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Plant Molecular Biology Reporter

, Volume 20, Issue 2, pp 149–159 | Cite as

Genotyping single nucleotide polymorphisms for selection of barley β-amylase alleles

  • Maxime Paris
  • Michael G. K. Jones
  • Jason K. Eglinton
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Abstract

A high-throughput single nucleotide polymorphism (SNP) genotyping system was developed and used to select barley seedlings carrying superior alleles of β-amylase. In the malting process, β-amylase is a key enzyme involved in the degradation of starch. Four allelic forms of the enzyme are found in barley, each exhibiting a different rate of thermal inactivation, or thermostability. The level of thermostability influences starch degradation, which determines the yield of fermentable sugars for alcohol production during brewing. Control of the fermentability level is important for barley breeding programs to allow targeting quality profiles of new varieties to suit end-user requirements. Alignment of the cDNA sequences encoding the 4 enzyme forms revealed 6 SNPs (cSNPs). The 4 alleles could be identified unambiguously by codominantly genotyping 2 of the cSNPs using a duplex single nucleotide primer extension (SNuPE) assay. Two genotyping primers with their 3 ends directly flanking the selected SNPs were annealed to the amplified target sequences and extended by single dideoxynucleotides complementary to the polymorphic nucleotides. Extended primers were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Making use of the inherent molecular weight difference between DNA bases, incorporated nucleotides were identified by the increase in mass of the extended primers. A cleaved amplified polymorphic sequence (CAPS) assay enabling broader classification of the alleles was also developed to facilitate the transfer of this marker to other laboratories. Plants carrying alternative β-amylase alleles were selected at the seedling stage for barley breeding.

Key words

barley beta-amylase MALDI single nucleotide polymorphism single nucleotide primer extension thermostability 

Abbreviations

cSNP

coding single nucleotide polymorphism

MALDI-ToF MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

SNP

single nucleotide polymorphism

SNuPE

single nucleotide primer extension

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Copyright information

© International Society for Plant Molecular Biology 2002

Authors and Affiliations

  • Maxime Paris
    • 1
  • Michael G. K. Jones
    • 1
  • Jason K. Eglinton
    • 2
  1. 1.Western Australian State Agricultural Biotechnology CentreMurdoch University
  2. 2.Department of Plant Science, Waite CampusThe University of AdelaideGlen Osmond

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