10/25/2016 - iowa state university
TRANSCRIPT
10/25/2016
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Doubled haploid technique in corn breeding
10/27/2016
Ursula Frei (Doubled Haploid Facility) 1218 Agronomy Hall 515-294-2756 [email protected]
Introduction Production of Doubled Haploid Lines Induction of Haploids Chromosome Doubling and self fertilization Why (doubled) haploids? The Doubled Haploid Facility at ISU
Production of haploid and doubled haploid plants
Pollen of same species “ig” in maize irradiated pollen (sunflower) “bulbosum” technique in barley haploid inducer lines in maize Wide hybridization (wheat, potato)
In vitro In vivo
Anther culture Microspore culture Gynogenesis (onion, sugar beet)
paternal
maternal
corn plant (diploid)
pollen (haploid)
egg cells (haploid)
Seed with diploid embryo
Androgenesis: Anther and microspore culture
• successful regeneration of haploids first reported in Datura in 1964 and tobacco (1969)
• highly effective and applicable to various plant species (barley, wheat, maize, rice, triticale, rye, tobacco, rapeseed and other species in the Brassica)
• problem: high genotype dependency within species and recalcitrant agricultural species such as leguminous plants, woody plants
• microspores and immature pollen grains can revert from the gametophytic to the sporophytic pathway
• initiated through various biotic and abiotic factors (pretreatment of flowering donor plants, developmental stage of microspores/immature pollen at collection, media composition, temperature and light regime…)
• haploids plants either regenerate directly from microspores (direct embryogenesis) or from callus (organogenesis) – embryogenesis is preferred.
http://www.mgki.hu/
Maize anther culture
Barely Microspore culture (Guasmi et al 2013)
Gynogenesis
• culture of various parts of the un-pollinated flower: ovules, placenta attached ovules, ovaries or whole flower buds • although successful in several plant species, applied mainly in onion and sugar beet • genotype dependent and influenced by growing conditions of the donor plant • in theory several cells of the embryo sac could develop into a haploid plant (egg cell, synergids, antipodal cells and
non-fused polar nuclei) • Egg cells develop directly into haploid plantlets (haploid parthogenesis) without an intermediate callus phase
Allium cepa Fayos et al. 2015
Production of doubled haploid plants using wide crosses
• Wheat: pollination with pollen of maize + tissue culture step: embryo rescue
• Barley: pollination of Hordeum vulgare with wilde relative H. bulbosum – tissue culture step necessary • Potato: pollination of Solanum tuberosum with wilde relative S. phureja – no tissue culture step necessary
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Maternal induction of haploids in corn
x o o
+ donor inducer
haploids ( 6-10%) hybrids (90-94%)
1st generation
Induction and haploid selection
RWS/RWK-76
- early flowering - makes readily side shoots - good pollen shedder, over several days - induction rate: 6-10%
- not adapted to Mid West growing conditions - susceptible to wind - low germination rate - difficult maintenance of parental lines and F1
STOCK6: Coe, 1959 CAUHOI: Chinese high oil inducer FIGH1: Bordes et al. 1997 KEMS:Shatskaya et al., 1994 KMS: Korichnevy Marker Saratovsky MHI: Moldavian Haploid Inducer PHI-3: Rotarenco - Procera PK6: Barrett, 2008 RWS: Roeber et al.,2005 UH400: Chang & Coe 2009 W14: Lashernes & Beckert 1988 W23ig: NSL30060 with ig ZMS: Tyrnov 1984
CAUHOI KMS
PK6
ZMS
MHI
KEMS
W23ig
W14
RWS PHI-3
UH400
FIGH1
Stock 6
Haploid inducing lines in maize
Haploid inducing lines in maize
• reasonable high haploid induction rate • tassel traits: abundant pollen shed over a longer
period of time • (hand-pollinations: side-shoots with additional
later tassels, medium height, easy bendable/not-breaking tassel)
• (for isolations: plant height)
• agronomic traits: seed set upon self-pollination, self-induction rate, germination rate, lodging, disease resistance
• one or more selectable marker for haploid identification
Ford, R. 2000: THE AMERICAN BIOLOGY TEACHER, 62: 181-188
Selectable Marker R1-nj
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Haploid selection
Endosperm
Embryo
H embryo F1 embryo Lethal out crossed or self-pollinated
Donor Inducer
Haploid selection
Haploid selection Haploid selection
• additional phenotypical marker – (PI1) red roots
Rotarenco, 2010
Alternative marker in haploid selection
Alternative marker in haploid selection
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Discrimination between haploid and diploid seed with the VideometerLab 3 (Videometer A/S, Hørsholm, DK)
Alternatives in haploid selection
• short turn-over time between seasons • visual haploid selection based on R1-nj by
hand is labor intensive time-consuming, error-prone
• selection rate: 1000/h (higher when selecting directly form the cob)
• goal: automation of haploid selection
Haploid Hybrid
-100
-50
0
50
100
Ke
rne
l W
eig
ht
(mg
)
Haploid Hybrid
-2
-1
0
1
2
Ke
rne
l O
il (
%)
Percent Oil Kernel Weight
Alternatives in haploid selection
High-throughput single-kernel NIR analyzer
• Data collection takes ~6 seconds per kernel
• Microbalance collects individual seed weights , ±1 mg
• Collects NIR spectrum, 907-1689 nm at 1 nm intervals
• Seeds can be indexed in microtiter plates
Alternatives in haploid selection
• biochemical differences between haploids and hybrids
Haploid Class Distance
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Hyb
rid
Cla
ss D
ista
nce
0.00
0.05
0.10
0.15
0.20
P1-102 Haploids (× 103)
P1-102 Hybrids (× 103)
P1-103 Haploids (× 103)
P1-103 Hybrids (× 103)
P2-20 Haploids
P2-20 Hybrids
Roger et al. 2011
non-destructive NIRS
Doubled Haploid Line production in corn
x o o
+ donor inducer
haploids ( 6-10%) hybrids (90-94%)
1st generation
Induction and haploid selection
2nd generation
Doubling and self pollination
Chromosome Doubling in Haploid Maize
- chemical treatment of germinated seed or young seedling with substances that interfere with cell division
- Substances: colchicine, antimicrotubule herbicides (amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin)
- There are different application mehods for chrosomsome doubglin agents: submersion and injection
Spontaneous doubling
Doubling by chemical treatment
- rare event during the first cell divisions – or later in the development of the plant
- the later spontaneous doubling occurs, the larger the percentage of plant tissue that is haploid (chimera).
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Chromosome Doubling in Haploid Maize
https://www.youtube.com/watch?v=V2jOEuZjjrg
Submersion
Chromosome Doubling in Haploid Maize
Injection
~ ¼- ½ ”
Partially fertile tassel…
Preparing the ear for pollination… Collecting pollen from single anthers….
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Pollination…
“In 2010, Pioneer generated more corn inbred lines via doubled haploid technology than we had produced in the 80 year history of our breeding program.”
http://www.pioneer.com/pv_obj_cache/pv_obj_id_40764A34C70F20BB2E738A227E9A4380E9CE0100/filename/DoubledHaploids.pdf
WHY DOUBLED HAPLOIDS?
100% pure inbred lines in 2 generations compared to 6-7 generations of subsequent inbreeding
time
efficiency higher selection intensity compared to selection in still segregating families
Speed in Line Development
Founder line 1 x Founder line 2
F1 Selfing
Inbred line
DH
Inbred line
QTL MAPPING: Speed, Accuracy
Type of Population
Strength Weakness
F2:3
• Speed of production • d and a estimates
• Heterogeneous families
RIL • Homogeneous families • Power of QTL detection
• Slow production
DH • Speed of producing homogeneous families • Power of QTL detection
• Laborious production process • Lower recombination (>RIL)
BC • Speed of production • Heterogeneous families • a and d confounded
Germplasm Enhancement in Maize (GEM) Mike Blanco, Candy Gardner
Exotic Maize Populations
Recurrent elite parent: B47 or Z51
BC1
DH
Isogenic BC1-DH lines - GEM
http://www.public.iastate.edu/~usda-gem/
Trait / Gene Stacking
X
Line 1 Line 2
or
Selfing (F2) DH Induction Goal: Fixation of target alleles
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
Probability for Fixation of Target Genes
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Some numbers: Requested # of DH lines: DHL= 100 Induction Rate: IR=0.06 Germination rate of donor population: GRD=0.95 Pollination rate in nursery: PR=0.85 Average number of kernels/cob: K=100 Germination rate of haploids: GRH=0.95 Haploids surviving treatment: SV=0.8 Haploids established in the field: F=0.95 Haploids shedding pollen: P=0.15 Pollinated haploids with seed set: S=0.95 Success rate: SR=0.10
200 donor plants for induction crosses, 16700 kernels have to be selected
1000 haploids have to be germinated, treated and planted out in the field
Service
Research projects
Inducer Development: development of new inducer lines with better adaption to the growing conditions in the Mid West development of inducer lines for specialty crops like popcorn and sweet corn development of inducer with additional selectable marker genes Haploid selection: automated systems for haploid selection: NIRS fine mapping and gene isolation for genes involved in induction ability Chromosome doubling: the ability for spontaneous doubling – a heritable trait increase the efficiency of colchicine application for chromosome doubling
Training
during summer season we have groups visiting
plant breeder and scientists from all over the world join us for 2-3 weeks during summer to learn DH technology hands-on
internships for undergraduate students