Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2009
Publication Date: 1/30/2010
Citation: Vinje, M.A., Duke, S.H., Henson, C.A. 2010. Utilization of Different Bmy1 Intron III Alleles for Predicting ß-Amylase Activity and Thermostability in Wild and Cultivated Barley. Plant Molecular Biology Reporter. 28:491-501. Interpretive Summary: The production of fermentable sugars from the starch stored in barley seeds is dependant upon the action of several enzymes that are made or activated by the seed during germination. One of the most important of these enzymes is beta-amylase. This enzyme’s contribution to mashing, the industrial process that results in the conversion of starch to fermentable sugars, is limited by its lack of stability at the high temperatures typically used for mashing. Significant effort is being spent to find germplasm containing a beta-amylase that is thermostable, which will then be used as a parent in malting barley breeding programs. A rapid selection tool is desirable for this purpose and specific segments of the gene encoding beta-amylase have been proposed to be used as specific markers of thermostability in beta-amylase. The work reported here demonstrates that none of the four sequences currently being used as markers for thermostability are capable of consistently and reliably predicting beta-amylase thermostability. The impact of this work is that breeder’s will no longer rely upon these ineffective tools for selecting malting barley.
Technical Abstract: Polymorphisms in intron III of barley (Hordeum vulgare L.) endosperm-specific beta-amylase (Bmy1) have been associated with beta-amylase activity and thermostability and are thought to have potential as a selective marker for breeding elite malting cultivars. The third intron of Bmy1 was sequenced in forty barley accessions. Four alleles were identified based upon insertion/deletions (indels) of 126-bp, 38-bp, 11-bp, and 21-bp. The Bmy1.a allele has the 126-bp, 38-bp, and 21-bp indels. Bmy1.b only has the 38-bp indel. Bmy1.c has the 38-bp, 11-bp, and 21-bp indels. Bmy1.d, found in only one accession, is missing the 126-bp and 38-bp indel. Thirty-nine accessions were assayed for ß-amylase activity and thermostability (percent remaining after exposure to 60'C for 10 minutes). Accessions with the Bmy1.a, Bmy1.b, Bmy1.c, and Bmy1.d alleles had averages of 26.7, 23.8, 38.5, and 56.1% residual activity, respectively. Each allele had statistically different thermostabilities according to the Kruskal-Wallis test. Accessions with Bmy1.a, Bmy1.b, Bmy1.c, Bmy1.d alleles had activity averages of 1181, 1460, 1058, and 2739 U/g flour, respectively. Each allele had statistically different activities using the Kruskal-Wallis test, except between Bmy1.a and Bmy1.c. These data support the use of intron III as a selective tool. However, when the data within each intron allele are analyzed, the conclusions are different. Using the least significant difference (LSD) test on the activities and thermostabilities within each allele (i.e. Bmy1.a, Bmy1.b, and Bmy.c), statistically significant differences were found. Therefore, we conclude that the third intron is not a reliable marker for use in selecting elite malting cultivars.