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					                    2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
                       EXECUTIVE SUMMARIES & RESEARCHER UPDATES

                                           Barley Improvement
                                    Kevin Smith, University of Minnesota

The overall aim of this research is to develop new barley varieties with acceptable malt quality, improved
disease resistance, and high yield potential. At present we are developing six-row spring and two-row and
six-row winter malting barley for the Midwest region. Our long term goal is to release winter barley for the
Midwest that will fit into cropping systems that are desirable to producers in the region such as a winter
barley-soybean double cropping system. This research will directly assist in AMBA’s mission to provide an
adequate supply of high quality malting barley. Barley improvement at the University of Minnesota is a
cooperative effort of the Departments of Agronomy and Plant Genetics, Plant Pathology, and Research and
Outreach Centers of the University of Minnesota. Specific breeding goals include high yield, enhanced
lodging resistance, resistance to Fusarium head blight (FHB), net blotch, spot blotch, stem rust, and Septoria
speckled leaf blotch (SSLB), and favorable malting and brewing characteristics. In addition, in our winter
barley program we seek to enhance winter hardiness and combine it with high yield and good malting
quality. To meet these objectives, we are conducting a comprehensive breeding and genetics research effort
funded by industry, state and federal grants. This AMBA proposal is designed to address research objectives
not funded through other sources, to maximize the effectiveness of resources, and to insure that germplasm,
information, and technological discoveries are translated rapidly toward the development of new malting
barley varieties.

In our winter barley programs, we have just made crosses in our two-row program and just planted our first
preliminary yield trials in our six-row program. In our six-row program, we are utilizing genomic selection
(GS) which should dramatically shorten the breeding cycle, which is the time from which we make a cross to
when a breeding line from that cross is selected to be used as a parent. We have just completed our third
cycle of selection in this program. Lines enter preliminary yield trials 2 years after selection and could be
entered into AMBA testing two years after that. We will harvest our cycle 1breeding lines in preliminary
yield trials next summer and have them evaluated for quality. Selected lines from this set would go to 2nd
year yield trials in the 2013-14 field season and then enter AMBA Pilot testing with the 2015 crop. Our two
row winter program is about three years behind the time line for the six-row program. Preliminary evaluation
of the GS predication accuracy for winter survival or low temperature tolerance (LTT) is encouraging. We
have worked out some of the bugs in the very tight timeline required to plant, genotype, make genomic
predictions, select parents, and make crosses in about a seven week period of time. Based on this success, we
will do two cycles of selection in our winter six-row program this year which compares to one cycle every
four years in our traditional breeding program.

We are also implementing GS in our spring six-row program. We just made selections for cycle 3. Our cycle
1 selections from that program were evaluated in 2nd year yield trials this year and will be considered for
entry into AMBA Pilot testing with the 2013 crop. Selections from cycles 2 and 3 would enter AMBA testing
in 2014 and 2015, respectively.

In the next funding cycle, we hope to continue to scale up our winter breeding programs and collect more
data on our genomic selection procedures to verify prediction accuracy and measure effectiveness.
We will also be introducing genetic diversity for LTT based on a large scale study that will identify the most
winter hardy barley lines from a world-wide collection of over 1,000 lines.

          Determining the optimum window for planting winter barley in the Northern Plains
                              Jochum Wiersma, University of Minnesota

Winter malting barley has potential benefits for producers, end-users, and the environment. Winter barley
has the potential to yield more than spring barley, avoid some diseases, reduce soil erosion during winter and
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early spring, and help diversify cropping systems. Thus, production of winter malting barley in the Northern
Plains could complement spring barley production and help support an adequate supply of high quality
malting barley for the malting and brewing industry. The success of winter barley in the Northern Great
Plains will largely depend on its ability to survive freezing temperatures. Cold hardiness in winter wheat and
other winter annuals is not a static condition; it’s controlled genetically and influenced by management
practices. Agronomic practices, including planting date and availability of standing crop residues that have
the ability to trap snow influence winter survival. A first step towards successful introduction of winter
barley in the Northern Plains is to determine a planting date window for winter barley that maximizes winter
survival. Our objective is to determine the effect of planting date on the winter survival and yield of winter
barley varieties with know differences in winter hardiness. Because of uniqueness of soils and climates
throughout Minnesota, trials will be conducted in unique environments and repeated over several years to
sample of range of fall weather conditions.

The optimum planting window for winter wheat into standing stubble in Minnesota is between September
10th and September 30th south of Interstate 94 and between September 1st and September 15th north of
Interstate 94. Planting delays have shown to increase winterkill and delay maturity the following growing
season. The proposed research will answer whether the same is true for winter barley. First year results
point to an optimum window much the same as winter wheat. The earliest planting date not only produced a
very lush fall stand but was also infected BYDV, both factors known to reduce winter hardiness. The latest
planting dates showed more winter injury for the less winter hardy cultivar Charles compared to McGregor.

Although this is not one of the objective of the current research, the opportunity to harvest a crop in early
July - as was the case in St. Paul in 2011 - may allow winter barley to be double cropped with, for example,
short-season soybeans or peas. Double cropping soybeans following winter wheat is common in the Eastern
United States but has not feasible in Minnesota to date; the earlier maturity and harvest of winter barley may
change that.

The very dry fall in 2011 caused uneven and delayed emergence in St. Paul. The no-till trial in Crookston
showed a very even emergence despite the dry conditions. Incidence and severity of BYDV at both locations
was lower in the earlier planting dates compared to the year prior. The advantages of no-till seeding to
protect the seedlings from lethal temperatures was showcased when mild temperatures in early January
caused most of the snow cover to disappear in the tilled field surrounding the trial in Crookston (Photo1).
Winter survival was excellent at all locations. The trials in St. Paul and Crookston were harvested and
samples are being processed.


                                       At this point in time, the emergence in Crookston is uneven and
                                       delayed as this summer's drought extended well into September.
                                       Although we have received substantial precipitation since October
                                       4th it may be 'too little too late' for a good emergence as temperatures
                                       in October have been well below average. Only some 224 Growing
                                       Degree Days (GDD) have been accumulated since the initial
                                       precipitation fell in the form of heavy wet snow on October 4th.

                                         Barley Trials in Wisconsin
                            Dean Volenberg & Matt Stasiak, University of Wisconsin

The lakeshore counties of Northeastern Wisconsin have historically produced malting barley. Over time,
other agricultural enterprises became more profitable and malting barley moved west in the United States and
north into Canada. Currently, there is renewed interest in malting barley in Wisconsin. This interest is
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
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favored by the Craft breweries that are looking to produce a unique local Wisconsin product. Presently, there
is little data on either winter or spring malting barley varieties for Wisconsin. The objective of this research
is to continue evaluation of winter malting barley selections from Dr. Kevin Smith, University of Minnesota
and also evaluate 2-row spring malting barley varieties -- Pinnacle, Newdale, and Scarlet in replicated field
trials at four different locations. Pinnacle, in a 2011 preliminary trial, showed promise, providing a suitable
malt quality profile. The replicated winter malting variety trial seeded on October 4, 2011 will screen
selections and named varieties for winter hardiness, yield, growth characteristics and malt quality will be
evaluated in cooperation with the USDA Cereal Crops Research Unit in Madison, Wisconsin. Similarly, the
spring malting barley trials will screen varieties for stand establishment, yield, growth characteristics, disease
susceptibility, and malt quality. This project will produce agronomic data and malt quality profiles on winter
and spring malting barley selections and named varieties for Wisconsin. The agronomic data from this
project will be completed by November, 2012 and malt quality data will be completed prior to 2013. This
project fulfills the primary objective of AMBA and also fills the strategic goal of quality evaluation for a
breeding program.

This is the second year of successfully establishing and producing winter malting barley in Northeast
Wisconsin. Yields of some entries produced over 100 bushels per acre in the presence of heavy disease
pressure from powdery mildew. The trial was conducted without foliar fungicide applications, making the
yields very impressive. Malt quality analysis has been completed by the Cereals Crops Research Unit in
Madison, Wisconsin. Successful winter malting barley production has been demonstrated at the plot-scale
level and future research needs to focus on field scale production. This will not only provide valuable
research data, but provide an opportunity to create awareness and build grower support for malting barley
production in Northeast Wisconsin.

Five varieties of spring malting barley were established in April at four different geographic locations that
vary climatically; Sturgeon Bay, Chippewa Falls, Alma, and Bayfield. In three of the four locations, field-
scale trials were established in cooperation with growers. The fourth trial was a large scale plot trial
conducted in Sturgeon Bay. Varieties included were Pinnacle, Stellar, Robust, Lacey, and Newdale. Pinnacle
in terms of yield, outperformed the other varieties. Malt quality analysis has been completed only for the
Sturgeon Bay site as of this report. Although varieties were established under excellent growing conditions in
April, drought and powdery mildew were likely the main factors depressing yields. Preliminary spring barley
trial results in Wisconsin during 2010-2011, and these current trial results, suggest that Pinnacle is a
good choice for Wisconsin. Moving forward, research objectives will be to evaluate fertility programs,
especially nitrogen, to maximize yield while maintaining malt quality.


                            Management and Epidemiology of Barley Diseases
                               Ruth Dill-Macky, University of Minnesota

This is an applied research program directed at the management of the diseases of economic importance to
the barley industry in Minnesota and the Upper Midwest. The project is instrumental in screening barley
germplasm for resistance to Fusarium head blight, net blotch, bacterial leaf streak and other diseases of
economic importance. This project; surveys commercial barley crops for disease; develops and maintains a
collection of barley pathogens; examines the structure and diversity of pathogen populations; examines the
epidemiology of barley diseases; conducts field and greenhouse tests to identify and introgress sources of
genetic resistance; and conducts experiments to develop and evaluate chemical, biological and cultural
control options for the management of barley diseases.

The screening of germplasm, conducted in collaboration with the Minnesota barley breeding program, serves
both to identify progeny suitable for advancement and/or release as commercial malting barley cultivars and
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provides information for the genetic characterization of resistance and the development of markers that may
be utilized in marker assisted selection (MAS). In addition to providing services to the breeding program
this program conducts independent research aimed at the development of effective and efficient techniques
for the identification of resistance to a number of barley diseases. Monitoring barley crops in the Upper
Midwest annually for the prevalence of diseases serves to identifying potential new and emerging threats to
the industry and insure against the unexpected upsurge of diseases that may impact barley yield and/or
quality. Studying the epidemiology of diseases and testing a variety of management options facilitates the
implementation of effective disease control practices for the diseases of economic importance to barley.

The development of new barley cultivars, like other studies undertaken in this project, are ongoing activities,
the scope of which extend beyond a one-year project. Annual outcomes from this project may however be
measured through the release of new malting quality barley varieties, such as Quest released in 2010, through
the numbers of lines screened for their reaction to various diseases, and through research outcomes published
in peer-reviewed scientific journals and extension publications including the variety trails bulletin.

In 2012 we had a successful year working on a number of diseases including Fusarium head blight (FHB),
net blotch and bacterial leaf streak (BLS). Our screening nurseries conducted in collaboration with the plant
breeding programs aided in the selection of germplasm with improved resistance that advanced the breeding
programs efforts to develop barley cultivars with resistance to multiple diseases prevalent in the Upper
Midwest. A study, funded by the USWBSI, examined integrated management of FHB in barley integrating
cultivar selection and fungicide treatments and demonstrated the efficacy of combined management practices
for disease control. We also examined the impact of BLS on yield in a field experiment in 2012. Preliminary
analysis of the data indicates that barley yields were not significantly impacted by BLS but that kernel
weights may be reduced by up to 5% by the disease.

Severe Barley Yellow Dwarf Virus (BYDV) and/or Aster Yellows (AY) developed in Minnesota's barley
crops in 2012. The high levels of BYDV and AY likely resulted from an early warm spring that facilitated
the arrival of vectors (aphids and leafhoppers) from southern states. Although this was likely an atypical
event, my program is working with our new extension plant pathologist, Dr. Madeleine Smith, to examine
ways to more rapidly determine the causal agent(s) of these virus- and phytoplasma-incited diseases.
Identification of the diseases facilitates better monitoring of the diseases and will improve out understanding
of the prevalence and relative importance of these diseases. Ultimately this work could lead to improved
control measures in future years.

I anticipate that the focus of our research effort in 2013 will be largely similar to 2012 with the exception of
the addition of a modest research effort to address BYDV and AY.


                      Breeding and Genetics of Six- and Two-rowed Malting Barley
                             Richard Horsley, North Dakota State University

The basic objective of the malting barley breeding program at North Dakota State University (NDSU) is to
develop and release improved varieties acceptable to barley producers in the northern Great Plains and to
those who use or process this barley. Because growers in the region have choices on which crops to grow on
their farms, it is our responsibility to develop improved malting barley varieties that are competitive with
other crops so barley is an economically viable choice for them to grow. Plant characters that receive the
most attention are those that affect quality of the grain, resulting in losses to producers and end users. These
characters include yield, grain protein, kernel plumpness, resistance to Fusarium head blight (FHB) and foliar
diseases, and resistance to preharvest sprouting (PHS). Our barley breeding project also conducts research
that leads to improved farming systems that promote production of barley with acceptable malting quality.
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Since the inception of our work on breeding for FHB resistance, a main objective has been to transfer FHB-
resistance from unadapted resistant accessions to our elite Midwest six-rowed malting barley germplasm. In
the 2011 fall greenhouse, 100% of the six- and two-rowed crosses made had a least one parent with improved
FHB resistance and lower DON accumulation. In 2011, four of the five six-rowed lines and two of the three
NDSU two-rowed lines submitted for AMBA Pilot Scale evaluation came from our FHB-resistance breeding
program. Over the next few years, our goal is to increase the frequency of lines with improved resistance to
100% from both programs.

The number of growers producing barley in North Dakota and the number of acres they produce has been
declining over the last 13 years. One of the reasons growers expressed for not growing barley was rejection
of their grain for malting due to quality issues. Development of varieties with improved resistance to FHB
will help in reducing the impacts due to disease, but growers also need to know the proper management
decisions they can make to increase the likelihood that their crop will be accepted. Two of the easier
management decisions a grower can make are what crop should precede barley and how should corn residue
be managed if corn precedes barley. Two separate studies were initiated in 2009 at four sites in North
Dakota to address these questions. Each of the studies requires two years to complete (i.e. one year for the
corn or previous crop treatments and the second year for the barley treatments) and our goal is to repeat each
experiment three times at each site over a four-year period.

In total, about 1,260 new entries (735 six-rowed and 525 two-rowed) were tested in replicated yield trials
(Preliminary Yield Trial; PYT) for their first time. Intermediate (IYT), Advanced (AYT), and Varietal Yield
Trials (VYT) were grown at Fargo, Carrington, McVille, Minot, Osnabrock, Tioga (Nesson Valley), and
Williston, ND. The six- and two-rowed VYT and AYT also were grown at Sidney, MT. There were 67, 20,
and 10 six-rowed experimental lines in the Intermediate, Advanced, and Varietal Yield Trials, respectively.
There were 75, 20, and 8 two rowed experimental lines in the Intermediate, Advanced, and Varietal Yield
Trials, respectively. Plant growth was uniform throughout the growing season at all locations, but below
average precipitation following heading limited yields. Foliar diseases were minimal at all locations due to
the dry growing conditions. Fusarium head blight (FHB) was observed at our Osnabrock research site, likely
promoted by very heavy dues during grain fill and crop dry down.

Six-rowed Malting Barley Program:
In January 2012 the NDSU Variety Release Committee recommended to released ND22421, with the
condition being acceptable brewing data from the 2010 crop. Since we have limited commercial scale
brewing data, I recommended in June 2012 that we wait on releasing the line until we have brewing data
from the 2012 crop. We produced a small Foundation seed increase in 2012 and will have sufficient seed for
release if we get acceptable malting and brewing data from the 2012 production. Averaged across our 2012
yield trials across North Dakota, ND22421yielded similar to Tradition (Table 1). The advantage ND22421
has over currently grown cultivars is its shorter and very strong straw (Table 2). This feature should make it
especially suitable for growers producing barley under irrigation or wanting a cultivar that they can straight
combine.

Five lines will be submitted for AMBA Pilot Scale evaluation in 2012. The lines ND26891 and ND27177 are
in their second year of evaluation. Each was rated satisfactory in 2011. ND28554, ND28555, and ND28993
are each in their first year of Pilot Scale evaluation. All entries accumulate less DON than Tradition and
Stellar-ND and ND28993 is a semi-dwarf line with very strong straw strength during the growing season and
at harvest (see attached tables).

Two-rowed Malting Barley Program:
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                           Page 6

After several years of Pinnacle being the top yielding two-rowed barley cultivar across the state, Rawson and
Haxby surpassed it in yield. This is the first time Pinnacle has been subjected to late season drought and high
temperatures across the state. We will have to see if this response continues.

Cargill contracted the two-rowed line 2ND25276 for production on about 300 acres in North Dakota. Grain
from this production will be used for the first year of AMBA Plant Scale evaluation. Across all locations in
2012, 2ND25276 had a 5% yield advantage over Pinnacle. Since 2006, average yield of 2ND25276 and
Pinnacle have been similar. Advantages of 2ND25276 over Pinnacle include lower concentrations of malt
beta-glucan and DON accumulation (Table 3), and improved resistance to the spot form of net blotch.
2ND25276 could be a candidate for release in January 2014 if we receive favorable commercial scale
malting and brewing results from the 2012 crop. We produced Breeder seed of 2ND25276 in Casselton last
summer and we will plan on having a small Foundation seed increase during the 2013 growing season.
Four NDSU developed two-rowed lines will be submitted for their first year of AMBA Pilot Scale evaluation
in 2012. The lines are 2ND27705, 2ND28065, 2ND28071, and 2ND28131. Compared to Pinnacle, each of
these lines yield more, have lower concentrations of malt betaglucans, and lower DON accumulation.
Especially promising lines are 2ND28065 because of the 13% yield advantage it has over Pinnacle and
2ND28131 because it is a semi-dwarf line with lower DON accumulation than Conlon. The semi-dwarf
character of the line will make it suitable for production under irrigation and/or straight combining.


                                    Barley Disease and Their Control
                             Robert Brueggeman, North Dakota State University

Diseases are the most important factor limiting yield and quality of malting barley production in the upper
Midwestern United States. The primary goal of the Barley Pathology Program at North Dakota State
University is to conduct research focused on the development of effective and durable genetic resistance to
barley diseases important to the economics of production in the state and surrounding regions. To ensure that
diseases effecting yield and quality of malting barley do not influence the choice producers make to grow
barley we are developing economically feasible methods and practical tools for disease control.

We are achieving this goal by working with the NDSU barley-breeding program (six- and two-rowed) to
screen experimental germplasm against a range of economically important diseases in the Upper Midwest.
The Barley Pathology Program screens material for both seedling and adult plant resistance in the
greenhouse and field. Our efforts also involve screening germplasm collections for new sources of resistance
as well as developing mapping populations to characterize resistance and develop molecular markers for the
rapid deployment of disease resistance. Collections of pathogen populations are being made to monitor
virulence spectra to ensure that any race changes in the population that threaten the effectiveness of deployed
resistance are detected. In addition we are developing a website that forecasts the probability of FHB
epidemics to assist producers in predicting when to apply fungicides. We also engage in applied research to
develop integrated pest management practices that include cultural practices, fungicide application and
genetic resistance. These activities are conducted in close collaboration with other programs at NDSU and
with pathology, molecular genetics and breeding programs from other institutions.

In the one year funding period, we will evaluate current elite breeding material and advanced lines in the
NDSU breeding program for resistance to four of the most important barley diseases; spot blotch, net form
net blotch, spot form net blotch and Fusarium Head Blight. These screening efforts help ensure that only the
elite lines with adequate disease resistance will be advanced. Crosses will be made between lines containing
new sources of net form net blotch and spot form net blotch resistance and stacked with two sources of Ug99
resistance to develop lines with resistance to all three diseases. Bi-parental populations will be produced and
advanced for mapping and molecular marker development. The susceptible parents in all crosses will be elite
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                           Page 7

malting barley varieties to expedite the process of deploying these resistance sources into adapted
Midwestern malting varieties. The virulence and genetic variability of major pathogens will be monitored to
look for shifts that indicate the need for new resistance genes in barley cultivars. The recent epidemic of a
new virulent form of spot form net blotch on the six-rowed malting barley variety “Tradition” was reported
to cause unprecedented yield losses of 50-75% in eastern Montana during the 2011 growing season. This is
an excellent example of why pathogen population monitoring is important. Genotype by sequencing will be
utilized on large population of Pyrenophora teres f. teres and Pyrenophora teres f. maculata to determine
the genetic diversity and virulence factors present in the pathogen populations in North Dakota and Montana.
This information will help determine what resistance genes to deploy and susceptibility factors to eliminate
to ensure durable resistance against the net and spot forms of net blotch.

Our efforts to screen barley lines for the breeders to improve and/or maintain current levels of resistance
while making gains in yield and quality are essential for the malting barley industry. We will help ensure that
cultivars are released with effective and durable resistance.

The barley pathology program has sustained its previous level of disease screening efforts by evaluating
and/or setting up disease nurseries containing the NDSU breeding program’s two- and six-rowed materials.
Field nurseries were set up to evaluate for reaction to spot blotch and Fusarium head blight. We also
continued our greenhouse screening for spot blotch and net form net blotch. To meet the challenges of newly
emerging diseases last year we expanded our screening efforts to include greenhouse screening for spot form
net blotch resistance and this year plan to continue this screening and include screening for barley leaf rust.
In collaboration with the NDSU breeding program the phenomics data of multiple diseases will be utilized
by Dr. Rich Horsley’s Ph.D. student for association mapping.

Research funding provided by the Triticeae Coordinated Agriculture Project has allowed us to identify eight
new sources of SFNB resistance from the world barley collection effective against a diversity of pathogen
isolates from around the world. These sources of resistance have been crossed with multiple adapted malting
barley varieties (Tradition, Lacey, Celebration, AC Metcalfe, Conlon and Pinnacle) to develop bi-parental
populations that will be utilized to validate resistance QTL identified by our association mapping analysis
and possibly identify new QTL. The association mapping data will be utilized to develop molecular markers
at six significant resistance loci that will be used in marker assisted selection to develop elite pre-breeding
lines. The progeny from the SFNB resistant lines and elite lines will be screened by marker-assisted selection
to develop adequate SFNB resistance through a rapid back crossing strategy. We have also introgressed stem
rust race TTKSK (aka Ug99) resistance from two sources (rpg4/Rpg5 and RpgSw645) into Tradition, Conlon
and Pinnacle and are developing markers that will enable us to combine resistance to both pathogens in these
elite backgrounds.

We have collected >150 NFNB and SFNB isolates from several geographically distinct locations in North
Dakota and in collaboration with Dr. Timothy Friesen have collected isolates from Montana. These
collections have been put through two rounds of single spore isolation and we are currently in the process of
phenotyping them on a barley differential set. DNA has been isolated from the isolates and we have begun
the process of genotyping using a new genotyping by sequencing (GBS) technology that we have developed
using the Ion Torrent Personal Genomics Machine (PGM) next generation sequencing technology. We have
invested ~$100,000 into buying this piece of equipment in collaboration with three barley researchers (Rich
Horsley, Tim Friesen and Robert Brueggeman) and it will give us great capability in genotyping both barley
lines and the pathogens that threaten production.

CHALLENGES
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In order to identify markers associated with disease resistance loci and QTL it is important that we develop
bi-parental mapping populations and have the resources to genotype them. Robust genotyping has been the
bottleneck because the cheapest technology available (Illumina Bead Express) still exceeds $60 per progeny
line. To overcome this research bottleneck access to the newest genotyping technology is needed at NDSU.
We removed this obstacle by organizing a group of barley researchers from The Departments of Plant
Pathology, Plant Sciences and the Northern Crops Research Unit-USDA to contribute towards the purchase
of an Ion Torrent PGM sequencer. A barley genomics/molecular pathology lab has been set up at NDSU
with the capabilities to run the GBS analysis bringing the cost down to ~ $17/ barley line or $15/pathogen
isolate. However, the large data sets we are now capable of generating has created a new bioinformatics
challenge. The funding to hire a technician to run the PGM and also to perform the down stream data
analysis is the major challenge facing NDSU barley pathology program at NDSU in 2013.


       Incorporation and Characterization of Barley Loci Associated with Stem Rust Resistance
                                           Enhancement
                          Maricelis Acevedo, North Dakota State University

Wheat stem rust is an economically important disease of barley everywhere barley is grown. To date only
two highly effective P.graminis f. sp tritici (Rpg) resistance genes have been identified. Of those, only
one, the rpg4/Rpg5 locus provides resistance to the highly virulent stem rust races of the TTKS (Ug99)
lineage (Steffenson et al 2009). Despite those resistance being effective against current stem rust
pathogen races, it is alarming to think that the entire barley crop depends on two major resistances against
a pathogen that is highly variable and constantly evolving. Newer races ofthe pathogen such as QCCJ in
the 90's in the US and the emerging Ug99 lineage in Easter Mrica serve as a reminder of the vulnerability
of the crop. Recently, evaluation of a double haploid population of a cross between the barley lines
Q21861 (stem rust resistant) and SM89010 (stem rust seedling susceptible) identified a "hot-spot" for
genes associated with an enhancement of stem rust resistance in the barley chromosome 2H (Moscou et
al., 2011). An earlier study, utilizing a different mapping population had also identified a region associated
with stem rust resistance in a second location of this chromosome (Druka et al., 2008). Surprisingly this
genetic region seems to not been actively utilized in breeding programs in the past.

The objective of the proposed project is to incorporate these two genetic regions to expand the genetic
basis of the stem rust resistance response in adapted barley cultivars with superior agronomic, malting, and
brewing characteristics which already combine the Rpgl and the
7pg-I!Rpg5 resistances. By incorporation of this "resistance enhancer" we will be able to increase the
resistance to Ug99 as well as local races. To be able to efficiently select and pyramid these resistances in
breeding programs, highly reproducible genetic markers tightly associated with the new resistance
enhancer regions need to be identified. This step is extremely important for an efficient incorporation of
these genetic regions since their effect seems to only
be noticeable in the presence of other genes with a larger effect on resistance. This project fits with
AMBA' s mission and primary goals of developing improved varieties with enhanced, longer lasting and
broader disease resistance, specifically Ug99 resistance.

If one year of funding is allocated, we will start the initial crosses of SM to adapted lines of the cultivars
Conlon, Pinnacle and ND25160 that Dr. Robert Brueggeman has already crossed (BC3) to a source of the
7pg4;Rpg5 resistance. After this initial cross, three additional backcrosses will be done to the adapted
cultivars carrying the additional7pg4/Rpg5 under greenhouse conditions. Initial rust resistance evaluation
to Ug99 will be done in Kenya during the spring season of2013 after the second backcrosses to the
cultivars have been performed. After the third backcross has been performed, preliminary evaluation for
agronomic traits will be done in summer on 2013 in field plots in Fargo.
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 9


The major concern for the success of this project is being able to obtain good disease reaction data that can
be utilized for the mapping and development of genetic markers associated with the rust resistance
enhancement trait. However, we have utilized the nursery for other projects without major problems in the
past so it is likely that we can accomplish our objective. Additionally, we will need to be able to develop a
good way of quantifying and measuring small differences in disease reaction. Do to the previous
experiences on similar types of resistance in oats and wheat rust systems we envision a successful
outcome.

Objective 1) Incorporation and pyramiding of genetic regions associated with enhancement of stem
rust resistance into adapted varieties.
All parental lines being utilized to incorporate additional stem resistance QTL into adapted malting barley
lines (as described in the executive summary) have been planted and will be crossed during the month of
November. An earlier attempt to make crosses during the field season was unsuccessful do to plant stress
caused by drought and high temperatures. After the initial crosses are accomplished, two additional
backcrosses to the adapted lines will be necessary before disease evaluation.

Objective 2) Identification/development of genetic markers highly associated with enhancement of
stem rust resistance to Ug99 and other Puccinia graminis f. sp. tritici (stem rust pathogen).
To identify candidate genes associated with the stem rust resistance enhancement, a total of 18 sets of PCR
primers were developed based on barley unigenes localized to the 2H region. A NCBI BLAST search of the
barley genome assembly produced alignments enabling the identification of intron-exon junctions. Eighteen
sets of gene specific exon spanning primers were developed to screen the 2H loci in Q21861, Conlon,
Pinnacle and ND25160. We are currently screening all parental lines to detect polymorphism that may be
associated with the stem rust resistance enhancement.

Next steps and challenges:

Marker development If the current approach of using unigene sequences to develop markers associated
with the stem rust resistance doesn’t provide the expected results, additional marker development will be
performed using single nucleotide polymorphism (SNP) evaluation between Q21861, Conlon, Pinnacle
ND25160 and SM89010. Since the region we are trying to saturate with markers has been previously
delimited to barley bins 16-18 (Moscou et al., 2011) a relatively low-cost SYBR-green assay couple with a
high resolution melt curve analysis approach will be utilized to detect SNPs. The thermo-cycler and software
necessary to develop this assay are available in Dr. Acevedo’s laboratory.

Disease screening: To confirm the usefulness of the markers developed, the new barley populations and
lines will be phenotyped at the adult stage in the international stem rust nursery in Kenya. As an initial step,
seedling test can be performed at the U of Minnesota facilities during the winter of 2013 but there are space,
time and disease phenotype limitations that can only be overcome by field testing.
Additional funding as stated in the initial grant proposal will be necessary to accomplish the field screening
and finalize genetic marker development currently under way.


                                 Malting Barley Germplasm Development
                                Lynn Gallagher, University of California-Davis

The barley breeding program at UC Davis will develop both six-rowed and two-rowed germplasm which
combines multiple disease resistances and malting quality with good agronomic performance in the Western
Region. The developed germplasm will be distributed to both private and public institutions which express
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                           Page 10

an interest in evaluating germplasm for breeding purposes. Disease resistances for which selection will be
practiced include the following: primary foci are 1) cereal yellow dwarf virus (CYDV); 2) barley yellow
dwarf virus (BYDV), adding genes Ryd2 and Ryd3 together; 3) stem rust (Ug99); 4) barley stripe rust, and
secondarily, net blotch, scald, leaf rust, and powdery mildew. The variety Madre Selva (MSsel) will provide
some resistance for the first three of these diseases. A second source of resistance CI 11797 will be used as a
donor of Ryd3. The genes Ryd2 and Ryd3 act in an additive manner, conferring a higher level of resistance
to BYDV than either gene acting alone. It is doubtful that the primary objective of developing malting barley
varieties broadly adapted for production areas in North America can be achieved without incorporating these
resistances. Within one year I will create new advanced lines having various combinations of disease
resistances that will be ready for evaluation in the 2012-13 crop season (November –June).
 The UC Davis barley breeding program also strives to increase secondary uses of barley through the
improvement of both feed and food (hulless) barley varieties. The breeding pipeline is now full of advanced
two-rowed and six-rowed lines which will be evaluated in the field for agronomic appearance and freedom
from diseases.

The UC Davis malting barley improvement will focus mainly upon two-rowed germplasm development
which combines disease resistance and malting quality in a good agronomic background. The main foci are
the following resistances: 1) cereal yellow dwarf virus (CYDV), 2) barley yellow dwarf virus (BYDV), 3)
stem rust (Ug99), and 4) scald. Other diseases include stripe rust, net blotch, leaf rust and powdery mildew.
The resistance gene Ryd3 is being incorporated into malting germplasm as rapidly as possible to increase
resistance to BYDV.

Advanced lines from the mapping population Butta 12 X Madre Selva appear to have combined CYDV
resistance with malting quality in a good agronomic background based on USDA Malt Lab results of
October 2012. The CYDV disease resistance of Madre Selva has been combined with the malting quality of
Butta 12. The resistant lines will be used in April 2012 crossings. The mapping population is being exposed
to the R. padi aphid viruliferous for CYDV to confirm initial observations. Additional money is needed to
inventory UCD gemrplasm at the molecular level for the presence of genes conferring resistance to these two
viruses.

Secondary uses of barley include the development of six-rowed feed, food (hulless or naked grained), and
forage types. Five forage types will be grown in half-acre plots by a private company which is cooperating in
an agronomic evaluation in the 2012-13 growing seaon. UC Davis germplasm has been distributed to BARI.
CO; Mt Vernon, WA; APB, Arizona; KARC, Ethiopia, and Egypt plus ICARDA-Morocco.



    Malting Barleys for the Intermountain west: two- and six-rowed spring and winter cultivar and
                                      germplasm development
                                Gongshe Hu, USDA-ARS, Aberdeen, ID

Our project mission is to 1) develop superior widely adapted spring and winter two-rowed and six-rowed
malt barley cultivars, and 2) develop improved spring and winter barley germplasm for use by public and
private barley breeding programs. In addition to cultivar and germplasm development, our work also
provides valuable information to industry, private and public breeders, and barley producers via multi-
location testing of lines from other breeding programs. Therefore we assist in meeting the mission of AMBA
by the development of improved cultivars and germplasm, and assisting other breeding programs in their
effort to release improved cultivars.
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EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 11

We are specifically addressing the development of improved winter and spring malting cultivars for the
Intermountain West (IMW) areas of Idaho, Montana, Oregon, and Washington with the goal of releasing
lines which not only meet industry standards for malt quality, but also exceed existing cultivars in agronomic
performance, benefiting both the malting industry and the barley producer. Included in these agronomic
characteristics are improved yield, lodging resistance, and disease resistance for both spring and winter
cultivars. In addition, improved winter hardiness is a major objective, especially for the two-rowed winter
program. AMBA funding allows us to evaluate lines at multiple locations leading to the development of
cultivars with wide environmental stability for use across large growing areas.

Specific goals in 2012-13 for the winter malt effort were to propagate enough seeds for the field planting in
2012 for the plant-scale evaluation of 02Ab17271 for targeted plant-scale evaluation production in the fall of
2012. Seed supplies were limited and so another increase is underway for actual planting for plant-scale
evaluation in the fall of 2012. The general goal in one-year funding period is to advance breeding materials
in all developing stages for the 2-row spring and winter types.

In addition to the 02Ab17271, other highlights in the Aberdeen program include: 1) another 2-row spring
malting line of 2Ab04-X010084-27. This 2-rowed spring malting barley was approved for the 3rd year of
AMBA pilot-scale evaluation. The current data indicated that this line is similar to Harrington in malting
quality except that the beta-glucan is significantly lower and yield potential is higher; 2) Two winter lines in
the second year of AMBA pilot scale evaluation: 02Ab431 and 02Ab671, Those two 2-row winter malting
barley lines showed better yield potential than Charles; 3). A 6-row winter malting line of 93Ab669 was in
the first year of AMBA pilot testing. 93Ab669 has a great yield potential.

Challenges in Aberdeen program
Major challenge in our program is short of a helper. There was a full time helper who was working the seed
preparation and cleaning. The helper was supported by USDA-NIFA Barley for Rural Development
earmarked grant. The grant expired on August, 2012 and will not be renewed due to Congressional
elimination of earmarks. Our basic funding is not sufficient to support the position.

                            Development of Improved Malting Barley Varieties
                                   Tom Blake, Montana State University

Improving yield and reliability while retaining Harrington-like quality and flavor is key to restoring the
health of American malt barley production. Barley acreage declined from more than 12 million acres in 1986
to currently about 2 million acres. Three states, North Dakota, Idaho and Montana, account for about 80% of
US barley acreage. If barley is to regain its historical position, it must be more competitive with its
traditional rotation crops, winter and spring wheat. This will depend upon the deployment and utilization of
more competitive barley varieties, and aggressive marketing efforts by the US malting and brewing
industries.

Hockett successfully entered commercial production in Montana in 2010. Hockett displaced Harrington in
2011 as the second most widely grown malt barley variety in Montana. MT010158 is in its final year of
plant scale evaluation with the 2011 crop, and will likely enter commercial production following a large
Foundation Seed increase in 2012.

The most efficient, lowest cost method for increasing the rate of germplasm improvement is through
enhanced germplasm exchange among capable barley improvement programs. In 2007, the MSU barley
improvement program volunteered to perform replicated yield trials for the 8 sets of lines submitted to the
USDA Barley CAP program from North Dakota, Utah, Washington, Montana, Minnesota, USDA-Aberdeen
and Busch Ag. Resources. These 768 entries were subject to Illumina Barley OPA1 genotyping, and the
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 12

agronomic datasets from MSU and the genotype data sets produced by Dr. S. Chao at the USDA-NDSU
small grains genotyping laboratory were made available to the barley community in January, 2008. Now
data from field experiments spanning the US are available from 2007-2010 (www.triticeaetoolbox.org). This
remarkable program is currently leveraging the investments made in each program for the benefit of all. We
collaborate extensively with the Malteurop barley improvement program, and are again providing evaluation
of early generation selections and advanced breeding lines. Those lines that demonstrated excellence in 2011
are being advanced to multi-location and statewide trials in 2012.

Hockett successfully entered commercial production in Montana in 2010. Hockett was seeded to 125,000
acres in Montana in 2012, up from 60,000 acres in 2011. Improving yield and reliability while retaining
Harrington-like quality and flavor is key to restoring the health of American malt barley production. Barley
acreage rebounded in 2012 with 3.3 million seeded acres nationally. If barley is to regain its historical
position, it must be more competitive with its traditional rotation crops, winter and spring wheat. Oilseeds
and pulses are providing additional competition for acres across the Northern Plains.

The most efficient, lowest cost method for increasing the rate of germplasm improvement is through
enhanced germplasm exchange among capable barley improvement programs. In 2007, the MSU barley
improvement program volunteered to perform replicated yield trials for the 8 sets of lines submitted to the
USDA Barley CAP program from North Dakota, Utah, Washington, Montana, Minnesota, USDA-Aberdeen
and Busch Ag. Resources. During the lifespan of the USDA-supported Barley CAP project, we tested 3070
spring barley lines in replicated, dryland and irrigated field trials. These lines were completely genotyped
using the Illumina Golden Gate single nucleotide polymorphism detection system by Dr. S. Chao (USDA-
NDSU). These datasets are available at www.triticeastoolbox.org . Over the past 18 months we have
learned how to extract and distill the information contained in the data from these very large field
experiments. When we compare the results of the agronomic trials with those of the analyses provided by
the USDA-CCRU micromalting laboratory, we see that while gains can be made in field performance, the
gains to be made in malting quality are substantially larger. I predict that over the next five years, lines will
be advanced to pilot scale evaluation that will combine Metcalfe’s nearly ideal malt profile with more
reliable dryland performance. We will make malting barley a more competitive alternative to its rotation
crops.
The growth of the craft brewing industry is now being followed by the development of craft malting. Small
malting systems are now operating in Washington, Nevada, Texas and Massachusetts, and many more are
being built. It is my hope that these small malting companies that will attempt to utilize locally-grown barley
and specialty grains will provide a platform to evaluate and release malting barley varieties across much
more of barley’s traditional growing regions, and that we can reclaim many of the acres our favorite crop has
lost.


             Making the switch - accelerated development of two-row winter malting barley
                                   Pat Hayes, Oregon State University

How this project will help meet AMBA’s mission and primary objective:
Public breeding programs have an evolving and critical role in helping AMBA achieve its mission to provide
the malting and brewing industries with an abundant supply of high quality malting barley. One important
contribution, highlighted in the recent survey of research priorities for AMBA members, is the development
of winter malting barley varieties. Oregon State University has historically focused on the development of
winter and facultative six-rows by integrating QTL mapping, marker assisted selection, and fundamental
research in low temperature physiology, quantitative disease resistance, and malting quality. In the interest
of brevity, in the remainder of this proposal we will use “winter” to describe fall-sown barley, although the
thrust of our efforts is on facultative. Given the malting and brewing industries increased preference for two-
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                             Page 13

row, the OSU program is shifting gears. Making the switch from six-row to two-row will require accelerated
and efficient selection. Acceleration will be achieved via doubled haploid production and efficiency via
genomic selection. Together, these approaches constitute doubled haploid genomic selection (DHGS). The
DHGS program will leverage genotyping resources from the Triticeae Coordinated Agricultural Project (T-
CAP) and contribute to national and international efforts to develop a more complete understanding of the
genetics of barley growth, development, and malting quality. The research will address AMBA’s primary
objective – ensuring that barley is a competitive crop – by incorporating malting quality into high yielding
winter habit varieties that provide growers with a sustainable and economically viable cropping option.
Making the switch does not mean abandoning the 25 years of effort invested in winter six-row. There is
excellent germplasm in the pipeline that will be tested, advanced, and released. Accordingly, 2012 – 2013
will require simultaneous activity in two-row and six-row. Therefore, in addition to the recurring breeding
program budget request we are making an additional special request to cover the costs of doubled haploid
production and assessment.

Three major issues are addressed: improving (1) malting quality, (2) winter hardiness, and (3) agronomic
performance:

Malting quality - We continually make progress in meeting AMBA malting quality expectations for winter
six-row, as summarized in our 2011 AMBA Progress Report. For the past 17 years we focused exclusively
on six-row winter malting barley, based on the stated preferences of our research sponsors. However, shifting
interests and needs have changed this orientation to focus on winter two-row. This shift is in alignment with
international interests and will simplify some of the challenges associated with six-row: kernel sizing and
beta glucan.
Winter hardiness - Oregon AMBA Pilot entries, and prospective entries, have winter hardiness levels
comparable to the agronomic check (Eight-twelve). Our fundamental research efforts will allow us to do
better. We have identified a new winter hardiness QTL in winter six-row lines developed by Stephen
Baenziger at the University of Nebraska (Lincoln). We have embarked on an extensive association analysis
of low temperature tolerance in a sample of ~ 1,000 accessions from around the world, supported by the T-
CAP. These resources will allow us, via genomic selection, to rapidly and efficiently accumulate favorable
alleles for this complex trait.
Agronomic performance - Winter malting barley has impressive yield potential. In western states - where
water is a principal limiting factor - dryland winter barley captures prevailing winter precipitation and
irrigated winter barley uses less irrigation than irrigated spring barley. Winter barley is also harvested earlier
than spring barley, which brings temporal diversity to the farm and reduces risk of weather-related damage at
a single harvest date. Winter malting barley has potential for other regions of the country, including the
Upper Midwest. We now have an integrated program with Dr. Kevin Smith, University of Minnesota.

Goals and outputs - A malting barley breeding program has five principal components (1) parent selection;
(2) crossing; (3) assessment and selection of cross progeny in preliminary, advanced, and regional trials; (4)
assessment of selected lines in the AMBA pilot and plant scale programs; and (5) variety release. In our new
winter two-row initiative we will take a new approach to the first three steps via DHGS. Our goals are to
maximize efficiency and productivity of the entire process.

One-year and long-term goals - Our one-year goal is to achieve excellence in the five steps of a breeding
program, as enumerated in the preceding section. Details are presented in the Experimental Plan section of
this proposal. Our long-term goals are to (i) ensure a steady stream of AMBA pilot and plant scale
candidates and (ii) provide the AMBA barley breeding community with effective tools for molecular plant
improvement. Our experience with DHGS will provide a useful template for all AMBA scientists and we
envision providing doubled haploid production on a for-fee basis.
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                                 Page 14

2012 was the year the Oregon program switched from six-row to two-row. Based on industry interest and
directives, we just said “no” to lateral florets. We have not abandoned six-rows entirely – all advanced lines
in the program with agronomic merit are included in the TCAP GWAS panels. Optimistically, one or more
varieties may come from that pipeline. Nor have we abandoned winter barley. We are still all about winter
barley, specifically facultative barley. Facultative barley has maximum cold tolerance and it can be planted at
any time. If it is planted in the fall, it has the cold tolerance “wiring” to survive. If it is planted in the spring,
the cold tolerance circuit is never “turned on” and therefore there is no metabolic cost to the plant.

The switch to two-row would be inconceivable were it not for: accelerated generation time, high throughput
genotyping, top shelf phenotyping, and innovative staff. We are achieving accelerated generation time via
doubled haploid production: we are also offering our services to other breeding programs. Genotyping is
afforded by the TCAP project, which allows us to leverage genetic resources for applied breeding objectives.
We have established a pool of field equipment that allows us to effectively manage trials and process seed.
Our staff has conceived new breeding methods (doubled haploid genomic selection), implemented improved
field phenotyping and data handling procedures, and established doubled haploid laboratory and greenhouse
delivery systems.

Thanks to a diverse portfolio of support for winter two-row malting barley research (current total =
$119,040), we are a vigorous public sector breeding program. Additional funding, however, will be
necessary to sustain and expand our activities. We are deeply grateful to AMBA for long-term funding and
for the additional one-time support in 2012 for launching the doubled haploid lab. AMBA funding lays an
essential foundation that we build on with the support provided by Busch Agricultural Resources, Great
Western Malting, and the grower commissions of Oregon and Idaho.

       Russian Wheat Aphid-Resistant Malting Barley Germplasm Enhancement and Evaluation
                         Dolores Mornhinweg, USDA-ARS, Stillwater, OK

Whenever insect pests impact grain yield of barley, Hordeum vulgare (L.), they affect malting quality. There
are three yield limiting aphid pests of barley in the US, Russian wheat aphid, Diuraphis noxia (Kurdjumov),
RWA; greenbug, Schizaphis graminum (Rondani), GB; and bird cherry-oat aphid, Rhopalosiphum padi (L.),
BCOA. RWA is a pest in the malting barley production areas of the western US while BCOA is more
widespread and common to all barley production areas. The major cause of yield loss with RWA feeding is
head trapping which results in reduced fertility and a severe decrease in plumpness of surviving seed. Not
only is less grain available for malt, but the quality of that grain is greatly decreased. When RWAs feed on
susceptible plants, the new leaves do not unroll and aphids build up in high numbers inside the unrolled
leaves where they are protected from contact insecticides as well as natural parasites and predators, wind and
rain. In years of severe or early infestation, chemical control can only be accomplished with repeated
applications of systemic insecticides. These chemicals are not only expensive to the grower; they could quite
possibly end up in malt produced from treated fields. The solution to these problems is resistant varieties.
116 unadapted germplasm lines have been developed in previous years as a part of this project after
screening the entire NSGC. Two lines (STARS 9301B and STARS 9577B) were officially released to
breeders. Inheritance studies, also accomplished in previous years by this project, have given barley breeders
valuable information on how to best utilize these germplasm lines in their breeding programs.

Negative effects on yield and malting quality are often associated with the use of unadapted germplasm in a
breeding program. A pre-breeding program was initiated to bring resistant genes from unadapted lines into
good malting quality backgrounds adapted to all the barley growing areas of the US. Due to the common
occurrence in aphid populations of biotype change, after which the aphid can damage previously resistant
lines, all 116 resistant sources have been utilized in this pre-breeding program in hopes of producing
germplasm with genetic diversity for resistance which could protect barley from any future aphid biotype
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EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                          Page 15

change. A total of 60 adapted RWA resistant germplasm lines have been released from this program. Thirty-
six of these lines were in malting barley backgrounds (17 two-rowed and 19 six-rowed). Four feed barley
cultivars have also been released.

Since the introduction of RWA (RWA1) in 1986, 7 new biotypes (RWA2 – RWA8), have been identified
and more are suspected. All 43 adapted spring germplasm lines were screened for resistance to RWA1-
RWA5. Their resistance held. STARS 9301B and STARS 9577B have been reported to be resistant to
RWA1 - RWA8 as well. It is critical that we stay informed on future biotype changes and continue to test
released resistant lines/cultivars to these biotypes as soon as they are detected. All germplasm releases will
be screened to new biotypes as they are identified. A set of 94 unadapted and adapted germplasm lines from
this program are currently being screened to other world biotypes.

The second phase of the pre-breeding program is to determine the inheritance and genetic diversity of
resistance in released lines. Inheritance studies will give breeders information on how best to utilize these
germplasm lines in their breeding programs. The genetic populations necessary for inheritance studies have
been developed in previous years. Inheritance studies are labor intensive and time consuming limiting the
number of studies to 1 or 2 within an acceptable screening environment any given year. An inheritance study
on resistant source R017 was completed in 2011/2012. Out of the 16 inheritance studies completed so far in
the project, this is the first single gene resistance for RWA found in barley and is the resistant source in
STARS 0505B, a winter feed barley. STARS 0505B and its unadapted parent were the only 2 lines found to
have resistance to the virulent Iranian biotype. Inheritance studies will again be the focus of research for
2012/2013. We propose to complete genetic studies on as many germplasm lines as time and environment
will allow.

Cooperative projects to identify genetic markers and map genes for RWA resistance in STARS 9301B and
STARS 9577B have verified that genetic diversity for RWA resistance does exist in these 2 lines.
Cooperative projects to identify genetic markers and map genes for RWA resistance in 6 more RWA-
resistant lines are very near completion. In the future, marker analysis will be combined with traditional
multiple allelism tests to determine genetic diversity and genetic control of RWA resistance in released
germplasm.

BCOA most often considered a crop pest because of its ability to vector yield devastating BYDV, has been
reported to cause yield reduction of up to 50% from aphid feeding alone. BCOA is adapted to barley
production conditions worldwide and can damage winter barley in the fall as well as winter and spring barley
in the spring. Yield reductions are most often attributed to a reduction in seed number and seed weight and
have been associated with BCOA damaged plants’ inability to withstand environmental stress including cold
temperatures. BCOA feeding negatively impacts the root system as well as above ground growth and can
reduce winter hardiness of cereals. With the new emphasis on developing winter malting barleys, resistance
to BCOA could become more vital to malting barley production. Resistance to BCOA would be essential to
protect malting barley from feeding damage, reduce BYDV incidence by a reduction in phloem feeding, and
reduce the negative impact on winter hardiness. Application of established seedling screening techniques to
identify BCOA resistance has not been successful because seedlings under standard conditions are
asymptomatic. A screening technique has been developed by the USDA-ARS, Stillwater, in which
seedlings show a varied response (from live to dead) to BCOA feeding. After years of testing a 1-4 rating
scale has been defined and experiments conducted to validate both the technique and rating scale. Close to
900 lines were screened in the spring of 2012 to identify homozygous resistant lines which will be used in
2013 to validate the screening technique and rating scale. Once the screening technique is validated in the
greenhouse, screening of the NSGC will begin. A selection of 166 most recent barley cultivars was screened
in late summer of 2011 and is currently being screened again. Analysis is ongoing but preliminary results
indicate large vulnerability of US barley to BCOA. The ultimate validation, planned for future years, would
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 16

be evaluation of grain yield and yield components of identified resistant lines under field infestation of
BCOA.

GB is more often a pest of winter feed barleys in the Southern Plains. Although GB research is conducted on
this project with respect to barleys for feed and ethanol, as a rule it does not concern malting barleys.

We have successfully identified homozygous BCOA-resistant lines and are currently harvesting seed. This
seed will be used in an experiment in late spring 2013 to validate the screening technique and rating scale
developed from this project. Once the validation is complete we will be able to screen all barleys in the
NSGC as seedlings in the greenhouse to identify BCOA resistance. While spring infestations of BCOA can
reduce grain yield, fall infestations can greatly affect winter survival of small grains which would compound
the problem of reduced winterhardiness in future winter malting barleys. A preliminary screening of 166 of
the most recent barley cultivars identified some resistance in spring barley cultivars Burton and Ishi and
winter barley cultivars Tambar 500 and Charles.

A set of 74 barleys resistant to US biotypes of RWA are currently being screened to other world biotypes of
RWA by cooperators in France to ascertain the threat to US barley production by another introduction event
such as the one which occurred in 1986. This is part of an association mapping project and the genes for
resistance, if identified, will be mapped. We are continuing inheritance studies to provide breeders with
information which will allow them to best utilize released RWA-resistant germplasm.


          Utilizing Marker Assisted selection for Malting Barley Improvement in Washington
                              Kevin Murphy, Washington State University

The overall goal of this project is to identify molecular markers that can be used to supportand enhance
selection of malting quality traits in the malting barley breeding lines in the WSU two-row spring barley
breeding program. Due to the genetic complexity of malting quality traits, and the expense involved in
measuring malting quality parameters, the use of marker-assisted selection (MAS) is particularly valuable for
malting barley improvement. Using molecular markers together with phenotypic data will speed up the
overall breeding process from initial cross to eventual varietal release. Specifically, it will allow for more
efficient tests for the presence/absence of relevant traits; 2) evaluate a larger number of genotypes in earlier
generations; and, 3) enable selection during off-season cycles, thus streamlining the breeding process and
increasing the rate of genetic gain.

The specific objectives of this project are 1) to identify, optimize and validate the molecular markers that will
allow for rapid, large-scale identification and scoring of traits of importance to malting barley production and
quality improvement, and 2) to integrate several years of phenotypic data based on multi-location trials of a
diverse set of cultivars and breeding lines with existing genotypic data. Specifically, these traits include malt
extract, diastatic power, grain nitrogen, alpha amylase activity, grain protein content, β-glucanase activity
(kilned), β-glucanase activity (green malt), kernel plumpness, β-glucan (barley), β-glucan (malt), test weight,
kernel weight, soluble/total protein, fine coarse difference, extract viscosity and polyphenol oxidase activity.
Other traits of interest include pre-harvest sprouting tolerance, herbicide tolerance, fermentability prediction,
and analysis of -amylase, β-glucan, phenolic compounds, and/or phytates.

Within the one-year funding period of this project, we have identified 48 single nucleotide polymorphism
(SNP) loci as molecular markers to complement the field-based selection program which focuses on
evaluations of malting and food quality traits. These SNP loci or markers were based on the linkage map of
the Oregon Wolfe Barley Mapping Population, which has integrated hundreds of several kinds of molecular
markers associated with malting quality12. This first set of SNP markers will be used to genotype an initial
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 17

set of 200 malting and food barley breeding lines. However, since this set of SNP markers had been
identified from a linkage map generated from one mapping population, we need to know if these markers can
be used for marker-assisted selection on our malting barley breeding lines. We anticipate that after this
initial step, we will test and validate the robustness of these markers and use these to genotype 1920 breeding
lines. Markers can be added to the initial set of SNP markers in response to the marker validation tests. We
will also integrate several years of phenotypic data based on multi-location trials of a diverse set of cultivars
and breeding lines with genotypic data to really fine-tune the set of markers we can use for MAS of malting
barley.

Within three years, we anticipate the establishment of a full set of molecular markers for genomic selection
that will increase the speed and improve the overall quality of the malting barley lines in, and varieties
released by, the WSU barley breeding program. These goals reflect a reasonable, outcome-based plan and
should be attainable within the project timeline of one to three years.

Methodology and Results:
A set of 48 SNP loci/markers (Table 1) were identified using the Barley Integrated Map for malting quality
(Szucs et al. 2009). This set of markers will be used to genotype 200 malting and food barley breeding lines
(Table 2). In coordination with the USDA-ARS Western Regional Small Grains Genotyping Laboratory, the
sequence file containing the SNP for each SNP locus was examined. Using these sequence files, the pre-
amplification and extension oligonucleotides have been designed. The oligonucleotides will be ordered and
paid for with funds from the T-CAP (Triticeae Coordinated Agricultural Project, around $1,500). The 200
lines will be grown and DNA extracted from the seedlings. The cost of the reagents for the genotyping
assays will be covered with AMBA funds ($750).

Update:
Instead of 200 lines, we grew 192 lines to the seedling stage and extracted DNA from the leaves. We
reduced the number of lines to 192 for ease of handling, since the plates used for the machines have 96 wells.
This number of lines also worked better for the set of SNP markers we used, which was 48. The
oligonucleotides were ordered and paid for with funds from the T-CAP ($1,500). The genotyping assay was
conducted in the USDA-ARS Western Regional Small Grains Genotyping Laboratory, with the cost of the
reagents covered by the AMBA funds ($750). Using the Sequenom® iPlex GoldTM Assay technology, we
genotyped the 192 lines with the 48 SNP markers we have identified earlier (Szucs et al. 2009). At present,
we have 18,480 data points. This data will be analyzed in the next couple of months, specifically for genetic
similarity of the lines using principal component analysis and the software STRUCTURE, and correlations
will be made between the genotype and phenotypic traits related to malting.

Other Barley Research and Future Direction of Program
Use MAS to augment field based selection of lines to be developed and released as feed, food and malting
barley; coordinate efforts with T-CAP in developing markers for MAS

Once the validity of the 48 SNP markers for malting traits have been established, we plan to use these
markers on a larger set of barley lines and cultivars to conduct association mapping of malting traits.

Breeding barley for imidazolinone resistance by a) continuing to backcross a ‘Bob’ herbicide resistant
mutant field testing for resistance, yield, and other agronomic and quality traits and b) transferring the gene
responsible for herbicide resistance to other barley cultivars adapted to WA

Update:
We crossed the ‘Bob’ mutant (resistant to imidazolinone) to two malting varieties, Radiant and Conrad, in
order to transfer the herbicide resistance gene. We had 37 crosses between ‘Radiant’ and the ‘Bob’ mutant,
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                           Page 18

and we obtained 380 seeds. We also had 35 crosses between ‘Conrad’ and the ‘Bob’ mutant, and obtained
454 seeds from these crosses. All these F1 seeds have been sown in the greenhouse and the F2 seeds will be
harvested and screened for herbicide resistance. Molecular markers are also being screened on 13 barley
genotypes for polymorphism, and will be used to identify the chromosomal location of the herbicide
resistance gene in barley.


                          Study of the Malting Quality of New Barley Selections
                                    Stan Duke, University of Wisconsin

The primary objective of the Malting Quality Analysis project at Cereal Crops Research Unit in Madison is
the timely return of accurate malting quality data of barley submissions to state and federally funded barley
breeders. This partnership allows them to select and advance promising lines with the release of improved
malting barley varieties as the ultimate goal. Barley subsamples are received from cooperating breeders; they
are characterized, micro-malted, and the subsequent malts are analyzed for quality using several ASBC
methods. The data are tabulated and returned to our cooperators who can then assess the suitability of the
lines for development of commercial malting varieties. A second major goal is the efficient support of
AMBA’s testing program through barley characterization, malting, and analysis of Pilot Nursery, MVBN,
WRSBN, and WMBT samples. We aim to provide complete data on these samples, ahead of the AMBA
Spring TC Meeting. A final major goal is to support biochemical and genetic investigations on barley and
malt, by supplying raw materials (barley and malt) or facilities access to interested researchers. A tertiary
aim is the promotion of malting barley acreage through support to interested cooperative extension agents
and growers.

The current number of samples submitted for analysis from the 2011 crop year is 5250: 4809 breeders’ and
441special (AMBA nurseries / WMBT). As of March 30, 2012, we have malted nearly 3640 of these
submissions, and analyzed nearly 3100. We expect similar numbers for the upcoming crop year and intend to
generate the data for our stakeholders in the same type of timeframe as in recent years.
We averted negative impacts on the timely return of malting quality data due to Federal budgeting issues,
and a reduced staff since October 2011. With the support of AMBA, and a full QA staff in place from the
start, we anticipate the 2012CY will rival the record setting efficiency of the 2010CY.

         The Malting Quality Analysis program at Cereal Crops Research Unit attained its major goal, for the
2011 Crop Year, of the efficient return of accurate barley and malt quality data to interested parties. Final
tallies included characterization, malting and quality analysis of 5014 submissions from national barley
breeders, characterization of 49 AMBA drill strip barley samples, characterization/malting/analysis of 77
AMBA Pilot Nursery samples, and the same for 78 Western Regional Spring Barley Nursery (WRSBN) and
104 Mississippi Valley Regional Spring Barley Nursery (MVBN) submissions, respectively. A report on the
malting quality of the 2011 MVBN was written and posted to our website, and a report for the 2011 WRSBN
is near completion. We received the first year of samples of the Winter Malting Barley Trial (WMBT),
coordinated by Dr. Kevin Smith (U. of Minnesota), from 6 locations, and these 108 submissions were
malted, analyzed, and data returned. In addition, we analyzed the quality of 16 (including ours) 2011 AMBA
Plant-Pilot Collaborative malt samples (AC Metcalfe and Legacy), and returned data.
         The Malting Quality Analysis team is in much better shape, staffing-wise, than at this time, last year.
Our AMBA-supported, UW Research Specialist, Andy Standish, attended LECO FP528 Nitrogen
Determinator training in St. Joseph, Michigan, which he found quite instructive, and we now have both full
time QA technicians fully proficient in LECO maintenance and troubleshooting. In addition, USDA-ARS
CCRU Research Leader, Dr. Cynthia Henson, was able to convert the Federal position of our Assistant
Maltster, Jordon Geurts, to a year-round position, from seasonal; this has greatly solidified our ability to
maintain malting efficiency.
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                              Page 19

        One potential quandary is the looming Federal Sequestration. If enacted, its consequences could
affect our output. However, the Antaris FT-NIR might lessen such negative effects, as we’ve developed
excellent calibrations for %moisture and %protein in barley and malt. Presently, this is a valuable back up
for our reference Moisture (Lindberg Drying Oven) and Nitrogen (LECO Dumas Combustion) ASBC
methods, which are the “gold standards”. However, if in a true budgetary, labor bind, the use of FT-NIR
results for barley/malt moisture and protein data, could become viable, and help us continue. (Efforts toward
additional calibrations such as hull-less barley/malt %moisture and %protein, and kernel hardness, are
continuing, time-permitting).
        Barley breeder submissions from the 2012 Crop Year are arriving daily, and we’ve received around
1,400 to date. About 900 samples have been malted and quality data returned on nearly 400. We’re
confident in our ability to maintain the pace of malting, analysis, and quality data return of recent years,
baring any of the aforementioned potential Federal budgeting issues.


    Measurement of Hormones and Other Metabolites Associated with Dormancy and Pre-Harvest
                                    Sprouting in Barley
                          Paul Schwarz, North Dakota State University

Barley dormancy and pre-harvest sprouting (PHS) both can have significant impacts on malt and beer
quality. The ideal situation is for barley not to sprout in the field, but to lose its dormancy soon after harvest.
The result of barley improvement programs that have targeted increased levels of amylase enzymes, has been
some lines that show significantly decreased dormancy, but also a predisposition to PHS. Research efforts
on the genetics of dormancy have identified several quantitative trait loci (QTL) (Li et al 2003, Han et al
1996, Takeda 1996 and Ullrich et al. 2002). It is typically accepted that the hormone abscisic acid (ABA) is
strongly involved in the mechanism of dormancy and that the gibberellin hormones are involved in
germination of barley (King 1976, Koornneef et al. 1982, Karssen et al. 1983, Seo et al. 2006, Jacobsen et al.
2002, Finch-Savage and Leubner-Metzger 2006). However, the actual mechanisms that regulate seed
germination are still largely unknown (He et al 2011). Difficulties in connecting the genetic data to
metabolic data are due to a number of factors. First is the fact that, up to 15 different gibberellins (GA1, GA3,
GA4…GA69) have been reported in germinating or developing grains (Jacobsen et al. 2002). Second is that
the quantification of GAs has been difficult and extremely time-consuming. The measurement of the ABA
and GA hormones in barley grains has typically been by GC-MS (Frank et al 2010, Okamoto et al.
2009,Widodo et al. 2009). However, the GC-MS methods require a significant amount of initial purification
by HPLC, and these complex multi-step methods (Figures 1 and 2) are not amenable for the study of large
numbers of samples, at various states of physiological maturity/dormancy.

A relatively new alternative is to couple HPLC with quadrupole time-of-flight (QToF) MS. This type of
instrument and associated software a permits the analysis of much more complex mixtures (including
overlapping peaks), increases sensitivity, and provides more accurate mass analyses. Derivativization of
compounds is not required for HPLC, and sample purification may not be required given the resolving power
of the instrument. HPLC QToF MS has been recently used to profile hormones and to investigate dormancy
in Macadamia integrifolia (Fletcher and Mader, 2007).

The Wheat and Barley Cereal Chemistry group in the Department of Plant Sciences at NDSU recently
purchased an Agilent HPLC QToF MS (≈$500,000). To our knowledge, this is the only such instrument in
the USA that is devoted only to the study of cereal grains. We propose to apply this technology to the
measurement of hormones and other metabolites associated with dormancy and pre-harvest sprouting in
barley, as it fits very closely with Dr. Horsley’s work on the genetics of PHS. The technology, however, will
also be of great value in other physiological studies, research on FHB and mycotoxins, and investigations on
malt flavor.
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                             Page 20

A recent objective of the Cereal Chemistry group in the Department of Plant Sciences at NDSU
(http://www.ag.ndsu.edu/plantsciences/research/cereal) is to expand work into the area of metabolomics. An
Agilent HPLC Quadraplole Time of Flight Mass Spectrometer (QTOF MS) was purchased in late
2011($500,000) and installed in March 2013. This complements existing GC-MS capabilities. Focus will be
on small molecules (metabolites) associated with various metabolic states, disease stress, or processing
conditions. The GC-MS is best suited to the study of non-polar compounds, while the HPLC QTOF MS to
more polar metabolites. Initial studies on barley were proposed to be the measurement of hormones and
other metabolites (e.g. phenolics) associated with dormancy and pre-harvest sprouting in barley, as it fits
very closely with Dr. Horsley’ s work on the genetics of pre-harvest sprouting (PHS).

Work conducted in the spring and summer of 2012 involved the evaluation of GC-MS for the measurement
of gibberellins (GAs). Both diazomethane and trimethylsilyl diazomethane were evaluated as methylation
agents, under various reaction conditions. However, it was found that all conditions resulted in the
production of artifacts. More specifically we believe the conditions of methylation resulted in the formation
of tautomers and dimers. As such multiple peaks were observed for only GA3, greatly obscuring the
interpretation of results. This is especially important when multiple GAs are present, as differences in the
masses are minimal.

Work on the use of HPLC QTOF MS for the measurement of GA3 and various phenolics began in the Fall
of 2012. First efforts focused on the development of an extraction solvent system. Current efforts involve
fine-tuning of the elution gradient (water and acetonitrile acidified with 0.1% formic acid). It appears that a
linear gradient from 95% water to all acetonitrile gives the best peak resolution. To date we have detected
and positively identified GA3 as well as vanillic, protocatechic, and syringic acids. Limits of detection are
being investigated, and associated with this, is the need to use excised embryos vs. whole seeds. A method
for excising embryos has been developed.

Once all analytical conditions are established, the goal is to evaluate differences in the metabolites present in
samples of Robust and Stander at various stages following physiological maturity. Actual analysis time will
only involve a few weeks. However, the processing of data through the Mass Hunter and Mass Profiler
Professional software will require several months. Current Mass Databases include largely mammalian
metabolites, and few that are specific to plants.


Investigations on Barley Diseases and Their Control
Brian Steffenson, University of Minnesota

Plant diseases are one of the most important constraints to barley (Hordeum vulgare) production and quality
in the United States. Our Cereal Disease Resistance Project is part of the Minnesota Barley Improvement
team led by Dr. Kevin Smith that develops six-rowed malting barley cultivars for the Midwestern United
States and beyond. However, we also conduct disease evaluations for other barley breeders (both spring and
winter programs) across the country. The primary mission of the Cereal Disease Resistance Project is the
control of economically important barley diseases. For many diseases, this goal is best achieved through the
development of cultivars with genetic resistance. Thus, the long-term goal of this project is to develop the
knowledge base, resources, and germplasm for achieving durable disease resistance in malting barley
cultivars. In conjunction with this goal, it is also essential to conduct barley disease surveys and monitor
pathogen populations for new virulence types. Our specific objectives for 2012/13 are to: evaluate breeding
lines for resistance to important diseases; survey commercial barley fields for diseases; collect pathogen
isolates from barley cultivars and determine their virulence phenotype; increase, maintain, and distribute
pathogen stocks for testing barley germplasm for resistance; and cooperate with other investigators
conducting research on malting barley and barley diseases. Our research goals all directly address AMBA’s
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                            Page 21

primary objective of developing malting barley cultivars, broadly adapted for producers in North America,
with improved agronomic and quality characters--in this case, the important trait of disease resistance. In
turn, the deployment of disease resistant cultivars will help ensure that an adequate supply of high quality
malting barley is available to the malting and brewing industry—the primary purpose of AMBA. All of the
objectives listed above can be completed within a one year period. We anticipate no obstacles to fulfilling
these goals.

Our project works in association with Dr. Ruth Dill-Macky to ensure that all barley breeding lines and
cultivars grown in the region have sufficient disease resistance. The emphasis, of course, changes with the
current pathogen threats. For the past five years, our project has led the national and international effort to
identify and incorporate resistance to African stem rust in barley germplasm. We have taken full advantage
of our special BSL-3 Plant Pathology Containment Facility to screen materials at the seedling stage and the
USDA-ARS-funded nurseries in Njoro Kenya or AMBA-funded ones in South Africa to screen materials at
the adult plant stage. We have established genetic populations with African stem rust resistant materials and
are mapping the genes through support from a USDA-ARS Specific Coop Agreement secured with
assistance through Mike Davis. We are also emphasizing research on bacterial leaf streak, which has caused
significant losses over the past decade. This disease became more prominent with the widespread use of
fungicides, which adequately controlled the leaf spotting fungi, but left an open niche for the bacterium to
infect the crop. In addition to these two diseases, we routinely screen for resistance to the important diseases
of stem rust (domestic races), spot blotch, and net blotch. We evaluate materials primarily from the
Minnesota Barley Improvement program; however, we also assist the Busch Agricultural Research Inc.
program and Miller-Coors program in these evaluations. Our goal is to ensure all barley cultivars, regardless
of their program of origin, are resistant to the major diseases in the region.

A future emphasis in our program for 2012 will be to initiate crosses against a new virulent isolate of the spot
blotch resistance, which was discovered in North Dakota. This isolate is virulent on all of the malting barley
cultivars in the region and threatens the crop. Since the mid-1960s, we have been fortunate to have durable
resistance to this disease. This project will be done in association with Shaobin Zhong at NDSU.

I have been working closely with Kevin Smith on developing winter barley varieties for the region. This
project has great potential for producers because winter barley often gives higher yields and better quality
than spring types--and is more efficient in water and N usage. I am doing more than just the pathology work
in this project. I am utilizing the variation present in the Wild Barley Diversity Collection to breed barley
with greater low temperature tolerance. There is great promise in this material as some accessions from
Central Asia have survived 4 Minnesota winters.

I continue to conduct my annual barley surveys, collect isolates and store them for long term use. This
information is critical for discovering potential new disease outbreaks as we have had numerous times over
the past two decades. We have the largest collection of barley pathogens in the world and are a repository
for individuals who wish to use them for detecting disease resistance. Also, we are a source of resistant
germplasm for individuals interested in breeding for resistance.

We continue to be actively involved in large cooperative project (BCAP and TCAP) that can greatly enhance
our efforts to develop resistant barley cultivars for not only our region but nationally and internationally. I
love working on barley and have contributed a substantial amount of my endowment funds (about ~$20,000
per annum) toward research on the crop. However, it is getting increasingly more difficult for me to keep a
technician fully funded on barley.

                              Winter Hardy Barley Germplasm Development
                               P. Stephen Baenziger, University of Nebraska
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                           Page 22



The purpose of this research is to develop the most winterhardy winter barley germplasm in North America
and to make it available to the U.S. barley community. As winter barley is becoming more important for
malting, a second purpose is to increase the allele frequency of malting barley genes in winter barley. This
germplasm will be used by winter malting barley breeders through the U.S. to enhance the reliability of
winter malting barley production.

The key aspect of this research will be the use of testing sites in Nebraska which have previously been shown
to reliably select for winterhardy winter barley, wheat, and triticale. These locations are unique in that they
consistently stress winter barley so that winter tender lines are lost, but are not so severe that every line is
winterkilled. Winterhardiness sites for winter barley often are too mild for effective selection so that you
keep everything or too severe so that you often lose everything. What is critical are sites that can
differentiate lines that are winter tender from those that are winterhardy. For over 70 years, Nebraska has
developed these sites and successfully created germplasm that has been used and studied by those most
interested in developing winterhardy barley lines.

This research by its nature is long-term. However, for the upcoming year we expect to continue our breeding
effort to enhance our selection of lines with superior winterhardiness and malting quality. We will plant our
F2 bulks at Lincoln, NE (a forgiving site for winter survival) to remove very winter tender lines and then
plant the harvested F3 seed in bulks at Lincoln, NE and Mead, NE. We will grow our F6 and later
generations experimental lines at Mead, Lincoln, and Sidney, NE. Mead NE is the site where we select for
wheat winterhardiness and our hope will be to develop winter barley with winterhardiness similar to winter
wheat. By replicating the F3 nursery and our F6 and later generations at Lincoln, we will ensure that we
avoid total losses of breeding material except in the most severe years. Sidney, NE is our drought prone
location and allows us to select lines with drought tolerance and winter hardiness. We began this breeding
methodology with AMBA’s support in 2010, so it is currently too soon to know the success of this effort.
Our main limitation will be variable climate which may affect our selections. To enhance the frequency of
malting barley alleles, we will increase our number of crosses to winter and spring malting barley types with
an emphasis on 2-row malting barley parents.

In 2011-2012, we expanded our testing to include the Winter Malting Barley Trial (WMBT), in addition to
the Uniform Winter Barley Winterhardiness, Uniform Winter Barley Yield Trial, and our routine breeding
trials (elite nursery (BVT), preliminary nursery, and observation nursery). Both the WMBT and BVT were
submitted to the USDA-ARS Cereal Crops Research Laboratory for malting quality. In Nebraska, the barley
lines from Michael Goetz and those from Euromalt had better malting quality. The Nebraska winter barley
lines were mediocre to very poor which was to be expected as they have never been selected for malting
quality, but it was surprising to find even mediocre lines. These lines will be crossed to elite 2-row and 6-
row malting lines. P-845 has been recommended for release. The pedigree of P-845 is Krasnodar
K304/NE90701. It is a high yielding grain type that also has excellent forage potential and is adapted to
central KS and south. It will be marketed by Paramount Seed Farms who have the exclusive rights to market
tour lines globally. Based upon 2012 seed sales, winter barley seems to be making a comeback in the
southern Great Plains where forage and grain for dairy cows is becoming more important as dairies are
moving into this region due to cost savings and freedom to operate issues. Barley, with its water use
efficiency and early season maturity thus escaping heat stress, compared to winter wheat, is an ideal drought
tolerant crop for this region. It also adds crop diversity in the agroecological landscapes and choice for
producers.

                Identification of Barley Metabolites that Influence Malt and Beer Quality
                                  Jessica Prenni, Colorado State University
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                             Page 23

Our recent investigations of barley and malt metabolite variation demonstrate a novel relationship between
barley grain metabolite content and malting quality. Numerous barley metabolites have been identified that
exhibit significant correlation with various malting traits related to beer quality. Based on these results, we
hypothesize that (1) a panel of barley grain metabolites can be established to predict malting quality and (2)
that these correlations will extend to beer quality traits such as flavor, aroma and stability. Thus, the long
term goal of the proposed work is to establish a panel of metabolites predictive of both malting and beer
quality which can be incorporated in barley breeding process.

We will test our hypotheses in two Aims. First, metabolite profiles will be collected for barley grain from 36
2-row varieties grown in 2011 and another 36 2-row varieties grown in 2012. This data will be combined
with our previous study (36 2-row varieties grown in 2009) and will provide three independent years for
which malt quality and metabolite associations can be performed. From this analysis we will validate our
previous barley grain metabolite-malting quality associations and develop a robust statistical prediction
method. Secondly, correlation between barley metabolites and beer quality will be investigated by the
analysis of eight barley varieties which were selected based on divergence in their metabolite profiles as
observed in our previous work. Specifically, these eight select varieties will be grown, malted, and brewed,
and subsequent metabolite profiling, malting quality assessment, and sensory analyses will be performed.
The data will be interrogated to determine correlations between metabolite content and beer quality.

The overall expected outcomes of this work are: (1) the development of a validated statistical model of
barley metabolite content predictive of malting quality and (2) preliminary information on the correlation of
metabolite content and beer quality. These goals can be met within the 1 year funding time frame.

We anticipate no significant obstacles with the completion of the proposed work in Aim 1. Our selection of
the varieties to be analyzed in Aim 2 is based on their divergence in metabolite profiles and the assumption
that there is a correlation between barley metabolite content and beer quality. To our knowledge this has not
been tested and warrants investigation. Due to practical limitations involved with growing, malting, and
brewing we are limited to the investigation of 8 varieties, which is admittedly small replication for statistical
rigor. However, success of this aim would lay the groundwork and provide justification for future, larger
scale studies to validate our initial findings. Furthermore, while our study is limited to commercial and in-
trail AB varieties we believe that the genomic and malting quality diversity represented in our population
will allow for our model to be translated to other breeding populations. Future studies will be focused on
evaluation of our methods and statistical model by expanding our study to include a broader population of
non-AB varieties.

Our aims for grant funded in Fiscal Year 2012 are to (1) develop and validate a statistical model to predict
malting quality using barley and/or malt metabolite profiles and (2) evaluate the relationship between barley
metabolites and beer quality.

For Aim 1, we characterized 218 metabolites (out of 27,420 total molecular signatures) that correlated to at
least 1 of 20 quality traits. The chemical classes included amines/amino acids, lipids, alkaloids,
polyphenolics, purines, small peptides, and others (including many unknowns). The set of 218 metabolites
was used to predict quantitative values for each of the 20 traits using a regression model. The metabolite set
successfully estimated the rank of varieties for many traits (e.g. high vs. medium vs. low fine extract
content). Future plans include extracting metabolites from barley and malt samples (2011 and 2012 growing
season) to determine (i) which of the 218 metabolites follow similar trends in the 2011 and 2012 datasets and
(ii) evaluate data from one year to predict another. We will extract and run the 2011 and 2012 samples later
this year.
2012/2013 AMBA RESEARCH GRANT PROGRAM PROPOSALS
EXECUTIVE SUMMARIES & RESEARCHER UPDATES                                                         Page 24

For Aim 2, current efforts are to coordinate malting and brewing with metabolite detection and beer quality
measurements. For this study, select barley varieties will be malted and brewed, and samples will be set aside
for metabolite and quality analysis from both fresh and stored beer. A diverse set of barley varieties was
chosen to maximize variation in beer quality without requiring different malting and brewing parameters. We
will then characterize barley and/or malt metabolites that correlate with downstream beer quality metrics
using the same techniques as Aim 1.

				
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