Mapping of barley (Hordeum vulgare L.) beta-amylase alleles in which an amino acid substitution determines beta-amylase isoenzyme type and the level of free beta-amylase

Abstract

The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding.