Eighty-two different accessions of barley from Nordic countries were obtained from NordGen (the Nordic Genetic Resource Center). The plants consisted of four groups: Landraces, old cultivars (1890-1940), cultivars (1941-1970) and modern cultivars (1971- present). Furthermore, accessions from the countries Norway, Sweden, Denmark and Finland were equally represented within each group. Two barley varieties, ‘Karl’ and ‘Lewis’, were received from the Germplasm Research facility of the Small Grains Collection, US(United States) and were used as controls. The ‘Karl’ control with accession number Clho 15487 has low GPC and the ‘Lewis’ control with accession number Clho 15856 has high GPC. ‘Karl’ and ‘Lewis’ are polymorphic in two SNPs (Single Nucleotide Polymorphism) positioned in NAM (Distelfeld et al., 2008). 

The barley plants were cultivated in a greenhouse for three months at Link ö ping University. Three replicates of each accession were grown and the plants were well watered and supplied with fertilizers. The seed size and maturation time was recorded during the maturation stage. The grains were harvested manually after three months of maturity. After 94 days, the height of the plant from soil surface to the top of primary spike was measured for each plant in a pot.The dried grains were grounded and threshed thoroughly to remove all chaff and were counted to obtain 30 grains to measure its kernel size. The grain samples were analysed for mineral concentration. Analyses of Nitrogen (Leco Corp, Lakeview Avenue, United States), and Iron and Zinc content was measured using Inductively Coupled Plasma atomic emission spectrometry at Agrilab AB in Uppsala.

The DNA extraction was performed using Qiagen DNAeasy Plant Mini Kit (Qiagen, Germany). Leaf tissue samples from the various different barley lines were collected and crushed thoroughly in the eppendorf tubes and the isolation was carried out according to the protocol in the kit.

The PCRs were run in a 20µl reaction volume consisting of 1U of 5U/µl Dream Taq DNA polymerase enzyme (Fermentas, Helsingborg, Sweden), 1x Dream Taq reaction buffer (with 20mM Mg), 0.2 mM dNTP, 0.2 µM forward primer, 0.2 µM reverse primer and 1µl DNA template. The amplification was run at 94ºC for 2min 30s in the initial denaturation step followed by 35cycles of   94°C for 30s, 58°C for 30s, and 72°C for 45s and a final extension step of 72°C for 7min.

For CAPS analysis, the PCR products were digested with 1U   Mwol (Fermentas) of in 20µl reaction volume for uhb6 and 1U TaaI(Fermentas) for uhb7.The PCR products were analysed by gel electrophoresis in 1% agarose prepared in 1XTBE. All gels contained SybrSafe (Invitrogen) for the detection of the DNA in UV-light. Finally, the results were viewed on the Gel documentation system (BioDoc Imaging system, UVP, Cambridge, UK)

Statistical analysis was performed for the obtained data using a General Linear Model-Univariate method and graphs were represented in the form of Error bars in the software SPSS statistics. Correlation and regression analysis was performed for the 82 barley lines.