Beer fans and brewers all know that getting the right mix is essential to a drinkable pint. For brewers and barley seed breeders, this mix is an essential brew of genetics with desirable varietal properties and suitability for the malting process. Through trait analysis, genetics shows the seeds containing the best sugars, appropriate protein content and enzymes that perform well during the brewing process, delivering a flavorsome drink with a satisfying head. A targeted genotyping by sequencing (GBS) approach helps determine varietal proportions in a barley seed mix with superior sensitivity and ease of use.
Barley brew crisis
In 2008, Uruguay was facing a crisis: the purity of its barley seed mixtures was in doubt, threatening the consistency of brewers’ products. Without controlling the exact varietal proportions contained in each batch of barley seeds, brewers had no way to ensure product quality.
For Carlos Azambuja and his team at Genia, a DNA analysis company, the solution was to develop a new method to determine seed lot purity through molecular methods. Quantifying the varietal components in a seed mixture through short tandem repeat (STR) analysis of individual seeds could provide data on varietal proportions within the seed mixes. However, in order to achieve low error rates and high accuracy, the team would have to analyze large numbers of seeds, as STR analysis did not have sufficient sensitivity to function well on pooled samples.
Error in small sample size
Azambuja’s team found that analyzing fewer than 100 seeds led to large margins of error. These margins could be reduced by analyzing larger numbers of individual seeds, but the workflows and costs were not feasible. In order to quantify low levels of contamination, the team needed to analyze tens of thousands of seeds and work with an approach suitable for pooled samples.
Furthermore, since the approach would require high sample numbers, turnaround time and cost became important considerations too. Eventually, after four years had elapsed, next-generation sequencing (NGS) provided the solution.
Each barley variety holds its own unique single nucleotide polymorphism (SNP) profile. Azambuja suggested that relative proportions of each variety could be calculated according to the SNP profile and allele frequencies in a pooled sample of the seed mix. For valid results, Genia needed a method that referenced all major brewing barley and native varieties in Uruguay and performed well for pooled samples.
NGS, with its high sensitivity, ease of customization and ability to target multiple markers in a single library prep, fulfilled all requirements on paper—but would it work in the lab?
Targeted GBS for barley seed success
In collaboration with Thermo Fisher Scientific, Azambuja created the AgriSeq panel to discriminate the 43 barley varieties used most frequently in Uruguay, Chile and Argentina. The research team chose a set of 400 SNPs from the commercially available iSelect 9k barley chip. Following in silico testing by Thermo Fisher Scientific, the researchers created a final panel targeting 369 SNPs.
For data processing, the Genia team created an algorithm based on Bayesian methods and the genotype frequencies to deconvolute the NGS data into varietal proportions in the mix.
The team extracted DNA from bulk pooled flour samples from 10,000 seeds for AgriSeq library constructions. They then processed the samples using an Ion S5 System, obtaining results in as few as two days.
Using this targeted GBS AgriSeq workflow, the researchers successfully measured seed mixture purity. They obtained a 99% confidence interval with less than 1% error to calculate purity levels ranging from 95% to 99%. The method was highly sensitive and reproducible, detecting contamination levels as low as 0.8%.
Seed mix clarification for the future
Varietal contamination at the seed level is a big problem for seed producers and growers, not just for malting barley but also soy bean and clover crops. Azambuja is confident that with the AgriSeq GBS approach, seed breeders now have a valuable higher-sensitivity tool that is easier to deploy.
Reflecting on the results of the study, Azambuja urges his peers to consider the AgriSeq technology. “The results demonstrate that the method is very reliable and reproducible. With development over time, this tool will become a gold standard in the industry,” he says.
If you would like to find out how Thermo Fisher AgriSeq technology can help with your plant and animal breeding programs, contact us here for a free consultation.
Learn more about AgriSeq targeted GBS solutions for examining varietal purity in malting barley in this Plant and Animal Genome Conference workshop with Dr. Azambuja.