| | Barley varieties have unique quality characteristics that affect how they respond to the malting process. To achieve uniform production of high quality malt, malting companies generally purchase specific barley varieties with varietal purity as a contract specification. Typically, 95 percent or greater purity is required. In order to meet these demands, robust means of variety identification are necessary.
Although certain morphological characteristics may aid in the visual identification of some barley varieties, such characters are subject to environmental influences and may vary along a continuum within varieties, making it exceedingly difficult, even for experienced grain inspectors, to determine the varietal composition of barley shipments. Therefore, many in the industry have turned to biochemical means of variety identification. The Variety Identification Unit of the Canadian Grain Commission (CGC) has used seed storage protein markers as tools for variety identification for several years. Varietal composition of a barley sample is determined through the analysis of multiple single kernels by electrophoresis and(or) by examination of a bulk ground sample using high performance liquid chromatography (HPLC). However, owing to the narrow gene pool of malting barley varieties and the limited amount of variation of seed storage proteins among varieties, it has become increasingly difficult to discriminate among all malting barley varieties registered in Canada using these methods.
To enhance our barley variety identification capabilities, we have also developed and implemented DNA-based methods. Two types of polymerase chain reaction- (PCR-) based DNA markers are currently in use. For six-rowed barley varieties, we use six microsatellite markers (also known as simple sequence repeats, or SSRs) that are multiplexed into two PCR sets. For two-rowed barley, we currently use 12 sequence-tagged-site (STS) markers that are multiplexed into three sets. We are also in the final stages of developing a more robust system for two-rowed barley that consists of eight SSRs multiplexed into two sets. For each marker system, we have constructed catalogues of DNA fingerprints of barley varieties registered in Canada. All 67 two-rowed varieties registered as of April 2003 can be uniquely identified. For six-rowed varieties, each of the 17 registered malting varieties can be distinguished from one another and from the 85 registered non-malting varieties. Most of the six-rowed non-malting varieties can also be uniquely identified. DNA-based tests are routinely used at CGC to supplement the less expensive, but lower resolution, protein-based methods for barley identification. DNA-based testing also permits the identification of malted barley. Protein is degraded by the malting process whereas DNA remains sufficiently intact for PCR-based tests.
Currently, biochemical testing for variety identification is applicable only in a laboratory setting and, with the exception of HPLC, is performed on single kernels. However, there is a strong demand for an automated and quantitative variety identification technology that can determine the varietal composition of a ground sample rapidly and preferably at the point of delivery. Such technology is not available at present, and likely will not be for several years. However, current indications are that when it is developed, such technology will likely be DNA-based and use single nucleotide polymorphisms (SNPs). SNPs are bi-allelic, highly abundant and hold promise for automation. In anticipation of a future demand for barley SNPs to be applied on a variety identification platform, we are engaged in a SNP discovery and characterization project. This project, a collaborative effort involving researchers at the CGC and Agriculture and Agri-Food Canada, was initiated in 2000 with the financial support of Automated Quality Testing Inc. (AQT).
Our approach to SNP discovery has been to obtain PCR primers, either publicly available STS primers (See et al., 2000), or primers that we designed based on alignments of expressed sequence tags (ESTs) of barley genes. DNA from a subset of eight barley varieties was used for PCR amplification. The PCR products were then sequenced and the aligned sequences were analyzed to identify putative SNPs (Figure 1). Putative SNPs were initially evaluated in the DNA of a single kernel of each malting variety using restriction of PCR products (PCR-RFLP), allele specific PCR, or SNaPshot primer extension assays (Applied Biosystems, Foster City, California). We are now investigating polymorphism within varieties using the DNA of 32 individual kernels from each malting variety.
Figure 1. SNP position within a DNA alignment of genomic sequences from eight two-rowed malting barley varieties.
Over 500 putative SNPs have been discovered in 55,190 base pairs of sequenced PCR products (Dusabenyagasani, 2003). Many of these SNPs are tightly clustered and, owing to linkage disequilibrium, do not provide additional information for variety identification purposes. However, we have identified a set of 70 SNPs that are nonredundant in the 35 malting barley varieties registered in Canada. The polymorphic information content (PIC = 1 -  pi2, where pi is the frequency of the ith allele) of these SNPs ranges from 0.1 to 0.5.
A key requirement of rapid SNP-based variety identification will be the ability to determine the frequency of SNP alleles in mixtures. We have investigated the Invader Assay (Third Wave Technologies, Madison, Wisconsin), a technology that shows some promise for the quantitative genotyping of SNPs. Not all SNPs were found to be suitable for the Invader Assay as only two of the six barley SNPs that we evaluated were correctly genotyped. Further investigation is required to evaluate quantification using this system. The development of SNP genotyping platforms is an evolving field and we will continue to explore those that show promise for quantitative genotyping of SNPs as they become available.
Acknowledgments
Automated Quality Testing Inc., Winnipeg, Manitoba, provided financial support for the SNP research project. Mathieu Dusabenyagasani and Sung-Jong Lee provided technical assistance.
References
Dusabenyagasani, M., D. Perry, S-J. Lee and T. Demeke. 2003. Genotyping malting barley varieties registered in Canada with SNP markers. Abstract and poster presented at the XI Plant and Animal Genome Meeting. San Diego, California.
See, D., V. Kanazin, H. Talbert and T. Blake. 2000. Electrophoretic detection of single nucleotide polymorphisms. BioTechniques 28: 710-716.
Tigst Demeke, Daniel Perry and Jean Mellish
Canadian Grain Commission, Grain Research Laboratory, Winnipeg, Manitoba
Presented at the 3rd Canadian Barley Symposium, June 19-20, 2003 |
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