 ml/min. The eluent consisted of water and methanol (Fisher, Optima® LC/MS grade) supplemented with formic acid (Fluka, LC/MS grade) in the following proportions: Solvent A = 95:5 water:methanol + 0.1% formic acid; Solvent B = 5:95 water:methanol + 0.1% formic acid. The separation method is described as follows: 0.1 min hold at 100% A, 14.9 min linear gradient to 100% B, 5 min hold at 100% B, 1 min linear gradient to 100% A, and 1 min hold at 100% A. A blank injection of water and 15 min chromatographic run was preformed between samples to eliminate possible carryover of analytes and to re-equilibrate the column. This cleaning method is described as follows: 0.1 min hold at 100% A, 2.9 min linear gradient to 100% B, 1 min hold at 100% B, 3 min linear gradient to 100% A, and 8 min hold at 100% A for equilibration. The flow rate for all steps was held at 0.15 ml/min.
Eluate was directed to a Q-TOF Micro quadrupole orthogonal acceleration time-of-flight mass spectrometer controlled with MassLynx software (Waters/MicroMass, Millford, MA, USA) using electrospray ionization in the positive mode (ESI+). Mass data were collected between 50 and 1000 m/z at a rate of two scans per second with a 0.1 second interscan delay. The voltage and temperature parameters were tuned for general profiling as follows: capillary = 3000 V; sample cone = 30 V; extraction cone = 2.0 V; desolvation temperature = 300°C; and source temperature = 130°C. Mass spectral peaks were centered during acquisition producing centroid data. Leucine Enkephalin was infused via an orthogonal ESI probe and baffle system (LockMass) which allowed reference ions to be detected for a single half-second scan every 10 s in an independent data collection channel. The standard mass was averaged across 10 scans providing a continuous reference for mass correction of analyte data.
Chromatographic and spectral LC-MS peaks were detected, extracted, and aligned using MarkerLynx software (Waters, Millford, MA, USA). Chromatographic peaks were detected between 0 and 18 min with a retention time error window of 0.1 min. Apex track peak detection parameters were used, automatically detecting peak width and baseline noise. No smoothing was applied. To reduce the detection and inclusion of noise as data, an intensity threshold value of 40 and a noise elimination value of 6 were used. Mass spectral peaks were detected between 50 and 1000 m/z with a mass error window of 0.07 m/z. The de-isotoping function was enabled to eliminate the inclusion of isotopic peaks. A matrix of features as defined by retention time and mass was generated, and the relative intensity of all features, as determined by area, was calculated for all individual samples. Potential effects of technical variability were minimized by normalizing the intensity values to the total ion current (TIC) such that the summation of all feature intensities in each individual sample were equal.
For microarray analysis, data were imported into Partek Discovery Suite software (Partek, St. Louis, MO), PC and 1-way ANOVA (random effects) (p < 0.01) analyses were performed. For 2DGE, PC, hierarchal clustering based on Euclidian distances, and 1-way ANOVA (random effects) (p < 0.05) analyses were carried out using Partek Discovery Suite software. Finally, the LC-MS feature matrix was mean centered and imported into SIMCA-P+ software (Umetrics, Inc., Umeå, Sweeden). PC analysis was performed using Pareto scaling. Hierarchal clustering based on Euclidian distance and 1-way ANOVA (random effects) (p < 0.05) analyses were performed using Partek Discovery Suite software. Fold change for both proteomics and metabolomics was calculated using Partek Discovery Suite.