10/25/2016

1

Doubled haploid technique in corn breeding

10/27/2016

Ursula Frei (Doubled Haploid Facility) 1218 Agronomy Hall 515-294-2756 ufrei@iastate.edu

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

10/25/2016

2

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

10/25/2016

3

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

10/25/2016

4

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).

10/25/2016

5

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….

10/25/2016

6

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

10/25/2016

7

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