How Does the MSU Barley Breeding Project Work

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How Does the MSU Barley Breeding Project Work?
We make 100 new crosses a year Using the plant
growth center, we're able to produce 200
recombinant inbred lines from each These 20,000
headrows are planted at Bozeman 2000 are
advanced to the next (F6) generation Of those
2000, 250 move on to yield trials at Bozeman and
Huntley Of those 250, 20 move on to statewide
multiyear yield trials Of those 20, perhaps one
will become a new variety So, of 20,000 lines
evaluated each year, 1 variety may result
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Genetic Analysis-Assisted Barley Breeding in
Montana

• Population size Standard Nursery is 64 entries,
  3 reps, lattice, 11 locations (6 dry, 5
  irrigated)
• Our largest standard nursery is 256 entries,
  planted at two locations
• Nonreplicated 3000 double rows
• 20,000 short headrows

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How Long Does it Take?
Shortest time from cross to release gtValier, 8
years Longest time from cross to release
gtChinook, 18 years Reason for difference in
time With Valier, we used modern genetic
evaluation tools, with Chinook we didnt, and the
malting industry demanded 6 years of evaluation
for a malting recommendation.
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Is an enlightened malting industry any more
likely to promote a new variety?
The malting industry must provide products the
brewing industry wants. The brewing industry,
(A-B, Miller, Coors), dictates price to the
malting industry and demands consistency above
all else. The malting industry is moving West
thanks to Fusarium head blight (scab).
International Malting Company, Great Falls, 12
million bu/yr.
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The Niche of the Local Plant Breeder
All Adaptation is Local. We can identify locally
adapted varieties. We do all phases of the
process. We can easily adjust objectives. We can
develop or reject strategic alliances, without
concern for shareholders.
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Have we been Successful?
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Serving Specialty Markets
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Hay Barley- Another Alternative
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Premium Feed Barley A Whole New Market Class
Tom Blake and Jan Bowman College of
Agriculture Montana State University
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Barley Why Bother?
Beef production is moving North Feedgrain
supplies are low Montana produces around 1
million calves per year Improving feedlot
efficiency could have tremendous impact on
Montana's economy

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Agricultural Cash Receipts, Montana 2000
1,806,371,000
Montana Agri. Statistics, 2001
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Cow-Calf Production System
Finishing cattle 70,000 head
Feeder calves 1,100,000 head
Total 1,531,000 head of cattle
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Primary Production Areas of Cattle
Feedstuffs
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Montana Barley , 2001
Other feed varieties
Harrington (45.5)
Westford ( 2.8)
Malt(57)
Gallatin (3.1)
Feed (43)
Baronesse (5.2)
B1202 (5.5)
Moravian 37 (3.7)
Haybet (10.2)
Other malt
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Feed Barley for Rangeland Cattle The Project
Supported by CSREES, a Division of USDA
Resources 2 Research feedlots- 16 pens per lot,
5 calves per pen fed from weaning to finishing on
experimental diets 10 cannulated calves for
digestibility trials (nylon bag, in situ) 20
single calf pens for feeding behavior studies 11
Research site barley variety testing system A
State-of-the-art barley genetics program
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Our Experience, Corn vs. Barley Varieties
In our experience, corn shows about 7-8
advantage over most barley varieties in ADG
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Variation for Feedlot Quality in Agronomically
Meaningful Germplasm
Average Daily Gain (Kg) Bozeman feedlot vs.
Havre feedlot. Twenty calves per variety at each
feedlot, feeding period 110 days Variety effect
is significant (Morex gt Baronesse, Lewis gt
Steptoe) Feedlot effect is significant (Bozeman gt
Havre)
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Trait Correlations for Feedlot Trials, 20
calves/treatment, 56 treatments, 21 cultivars.
ADG vs ISDMD R2 -0.36, P 0.007
ADG vs NEm R2 0.32, P .03 (Same for NEg)
ADG vs DMI R2 0.48, P 0.0003
ADG vs Starch intake R2 0.53, P 0.0006
ADG vs DMD R2 -0.33, P 0.07
DMI vs Grain starch R2 -0.29, P 0.04
DMI vs Particle size R2 -0.31, P 0.04
DMI vs NEm R2 -0.71, P 0.0001
(Same for NEg)
Starch intake vs Grain ADF R2 -0.45, P
0.008
Starch intake vs NEm R2 -0.57, P
0.0002 (Same for NEg)
DMD vs ISDMD R2 0.52, P 0.002
DMD vs Particle size R2 -0.68, P 0.0001
Grain NEm vs ISDMD R2 -0.59, P 0.0001
Grain NEg vs ISDMD R2 -0.60, P 0.0001
Grain NEm vs grain starch R2 0.34, P
0.02
Grain NEg vs grain starch R2 0.37, P
0.01
Grain starch vs Grain ADF R2 -0.48, P
0.0003
Data sources Dan Hinman, U. Idaho, Jan Bowman,
MSU/MAES
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Prediction equations derived from the Montana and
Idaho decade of feedlot trials
ADG -0.256 1.086(NEm) - 0.029(Grain ADF) -
0.011(Grain starch) -
0.000059(Particle size) R2
0.75, P 0.0001
FE gain/feed -10.27 10.60(NEm) 0.0195(Grain
starch) 0.0067(ISDMD)
R2 0.66, P 0.0001
NEm 2.554 0.0062(Grain starch) - 0.012(ISDMD)
R2 0.42, P 0.0001
20
Average Daily Gain Predictions from previous
slide ADG equation, based on grain composition
and DMD
PS
DMD
Starch
ADG
NE
(Kg)
m
2.03
47.9
54.6
2.84
Valier
1083
Lewis
1081
48.6
54.0
2.56
1.70
1.40
Baronesse
1073
52.9
53.6
2.25
1.45
Chinook
1074
46.0
55.7
2.35
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The 1998-99 Valier Feedlot dataset- one location,
one year, 20 calves/treatment
Baronesse 3.57 lbs/day Lewis 3.41
lbs/day Valier 3.84 lbs/day
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Feed Barley Selling Grain or Putting Weight on
Calves?

• Using grain in-state reduces transportation costs
• Producing beef in-state reduces morbidity,
  mortality and encourages provenance accuracy
• The Foreign Worker initiative may make labor
  costs more reasonable

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Genetics Tools and Approaches Used By the MSU
Barley Improvement Project
Genetic Mapping RFLPs, STSs, AFLPs, -gt
SNPs Linkage Analysis -gt Quantitative Trait
Dissection Surprise 1 Most Traits are
Easy Surprise 2 More useful variation exists
than we had dreamed
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Finding Diversity in the World Collection
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1H
2H
5H
6H
7H
E1H8-221
E1H8-246
E1H5-098
E2H7-491
E1H1-242
0.0
0.0
0.0
0.0
0.0
HorB
15.3
E2H8-234
12.1
E1H1-277
22.0
KV19
E1H2-231
28.3
17.5
E1H1-405
32.4
HorC
cMWG682
37.0
28.4
ABG380
36.6
Bmac144b
EBmac684
45.3
31.2
E4H8-396
37.9
Bmag0135
49.2
E1H4-359
51.8
Hvbkasi
37.9
E4H8-299
40.6
E2H7-313
43.9
Bmag0914
53.9
E2H2-133
54.8
E1H1-308
52.4
E3H4-263
63.6
HorD
E1H3-127
64.5
57.6
ABC255
72.0
E2H2-127
E2H2-216
66.6
66.5
E2H4-338
E2H5-328
77.1
70.5
Bmac0154
cMWG699
68.3
81.2
E3H4-421
77.8
E3H2-115
79.3
Bmag0211
70.8
Vrs1
84.3
E4H8-496
87.8
E3H4-412
79.7
E1H3-197
88.2
Bmag347
74.2
E2H7-418
91.1
E1H4-139
92.6
nud
90.3
Bmag872
MWG503
75.9
100.2
E1H5-215
102.0
EBmac0602
E1H4-413
100.7
102.7
EBmac501
E3H4-130
78.0
109.9
ABG396
104.9
E3H4-236
102.3
Bmag718
E2H8-354
79.5
113.8
E3H4-426
115.6
E1H4-470
105.7
ABG10
E2H8-163
E4H8-383
121.5
80.4
117.1
E1H8-217
107.0
E4H8-337
128.6
E2H4-305
Bmac144a
84.6
121.7
Bmac0067
136.3
E1H1-330
108.6
E3H5-230
E1H3-113
89.0
139.6
E1H4-257
141.1
TB19-20
114.5
E3H4-318
E1H8-139
92.4
142.4
E1H8-213
128.0
Bmag0770
152.4
E3H4-198
98.3
HVM54
154.7
E1H1-291
137.0
E2H8-138
100.7
ABG613
Bmag500b
165.8
166.3
E1H1-233
146.9
E3H2-341
109.9
Bmag500a
173.8
E1H3-117
147.4
E2H4-186
176.1
E1H1-263
174.0
Bmac0316
185.3
ABC305
175.9
Bmag0900
190.6
E2H2-332
201.3
MWG514
210.6
E2H7-092
E1H5-355
220.8
221.7
Morphological Protein STS SSR AFLP
E1H8-272
233.2
E3H8-186
248.4
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DMD, one Major gene On Chr. 2 Source
Unutilized 6-rowed Hulless Barley lines From
W.Asia BC2 F2 pops Available 2005
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Chr 2 ppd1, Pleiotropy
Chr6 Main Effect, near Hvm74
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Chemical Composition of Major Feedstuff Grains
Crude protein Crude fiber
Starch Corn Dent 10.4
2.5 72.2
Opaque 12.6 3.2
ND Wheat Hard
14.2 2.3
63.4 Soft 11.7
2.1 67.2 Barley
13.3 6.3
64.6 Oats 12.8
12.2 41.2 Sorghum
12.4 2.7
70.8
NCR publications, 1982
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Insitu Dry Matter Digestibility of Major
Feedstuff Grains
of DMD
Digestion Time
Herera-Salaana, 1990
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If the Rumen Digestibility is too fast
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Standardization of Insitu DMD Method
Diet 79 up to 83 1-2 times/day
Adaptation Period 2 weeks
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DMD of Montana Adapted Feed Barley
Bowman,
personal
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Prediction of ADG
Predicted ADG
r 0.866 P lt 0.001
Observed ADG
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Predicted NEg for Valier/WC1064 F4 Families
NEg (Mcal/Kg)
NEg1.784 0.0067(Starch ) - 0.011( DMD)
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Predicted Feed Efficiency for Valier/WC1064 F4
Families
Feed efficiency
Feed efficiency -10.2710.6(NEm)0.0196(Starch
)0.0067(DMD)
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AFLP markers

• M-CAC P-GCT(FL1401SFL1415S)

• Genographer

• S

• M P

• FL1412S(445bp)

• FL1408B(310bp)

• B

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Sequence Data Analysis
Steptoe
AGAGAGGCTGATGACCCTGACCGGTGTGT
n
ATGGGTTCTGGAAGTACGTGAGCAACCTGGAGAGGCG
CGAGACTCGCCGTTACCTGGAGGATGTTCTACGCTCT
CAAGTACCG
T
ACCTGGTAGG
Morex
GAGAGGCTGATGACCCTGACCGGTGTGTATG
n
GGTTCTGGAAGTACGTGAGCAACCTGGAGAGGCGCGA
GACTCGCCGTTACCTGGAGATGTTCTACGCTCTCAAG
TACCG
C
AGCCTGGTAGGTT
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M 1 2 3 4 5 M 6 7 8 9 10 M11 12
13 14 15 M
410

316
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15
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Single and Multiple SNP Detection
ABC255, ABG65 and
ABC305 from right
To left, with the three
Multiplexed in the
Leftmost group.
Detection was done
On our ABI377.
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ABI 377 Sequencing Gel Image
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The Staden Package- Cambridge
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Multiple Alignments, Polymorphism Detection
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An Interesting Polymorphism
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A Common Transition Polymorphism
Transition
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Types percentage of polymorphisms
52.9
29.4
11.7
5.8
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Types and percentage of polymorphisms
62.1
31.0
6.8
0.0
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SNP Primer Identification

• Steptoe5GCTGATGACCCTGACCGGTGTGTATGGGTTCTGGAAGTAC
  GTGAGCAACCTGGAGAGGCGCGAGACTCGCCGTTACCTGGAGGATGTTAG
  CCTGGTAGG 3
• Morex5AGGCTGATGACCCTGACCGGTGTGTATGGGTTCTGGAAGTAC
  GTGAGCAACCTGGAGAGGCGCGAGACTCGCCGTTACCTGGAGGATGTCAG
  CCTGGTAGG 3

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Primer Annealing Specificity
5
3
5
3
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SNP Validation
Steptoe
TGTTCTACGCTCTCAAGTACCGTAGCCTGGTAGGTT ACAAGATGCGAGA
GTTCATGGCATCGGACCATCCAA
Morex
CGNCG
TGTTCTACGCTCTCAAGTACCGCAGCCTGGTAGGTT ACAAGATGCGAGA
GTTCATGGCGTCGGACCATCCAA
GCNGC
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Restriction Digestion Test
Steptoe
Morex
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Multiplexed Samples Gel
Heterozygote
ABC 255
ABG 65
ABC 305
Le, Kl, Lo, Br, St, Mx
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Peak Intensity
Homozygotes
Heterozygote
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Slide Arrayer
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Slide Scanner
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Three SNP detection slides 12 loci, simultaneous
primary and secondary Amplification. Loci 1,2
monomorphic Locus 3 fam and hex were fluors.
Filters used Distinguish Cy3 and Cy5. Locus 4
monomorphic among these genotypes. Locus 6
monomorphic Loci 7,8 informative. Locus 9
monomorphic Locus 10 informative Locus 11
failed Locus 12 monomorphic
steptoe
morex
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• The Objective
• A set of informative, mapped SNPs
• A robust, multiplexed, parallel technology
• Substantial reduction in mapping costs

Ch1 Ch2 Ch3 Ch4 Ch5 Ch6 Ch7
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How do we get there at a reasonable cost?

• Integrate polymorphism data within Graingenes
• Exchange Primers
• Exchange microarrays
• Get somebody to do a lot of sequencing and
• primer synthesis for us

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Microarray-based Genotyping
1 2 3 4 5 6 7
Steptoe Mona Katrina Alexis Karl Morex H.
spontaneum

• Markers
• ABC701
• ABC305
• ABG65
• ABC255
• ABC153
• ABG379
• 7. ABC160