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ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Research Project #438246

Research Project: Integrated Analysis for Identifying Barley Lines with Superior Malting Quality

Location: Cereal Crops Research

2021 Annual Report

Objective 1. Resolve the genetic basis of dormancy and Preharvest Sprouting (PHS) in malting barley. Objective 1.1: Demonstrate that domestication of barley for improved malting has increased the frequency of mutations within genes that affect dormancy and PHS. Objective 2. Evaluate and report intrinsic malting quality parameters of commercially viable barley cultivars as part of a Congressionally-directed mission of service (non-hypothesis driven). Objective 2.1: Evaluate and report intrinsic barley malting quality parameters used to guide breeder selection for superior variety development and evaluation of new varieties.

We plan to test preharvest sprouting on three unique panels of barley that include lines of contemporary, heritage, and globally diverse origin to reveal the extent of the genetic contribution to dormancy (or nondormancy) together with the screening of these lines for precocious germination on intact heads with the goal of developing genetic resources to reduce preharvest sprouting in North American varieties. The first phase of the approach will examine associations between PHS susceptible phenotypes and allelic variability by using each of the three panels as subjects of genotypic and phenotypic evaluation to 1) to assess sequence variation through a targeted sequencing approach of genes associated with PHS and 2) to develop preharvest sprout scores using mist treatments and germination assays. The second phase will employ a gene discovery-based approach using GWAS on genotyped lines in each of the three populations to identify SNPs (or combinations of SNPs) that can uniquely identify plants carrying alleles that influence PHS related traits. These QTL will fortify our developing panel of novel alleles with additional mutations associated with PHS. Finally, molecular markers targeting SNPs tightly associated with PHS will be developed with the intent of deployment for Marker Assisted Selection (MAS) in malting barley breeding programs. Processing and test procedures are based on the ASBC Methods of Analysis, with some slight modifications and improvements. A base calculation of steep time is generated by determining the average kernel weight (mg) of the sample and using an empirically-determined relationship to yield a base steep time in hours. Slight final adjustments to moisture levels are made prior to moving the samples to the germinator. At this point, we treat all of the submissions equally, using a standardized malting protocol that has been used reliably since 1998. Samples are germinated at 16ºC, 100% humidity, with intermittent turning. An intermediate weight for each sample is taken and a final water adjustment to maintain 45% moisture is made if necessary. After 5 days of germination, the “green malt” is moved to the kiln and the samples are dried for 24 hours, starting with a temperature of 49ºC for 10 h, and rising to 85ºC for last 3 hours. Once the samples are steeped, malted, kilned, and cleaned (rootlets and emergent acrospires removed), they are stored to allow moisture equilibration and sample aging. Samples are subjected to industry standard protocols by the American Society of Brewing Chemists (ASBC). Malts are ground and mashed (ASBC Malt-4) and analyzed for % extract (Malt-4), soluble protein (Wort-17), color and clarity (Wort-9), free amino nitrogen content (Wort-12) and beta-glucan levels (Wort-18). Malt grist is extracted into salt water at 20ºC and the diastatic power (Malt-6) and alpha-amylase activities (Malt-7) are determined. Total nitrogen contents of the barley are determined using FOSS Nova NIT and malt are determined on a LECO Corp. FP528 Nitrogen Analyzer utilizing the Dumas total combustion method (Malt-8).

Progress Report
Progress was made on both objectives in the approved research project. We were able to complete the DNA sequencing of three genes associated with dormancy in over 300 different malting and feed barley varieties. Samples from last year’s field season in Montana were pressured to preharvest sprout in mist chambers and those results were tied into the sequencing data. The study identified distinctly different genetic groups of barley varieties with members within each group having identical mutation patterns. The allelic variations characteristic of these groups were strongly associated with Preharvest Sprouting tendencies. Thus, we believe we’ve identified a suite of genetic mutations that give rise to much of the preharvest sprouting issues in our north American germplasm. We began micromalting and analyzing the malt characteristics from barley varieties that show measurable differences in their dormancy requirements to unravel the association between positive malting attributes and germination energy. Recently, in collaboration with a scientist at University of Wisconsin Madison, we coordinated the planting of 180 barley varieties from a genetic diversity panel with the intent of providing subject material for analysis of water sensitivity in the kernel and germination energy. In collaboration with another co-located SY from NP 301, we undertook an examination of Reactive Oxygen Species (ROS) during malting and the role of the Respiratory Oxidative Burst Homologs (RBOH) gene family in this process. This study identified that HvRBOHs are strongly induced during malting. Furthermore, genetic compensatory mechanisms were invoked in response to RNAi suppression of highly induced HvRBOHs during malting suggesting the ROS and NADPH oxidase enzyme activity is crucial during the malting process. The malt quality lab worked diligently to progress through the highest priority samples to meet stakeholder deadlines. Staff from the malt quality lab, working under maximum teleworking posture and facility occupancy limits, completed the malting and subsequent quality analysis of the “pilot” program samples in time to meet the stakeholder’s annual quality evaluation meeting held earlier this spring. Currently they are working to catch up FY19 samples with the hopes we can still provide some quality metrics for those scientists and trade association customers who submitted them prepandemic.


Review Publications
Mahalingam, R., Graham, D.L., Walling, J.G. 2021. The barley (Hordeum vulgare ssp. vulgare) Respiratory Burst Oxidase Homolog (HvRBOH) gene family and their plausible role on malting quality. Frontiers in Plant Science. 12. Article 608541.