46th Illinois Corn Breeders School, Champaign, 1st of March 2010 1
Andrés Gordillo1,2 and Hartwig H. Geiger1
1University of Hohenheim
Institute of Plant Breeding, Seed Science, and Population Genetics
70593 Stuttgart, Germany
2AgReliant Genetics LLC, Lebanon, IN
Optimum Hybrid Maize Breeding Strategies Using Doubled Haploids
46th Illinois Corn Breeders School, Champaign, 1st of March 2010 2
Outline
• Introduction
• In vivo induction of maternal haploids
• DH-line based breeding schemes
• Software MBP for optimizing the allocation of breeding resources- Features- Selected results
• Summary and conclusions
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Applications of the DH technology
• Marker-trait association studies
• Marker-aided introgression of genes
• Genetic engineering
• Molecular cytogenetics
• Hybrid breeding
The DH technology has become anindispensable tool of modern maize research and breeding
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Advantages of DH lines in hybrid breeding
• Maximum genotypic variance in line-per-se and testcross trials
• High reproducibility of early-testing results
Increased selection gain
• Complete homozygosity from the very first generation
Perfect compliance with DUS criteria for variety protection, short “time to market”
Reduced nursery expenses, simplified logistics Facilitates marker-assisted selection and
backcrossing
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In vivo induction of maternal maize haploids
For review see Geiger (2009) in Handbook of Maize. Springer, New York
• Pollination of maize plants with specific genotypes called inducers, which leads to kernels with a haploid embryo and a regular triploid endosperm
• Widely used for line development in commercial hybrid maize breeding
• Increasingly used in research
• Only moderate influence of donor genotype and induction environment compared to in vitro haploid induction
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About 10%-15% of haploid kernels
InbredA
InbredB
X X
X
Inducer
XF1 Inducer
Colchicinetreatment
Haploid seedlings (n)
Doubled haploid plants (2n)
Selfing
Doubled haploid lines
Donor
In vivo haploid induction in
maize
About 8-12% of haploid kernels
Treatment with doubling agent
Selfing
Haploid seedlings (n)
Doubled haploid plants (n)
Doubled haploid lines
InducerF1
InducerDonor
Inbred A
Inbred B
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Haploid-identification markers (1)
Markers expressed before chromosome doubling
Dominant grain color marker gene R1-nj(in conjunction with mutant pigmentation genes A1 or A2, and C2)
Causes pigmentation in the aleurone (endosperm) and inthe scutellum (embryo tissue)
Needs donor with colorless seeds
Expression may be suppressed by inhibitor genes (e.g. C1-I)…carried by the female parent
• Dominant color marker genes expressed in the primary rootand coleoptile (e.g. Pl1 in conjunction with B1)
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Haploid-identification markers (2)
„Red crown“ marker R1-nj
Endosperm
Embryo
H embryo F1 embryoLethalOutcrossed or self-pollinated
Donor InducerDonor
(r1-nj r1-nj)Inducer
(R1-nj R1-nj)
(After Röber 1999)
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Outcrossed or self-pollinated
InducerDonor
X
„Red crown“ marker R1-nj (3)
H embryo F1 embryoPhoto F.K. Röber
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Chromosome doubling
• Immersion of seedlings in colchicine (Gayen et al. 1994,Deimling et al. 1997, Eder and Chalyk 2002)
70 – 80% of the seedlings survive 10 – 40% of the surviving plants produce selfed seed
• Due to high toxicity of colchicine, most breeding companies are interested in less hazardous substances
Herbicides, e.g. Pronamid, APM, Trifluralin, Oryzalin
Nitrous oxide gas (Kato 2002)
-> not suited for large-scale haploid induction
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Seedling ready for reducing the root and clipping the tip of the coleoptile
Photo F.K. Röber
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Transplanting colchicinised juvenile plants into the field
Photo F.K. Röber
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DH-line observation nursery
Photo W. Schmidt
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Season
Experimental hybrids
2nd-cycle breeding
F1 inducer
< H / DH >
× T
D2L • T
DHL × T’
D2L • T’
L×L’ L’’×L’’’…
L >
W
W
W
W
S
S
S
S
1
2
3
4
1
4
2
3
F1 inducer
< H / DH >
DHL per se
DHL× T
TC1
× T’
TC2
L×L’ L’’×L’’’…
< DH
W
W
W
W
S
S
S
S
Standard schemeYear
Experimental hybrids
2nd-cycle breeding
F1 inducer
< H / DH >
D2L • T
× T’
D2L • T’
L×L’ L’’×L’’’…
F1 inducer
< H / DH >
T × DHL
TC1
DHL× T’
TC2
Accelerated scheme
DHL per se
Two-stage line development (LD) schemes
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Integration of genome-wide selection (GS)
Year Season
1 W
1 S
2 W
2 S
3 W
3 S
F1 Inducer
H / DH
T × DHL
TC1DHL per se
Recombination
F1 Inducer
Recombination
F1 Inducer
4 W
4 S
GS
GS
PS+GS
GS
GS
DHL
H/DH
H/DH
< A × B >, < C × D >, …
Year Season
1 W
1 S
2 W
2 S
3 W
3 S
F1 Inducer
H / DH
T × DHL
TC1DHL per se
Recombination
F1 Inducer
Recombination
F1 Inducer
4 W
4 S
GS
GS
PS+GS
GS
GS
DHL
H/DH
H/DH
< A × B >, < C × D >, …
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1 W Recombination
1 S Induction
2 W H / DH
2 S DHL per se
3 W DHL T
3 S TC1
4 W Recombination
4 S Induction
5 W H / DH
5 S DHL per se
DHL T’
TC2
Top lines
Experim. hybrids
2nd cycle breeding
RS
cycl
e t
RS
cycl
e t+
1
N0
N1
NRS N2
n
Integrated recurrent selection (RS) and parent line development (LD)
Year Season
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• Maximizes the expected genetic gain per year for a given annual budget and a limited relative annual loss of genetic variance.
• Allows to optimize 1-, 2-, and 3-stage testcross selection procedures for alternative breeding schemes.
• Allows the user to specify the tester type (e.g. pure line, single cross, population) for each testcross selection stage separately.
• Accounts for detailed monetary costs of each individual breeding step.
MBP (Version 1.0)Software for optimizing Maize Breeding Plans based on DH lines
(Gordillo & Geiger 2008)
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MBP is applicable to :
• Line development (LD) in hybrid breeding
• Recurrent selection (RS)
• Integrated RS/LD approaches
RS is treated as an integral part of LD
• Interlinking successive staggered breeding cycles
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• Estimates of variance components and genetic correlation coefficients
• Haploid induction parameters
• Costs of the individual breeding steps
All variables are based on data from collaboratingbreeding companies and can be varied by the useraccording to his genetic, technical, and financialresources.
MBP: Quantitative genetic and operational input variables
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MBP: Gain criterion
The gain criterion is the expected genetic gain in
GCA for an index composed of the testcross
performance for grain yield and dry matter content.
Arbitrary index weights may be chosen by the user.
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Selected MBP results: 1. General program specifications
• Annual budget: US $ 750,000
• Proportion of lines pre-selected for per se performance: 50%
• Single-cross tester(s) at all selection stages
• Yield trials: multiple locations, unreplicated
• Three finally selected lines per LD cycle (NLD = 3)
• Annual loss of genetic diversity restricted to 2%
• Gain criterion:
I = Grain yield (Qx/Ha) + 2.5 Dry matter content (%)
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MBP results: 2. Standard vs. accelerated two-stage LD scheme
a N, T, L = Number of DH-lines, testers, and locations, respectivelyIndices refer to test stages 1 and 2
Note: In the Accelerated Scheme, the per se evaluation of DH lines is not before but in parallel to the TC1
7146 vs. 5680 lines are evaluated per se in the standard vs. accelerated scheme, respectively
High-input, short-cycle breeding procedures maximize the genetic gain per year.
Scheme Optimum allocationa Genetic gain for yield (kg ha-1)
NLD N1 N2 T1 T2 L1 L2 per cycle per yearStandard(4 ys) 3 3573 57 1 7 5 20 812 203
Acceler.(3 ys) 3 5680 79 1 6 3 19 786 262
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MBP results: 3. Standard vs. accelerated 1- and 2-stage RS
Annual loss of genetic variance restricted to 2%
The accelerated version is more efficient than the standard scheme.
One-stage RS is superior to two-stage RS.
In the most efficient RS scheme more than 50 DH lines need to berecombined to comply with the loss-of-genetic-variance restriction.
Scheme Optimum allocation Genetic gain for yield (kg ha-1)
NRS N1 N2 T1 T2 L1 L2 per cycle per year1-stage RS:
Stand. (3 years) 43 2481 - 2 - 6 - 504 168Acceler. (2 years) 57 3900 - 1 - 8 - 442 210
2-stage RS:
Stand. (4 years) 32 3821 225 1 3 4 12 624 156Acceler. (3 years) 39 5574 274 1 3 3 12 576 192
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MBP results: 4. Integrated RS/LD standard breeding scheme; influence of the weights for RS and LD
• One-stage testcrossing in RS• Two-stage testcrossing in LD• Annual loss of genetic variance restricted to 2%
Weights given to RS and LD, respectively, considerably influence the optimum allocation but hardly affect the maximal genetic gain per year.
Genetic gain for yield (kg ha-1)
Optimum allocation per yearwRS : wLD NRS NLD N1 N2 T1 T2 L1 L2 GRS PLD
0 : 1 34 3 3573 57 1 7 5 20 143 204
0.5 : 0.5 32 3 3805 153 1 4 4 14 155 202
1 : 0 32 3 3831 225 1 3 4 12 156 199
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MBP results: 5. Influence of population size on the long-term progress in line development
0
1000
2000
3000
4000
5000
6000
0 1 2 3 4 5 6 7 8 9 10
Nrec = 10 (Δσ2g = 7.1%)
Nrec = 15 (Δσ2g = 4.5%)
Nrec = 30 (Δσ2g = 2.1%)
Cum
mul
. sel
ectio
n ga
in fo
r yie
ld [k
g ha
-1]
No. of selection cycles
No. of hybrid parent lines selected per cycle:
NLD = 3
Number of recombined lines
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Relative importance of recurrent selection and parent line development
RU
H / DH
D1LTC1
RU
H / DH
D1LTC1
RU
H / DH
D1LTC1
RU
H / DHTC2
TC2
TC2
1 RS cycle = 3 yrs
1 PLD cycle = 4 yrsn
n
n
RU
H / DH
D1LTC1
RU
H / DH
D1LTC1
RU
H / DH
D1LTC1
RU
H / DHTC2
TC2
TC2
1 RS cycle = 3 yrs
1 PLD cycle = 4 yrsn
n
n
Perf
orm
ance
Time
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Summary and Conclusions (1)
• In vivo techniques of haploid induction have becomestandard tools in maize breeding and research.
• Major advantages of DH lines in hybrid breedingincludemaximum genetic variance from the very first
generationperfect compliance with DUS criteriashort time to marketsimplified logistics reduced expenses for selfing and maintenance
breeding.
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Summary and Conclusions (2)
• Genome-wide selection can effectively be integrated in DH-line based breeding schemes.
• A software package “MBP (version.1.0)” has beendeveloped to optimize the allocation of breedingresources and to determine the relative merits ofalternative breeding schemes.
• High-input, short-cycle, breeding schemes are expected to provide maximal annual genetic gain.
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Summary and Conclusions (3)
• The long-term genetic gain in LD builds up on thecumulative genetic gain from RS.
It is advisable to weight RS higher than LD whenoptimizing combined RS/LD breeding schemes
• Increasing the weight for RS leads to a considerableincrease in the gain from RS, while it hardly affects thegain in LD.
• Sizable numbers of selected DH-lines need to be recombined to preserve enough genetic diversity for subsequent breeding cycles, especially in case of short-cycle (accelerated) schemes!
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Acknowledgements
• German Bundesministerium für Wirtschaft und Arbeit BMWA (AiF grant No. 13991)
• Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V. (GFP)
• Südwestdeutsche Saatzucht GmbH & Co. KG (SWS)
• Monsanto Agrar Deutschland GmbH
• KWS SAAT AG
• F. K. Röber (SWS)
• E. Holzhausen (Monsanto)
• M. Ouzunova and W. Schmidt (KWS)
• G. Seitz (AgReliant)
• S. Koch (University of Hohenheim)