The third intron of barley (Hordeum vulgare L.) β-amylase 1 (Bmy1) is extremely polymorphic. The use of specific insertion/deletions (indels) in the third intron as markers for cultivar development has been recommended based on associations with β-amylase activity and thermostability. The third intron of Bmy1 in 40 barley genotypes was sequenced and aligned with 15 Bmy1 intron III sequences from GenBank and four alleles (Bmy1.a, Bmy1.b, Bmy1.c, and Bmy1.d) were identified based on indels of 126, 38, 11, and 21 bp. β-Amylase activity and thermostability were assayed in 22 North American cultivars and 12 wild barley genotypes. Cultivars carrying the Bmy1.a and Bmy1.b alleles had β-amylase activity ranges calculated on a fresh weight (FW) basis of 1.8-and 1.5-fold, respectively, and thermostability ranges of 8.8-and 1.2-fold, respectively. β-Amylase activity calculated on a protein basis yielded a 2.4-and 1.4-fold range for Bmy1.a and Bmy1.b, respectively. Significantly different activities were observed in cultivars carrying either Bmy1.a or the Bmy1.b allele when calculated on a FW basis and the Bmy1.a allele when calculated on a protein basis. Significantly different thermostabilities were observed in cultivars carrying the Bmy1.a allele. Wild barleys were found to carry Bmy1.a, Bmy1.b, and Bmy1.c alleles with β-amylase activity ranges calculated on a FW basis of 1.7-, 1.7-, and 2.6-fold, respectively, and thermostability ranges of 1.3-, 1.4-, and 2.1-fold, respectively. β-Amylase activity measured on a protein basis identified a 1.3-, 1.4-, and 2.1-fold range for Bmy1.a, Bmy1.b, and Bmy1.c, respectively. Significantly different activities were found in genotypes with any of these three alleles when calculated on a FW basis yet only in those with the Bmy1.c allele when calculated on a protein basis. Significantly different thermostabilities in genotypes carrying either the Bmy1.b or Bmy1.c allele were observed. In the germplasm studied here, the Bmy1 intron III alleles are not reliable predictors of β-amylase activity and thermostability.

Malting quality data were collected on malts from three barley (Hordeum vulgare L.) breeding program trials. We tried to identify causal polymorphisms in the Bmy1 intron III and coding regions for use in marker‐assisted selection. Abundant malting quality variation exists in the spring barley germplasm despite the parents having identical Bmy1 intron III and coding regions. After complete Bmy1 sequencing, no polymorphisms associated with malting quality phenotypes, indicating the genetic basis for the observed variation resides outside Bmy1. Complete allele sequencing identified one winter barley parent that had a novel Bmy1 allele (Sd1a) based on amino acid substitutions that are candidates as causative agents for the phenotypic variation. Marker‐assisted selection against the Sd1a allele could be effective in improving diastatic power (DP). The Sd1a allele is associated with low DP and is present in only three of the 51 lines, presumably due to preceding generations being selected for high DP. Selection for DP has subsequently eliminated the Sd1a allele from this breeding program. This research shows the importance of having complete allele sequences and knowledge of functional polymorphisms in target genes before using marker‐assisted selection.

Two barley (Hordeum vulgare L.) β-amylase genes (Bmy1 and Bmy2) were studied during the late maturation phase of grain development in four genotypes. The Bmy1 and Bmy2 DNA and amino acid sequences are extremely similar. The largest sequence differences are in the introns, seventh exon, and 3' UTR. Accumulation of Bmy2 mRNA was examined in developing grain at 17, 19, and 21 days after anthesis (DAA). One genotype, PI 296897, had significantly higher Bmy2 RNA transcript accumulation than the other three genotypes at all developmental stages. All four genotypes had Bmy2 mRNA levels decrease from 17 to 19 DAA, and remain the same from 19 to 21 DAA. Levels of Bmy1 mRNA were twenty thousand to over one hundred thousand times more than Bmy2 mRNA levels in genotypes Legacy, Harrington, and Ashqelon at all developmental stages and PI 296897 at 19 and 21 DAA. PI 296897 had five thousand times more Bmy1 mRNA than Bmy2 mRNA at 17 DAA. However, Bmy2 protein was not found at 17 DAA in any genotype. The presence of Bmy2 was immunologically detected at 19 DAA and was present in greater amounts at 21 DAA. Also, Bmy2 protein was found to be stored in mature grain and localized in the soluble fraction. However, Bmy1 protein was far more prevalent than Bmy2 at all developmental stages in all genotypes. Thus, the vast majority of β-amylase activity in developing and mature grain can be attributed to endosperm-specific β-amylase.