Chromeleon 6.8 chromatography data system

DHI modified samples were diluted to 1:5 v/v with 0.5% aqueous formic acid, and then filtered through a 0.22 μm membrane filter before injection. UPLC analyses were carried out by using a Dionex Ultimate 3000 series UPLC system coupled with a diode array detector (DAD). The chromatographic fingerprints were obtained from a Welch Ultimate XB-C₁₈ column (4.6 mm × 250 mm, 5 μm). The gradient elution was performed as previously described by using acetonitrile (A) and water with 0.5% formic acid (B) at the flow rate of 1 mL/min44 . The program was as follows: 0–15 min, 2–10% (A); 15–20 min, 10–17% (A); 20–45 min, 17–28% (A); 45–48 min, 28–60% (A). An aliquot of 5 μL of each sample solution was injected into the UPLC–DAD system and detected at 288 nm. Peak areas were calculated with a Chromeleon 6.8 Chromatography Data System (Dionex) and cluster analysis of the nine DHI samples was conducted in SPSS 18.0 based on the peak areas. The clustering method was between-groups linkage, and the distance calculating method was Pearson’s correlation.

For nutrient analysis, four dispersal units of wild emmer wheat were dissected into caryopses and floral bracts, namely, glumes, lemmas and paleas (GLPs) followed by incubation in 600 μl of ultrapure water (Milli-Q) for 12 hours on a cooled orbital shaker (4°C). Samples were centrifuged (14,000 rpm, 4°C) and the supernatant was filtered (0.22 μm spin filter) and diluted with 4.8 ml of Milli-Q water and subjected to nutrient analysis. Three measurements were performed for each chemical element. The content of K, P, Ca, Mg, S and Zn released upon hydration from wild emmer wheat dispersal unit was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using ICP-720-ES (Varian Inc., USA). Soluble anions were detected and identified by Ion chromatography (IC). IC was performed by ICS-5000 instrument (Dionex, Thermo Fisher Scientific). Obtained data were analyzed by Chromeleon 6.8 chromatography data system (Dionex, Thermo Fisher Scientific).

For nutrient analysis, 10 mg of ground S. alba and A. thaliana pericarps and 10 mg of S. alba seed coats were incubated in 300 µL ultrapure Milli-Q water at cooled orbital shaker (4 °C). Analysis of nutrients released from seeds was performed as follows: 20 seeds of S. alba and 10 mg of A. thaliana seeds were incubated in 600 and 300 µL, respectively, of ultrapure milli-Q water. All samples were incubated for 12 h at 4 °C followed by centrifugation (14,000 rpm, 4 °C), the supernatant was collected and filtered using 0.22 μm spin filter. 200 μL of the filtered supernatant were diluted with 4.8 mL of ultrapure Milli-Q water and subjected to nutrient analysis. Three measurements were performed for each chemical element. The content of macro- and microelements released upon hydration was determined by inductively coupled plasmaoptical emission spectroscopy (ICP-OES) using ICP-720-ES (Varian Inc., Palo Alto, CA, USA). Soluble anions were detected and identified by Ion Chromatography (IC). IC was performed by an ICS-5000 instrument (Dionex, Thermo Fisher Scientific, Waltham, MA, USA). Obtained data were analyzed by Chromeleon 6.8 chromatography data system (Dionex, Thermo Fisher Scientific).