Need for a Public Doubled

Haploid Facility ?

Thomas Lübberstedt, Candy Gardner, Mike Blanco,

Uschi Frei, Elisabeth Bovenmyer
 Structure

- DH Production in Maize: How ?

- Why (Doubled) Haploids ?

- Who uses DHs ?

- Need for a Public DH Facility ?

 Technologies
1) Haploid production
- In vitro versus in vivo
- Maternal versus Paternal

2) Haploid identification
- Markers: Morphological or molecular
- Cytological / flowcytometry

3) Genome doubling
- Colchicine
- Other
In vitro: Anther Culture
 In vivo Haploid Induction
F1

X X
+ +
Maternal Paternal
Inducer Inducer (ig)
X

, , , , , ,
F2
 Workflow & Bottlenecks Maternal DH Induction
Step Challenge

Induction cross - Inducer availability

- Induction rate
Haploid (embryo) selection - Visual scoring
- Background effects
- Inducer DNA introgression ?
Genome doubling - Toxic Colchicine
- Alternative procedures
- Background effects
Selfing of double haploids - Chimera -> partial fertility
Identification of Maternal Haploids
Donor Inducer
Endosperm

Embryo

Outcrossed or Lethal H embryo F1 embryo

self-pollinated

Geiger 2009
 Comparison DH Approaches in Maize
Approach Strength Weakness

In vitro - No need of inducer - Low induction rate

- Genotype dependency
- Need of tissue culture

In vivo – paternal - Simple inheritance - Low induction rate (2%)

- cms conversion

In vivo – maternal -Limited genotype - Background effects

dependency -Complex inheritance
- Induction rate (10%)
Why (Doubled) Haploids ?
 Speed in Line Development

Founder line 1 x Founder line 2

F1
Selfing DH

Inbred line

Inbred line
Haploid Induction & Doubling of
Allelic Diversity Materials

Andrew Smelser et al., Plant Introduction Station, Ames

 QTL MAPPING
Type of Strength Weakness
Population
F2:3 - Speed of production - Heterogeneous families
- d and a estimates

RIL - Homogeneous families - Slow production

- Power of QTL detection

DH - Speed of producing - Laborious production process

homogeneous families - Lower recombination (>RIL)
- Power of QTL detection
BC - Speed of production - Heterogeneous families
- a and d confounded
 Trait / Gene Stacking
Line 1 Line 2

X Probability for Fixation of Target Genes

No. of genes F2 DH
1 0.25 0.5
2 0.0625 0.25
4 0.004 0.0625
8 0.00002 0.004
16 0.00000000002 0.00002
or

Selfing (F2) DH Induction

Goal: Fixation of target alleles

Usefulness in Line Development

Seitz 2005
Rapid Line & Population Improvement

Gordillo & Geiger 2008

 Conclusions
- Homozygous lines are obtained in short time

- Maximal genetic variation among lines: increased selection gain

- Reduces population size for gene pyramiding substantially

- Increases power for QTL or QTP detection

- Increases labor for line development – depending on technology

Who uses DHs ?
 DH Application in Plant Breeding
Maize: All major breeding companies (in vivo DH induction)

Other species (Tuvesson et al.): Rye, Oat, Triticale, Potato, Cabbage

Tuvesson et al. 2007, EU-COST

Need for Public DH Facility ?
 Pro and Contra Public DH Facility (Maize)
Contra:
- Inducers are available in maize
- Procedures are published
-> Everyone can do it

Pro:
- Users might need DH materials only once or once in a while
- Induction procedure requires know how (analogy to transformation)

- Technology advance might make procedure more accessible

- Possible platform for expansion into other species
=> Research opportunities in maize and other species

And: Training of future plant breeders !! Technology & Application of DHs

 Technological Challenges
Questions relating to inducer:

- Alternative/improved marker systems

- Increased induction rate
- Adaptation to US climate / photoperiod
- Genetics / biology of induction mechanism
Colored Seed
Partial Questions relating to genome doubling:
Fertile
- Genetic background effect ?
- Optimized procedure (with colchicine)
- Procedure without colchicine ?
- Increased doubling / success rate

Smelser et al. 2009

 Conclusion
Successful example of research oriented service facility at ISU:

http://www.agron.iastate.edu/ptf/index.aspx

New DH facility at ISU will soon be launched, if interested contact:

Thomas Lübberstedt
515-294-5356
THOMASL@IASTATE.EDU

Collaborators:
Candy Gardner, Mike Blanco, Uschi Frei
 Costs ?
Service On Campus Fee Off Campus Fee

a) Haploid Seed Induction

a) Selected Haploid Seed
a) Doubled Haploid Seed Production
(5-20 kernels)

a) Doubled Haploid Line (200 kernels)

- Summer / Winter pricing

- GM seed ?