Barley (Hordeum vulgare L.) seeds are a rich source of β‐glucans that can help reduce blood serum cholesterol content in human diets. However, high levels of this polysaccharide in the seeds causes inefficiencies such as slow lautering during the brewing process due to the higher wort viscosity. Thus, depending on the end use, barley breeding programs have opposing goals with respect to β‐glucan. Discovery of new alleles for β‐glucan content in exotic barley germplasm could have significant commercial importance. We leveraged the Wild Barley Diversity Collection (WBDC; N = 314), grown at University of California‐Davis experiment station, to characterize both the variation in β‐glucan content found in this wild barley subspecies (Hordeum vulgare spp. spontaneum) and to reveal chromosomal regions associated with this trait. Grain β‐glucan content ranged from 2.56 to 11.73% in the WBDC. Within structured subpopulations, geographical coordinates as well as annual precipitation data of original collection sites showed associations with differing levels of β‐glucan content. Genome‐wide association study identified a total of 13 quantitative traits loci (QTL) spread across the seven barley chromosomes that explained most of the variation in β‐glucan content in the WBDC. Previously reported genes associated with β‐glucan content such as β‐glucan synthases and hydrolases (e.g., callose synthase) were identified within these QTL regions. Genes such as Patatin and Nudix hydrolase provide novel targets for manipulating grain β‐glucan in barley. RNA‐seq data from both developing and germinating barley seeds for the annotated genes in the QTL regions provide further corroboration for a functional role for these genes in modulating seed β‐glucan levels.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize transcript abundance. The most popular gene targets for RT-qPCR are housekeeping genes because they are thought to maintain a static transcript level among a variety of samples. However, more recent studies have shown, several housekeeping genes are not reliably stable. This is the first study to examine the potential of several reference genes for use in RT-qPCR normalization during barley malting. The process of malting barley mechanizes the imbibition and subsequent germination of barley seeds under controlled conditions. Malt quality is controlled by many pleiotropic genes that are determined by examining the result of physiological changes the barley seed undergoes during the malting process. We compared the stability of 13 reference genes across both two-and six-row malting barleys (Conrad and Legacy, respectfully) throughout the entirety of the malting process. Initially, primer target specificity, amplification efficiency and average Ct values were determined for each of the selected primer pairs. Three statistical programs (geNorm, NormFinder, and BestKeeper) were used to rank the stability of each reference gene. Rankings were similar between the two- and six-row with the exception of BestKeeper’s ranking of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A consensus ranking among programs was determined using RefFinder. Our results show that Actin (ACT) and Heat Shock Protein 70 (HSP70) were the most stable throughout micromalting, while GAPDH and Cyclophilin (CYP) were the least stable. Two reference genes are necessary for stable transcript normalization according to geNorm and the best two reference genes (ACT and HSP70) provided a sufficient level of stability.