1
Exploiting Wheat’s Distant Relatives Ian and Julie King
The Challenge: To produce high yielding superior wheat varieties that meet the
needs of an increasing global population – breeders need genetic variation to achieve this
The Challenge: To produce high yielding superior wheat varieties that meet the
needs of an increasing global population – breeders need genetic variation to achieve this
Relatively little genetic variation is available in modern wheat varieties
The Problem
The Challenge: To produce high yielding superior wheat varieties that meet the
needs of an increasing global population – breeders need genetic variation to achieve this
How do we overcome this?
Relatively little genetic variation is available in modern wheat varieties
The Problem
UK consortium to increase the gene pool of wheat
PILLAR 1
Landraces
PILLAR 2
Synthetics
PILLAR 3
Wild
relatives
PILLAR 4
Elite
PHENOTYPING
GENOTYPING
BBSRC FUNDED PLANT
BREEDERS
Nottingham
London
Ian Julie
University Park
Jubilee Campus
Kings Meadow Campus
Sutton Bonington Campus
University of Nottingham Malaysia Campus
University of Nottingham China Campus
X
Hybrid
Wheat Ancestral species/distant relatives
• Distant relatives provide a vast reservoir for most if not all agronomically important traits • Interspecific hybrids provide the starting point for introgressing genes into wheat from its distant relatives
(Sears 1981)
How does introgression occur? Via homoeologous recombination between the chromosomes of wheat and those of the distant relative at meiosis in the gametes The Ph1 locus has to be removed before homoeologous recombination can occur. This is achieved by crossing to a line in which the Ph1 locus has been deleted – ph1ph1
Screen lines cytologically and for disease resistatance
(Sears 1981)
1) The identification of introgressions is difficult and time consuming
Why has introgression not been used more widely?
2) Introgressions are frequently very large and carry deleterious genes
Once a large alien chromosome segment had been introgressed into wheat it was very difficult to reduce its size further by removal of the Ph1 locus - difficult to remove deleterious genes
ph1/ph1 ph1/ph1
Identify plants with overlapping alien chromosome segments, in
which the target gene lies within the overlap and make a hybrid.
In the presence of Ph1 the alien and wheat chromosome segments will not recombine at
meiosis. However, the alien chromosome segments that overlap will giving rise to progeny
with small alien chromosome segments that carry the target gene but lack deleterious genes.
Wheat
Alien
Ph1/Ph1
X
T T
(Sears 1955 and 1981)
2) Introgressions were frequently very large and carried deleterious genes
(Sears 1955 and 1981)
2) Introgressions were frequently very large and carried deleterious genes
Need Genetic Markers
(Sears 1981)
Need for high throughput techniques to identify and characterize introgressions
1. Transfer of an entire ancestral genome to wheat in overlapping segments
Germplasm Development Programme – Start date 2011
Triticum urartu
Rye
Aegilops speltoides
Thinopyrum bessarabicum
Thinopyrum elongatum Aegilops mutica
Distant relative
Wheat
Introgressed
segments
increasing
in size from
telomere down.
Introgressed
segments
increasing
in size from
telomere up.
Ideogram of an introgression series of a single wheat chromosome, were synteny has been maintained
Identify plants with overlapping alien chromosome segments, in
which the target gene lies within the overlap and make a hybrid.
In the presence of Ph1 the alien and wheat chromosome segments will not recombine at
meiosis. However, the alien chromosome segments that overlap will giving rise to progeny
with small alien chromosome segments that carry the target gene but lack deleterious genes.
Wheat
Alien
Ph1/Ph1
X
T T
• Acid soil tolerance
• Drought
• Salinity
• High lysine
• Winter hardiness
• Disease resistance
• Powdery mildew
• Stem rust
• Stripe rust
• Leaf rust
• Boron tolerance
• High pollen load
• Out crossing
Utilize rye for improving wheat production
Thinopyrum bessarabicum
Highly salt tolerant (Forster;
King)
Thinopyrum elongatum
Yield, biomass, photosynthetic capacity, salt tolerance
Aegilops speltoides
Disease and insect resistant
Aegilops umbellulata
Resistance to leaf rust (saved the US
wheat crop late 50s early 60s), bread
making quality – (Sears)
Triticum urartu
Wheat A genome donor
photosynthetic capacity
A
B
D
Wheat ph1/ph1
X
Wild relative (R)
ph1
X A
B
D
Wheat Ph1/Ph1
High throughput screening of 1000’s of BC1 and
subsequent backcross progeny to identify
recombinants
Wheat/ancestral introgression
- Recombinants
Isolation of homozygous introgressions Phenotyping platform
A
B
D
Wheat ph1/ph1
X
Wild relative (R)
ph1
X A
B
D
Wheat Ph1/Ph1
High throughput screening of 1000’s of BC1 and
subsequent backcross progeny to identify
recombinants
Wheat/ancestral introgression
- Recombinants
Isolation of homozygous introgressions
17,000 + crosses in under 3 years!
Phenotyping platform
Distant Relative Parent Seed Numbers
BC1 Seed BC2 Seed BC3 Seed
Aegilops speltoides 118 1509 6242
Secale cereale 222 234
Thinopyrum bessarabicum 504 2242
Triticum urartu 118 1317
Thinopyrum elongatum 1000 1828
Aegilops mutica 40 764
Thinopyrum intermedium 42
Thinopyrum ponticum 76
Triticum timopheevii 110 972
Aegilops caudata 7 42
Secale iranicum 3 42 515
Secale anatolicum 1
Secale vavilovii 2 27
Secale montanum 1 14
Triticum triaristata 3 66
Triticum macrochaetum 3 36
Aegilops comosa 1 25
Shrivelled grain culture – dry grains
Thynopyrum bessarabicum x Chinese Spring Mutant 84
Triticum urartu x Chinese Spring Mutant 84
Secale cereale x Chinese Spring Mutant 84
(Summer season 2011)
Shrivelled grain culture – dissection
Shrivelled grain culture – growing plants
Ancestral/wheat crossing – F1 hybrids
Backcrossing – BC1, BC2 – Recombinants
Colchicine treated F1 hybrids - Amphidiploids
Amphidiploid seed multiplication
Marker development , sterile culture of
embryo/grain
Gustafson Glasshouse
Gustafson Glasshouse MK2 Crossing all year round
KU37
Ttd140
Triticum urartu
Thinopyrum bessarabicum
Rye (Secale cereale)
Aegilops mutica
Th. elongatum
Ae. caudata
Th. intermedium
Th. ponticum
Ae. varabilis
Ae. speltoides
Ae. tauschii 232
Ae. tauschii 320
Ae. tauschii JIC2220007
Ae. tauschii Ent-392
Ae. tauschii Ent-414
Ae. tauschii Ent-336
Ae. tauschii Ent-088
Progenitors/relatives
Watkins 34
Watkins 141
Watkins 209
Watkins 292
Watkins 352
Watkins 468
Watkins 729
Watkins 126
Watkins 199
Chinese Spring
Landraces Avalon
Cadenza
Rialto
Savannah
Xi19
Alchemy
Robigus
Hereward
Paragon
Pavon 76
Eite Cultivars
Used Exome sequencing for SNP discovery
132K feature Nimblegen capture array based on wheat cDNAs
• Mean exome sequence coverage per variety = 48X
• ~100,000 SNPs between 10 elite cultivars
• ~290,000 SNPs between elite hexaploid cultivars and 9 landraces.
• ~650,000 SNPs between hexaploid wheat and wheat relatives including Rye, Thinopyrum sp., Aegilops sp. and
Triticum urartu.
SNP discovery results:
Chinese Spring (F)
Aegilops speltoides (M)
X
F1 BC1 BC2 X Paragon X Paragon
Recombination in Aegilops speltoides BC1s
Marker analysis of 22 BC1 recombinant plants –
Sacha Allen and Keith Edwards, Bristol
Introgressed Ae. speltoides segments (blue) in 5 chromosomes
Introgressed Ae. speltoides segments (blue) in 12
chromosomes
Size and position of introgressed segments will be characterised in detail.
KASP
BC1 Aegilops speltoides – 22 genotypes sent to Bristol for primer validation and genotyping. All 22 genotypes had segments of Ae. speltoides present. Least number of segments = 5 Highest number of segments = 12 • Need to make more backcrosses to isolate lines with single introgressions • Far more introgressions than expected
KASP
Isolate genotypes with a single introgression
BC1 Aegilops speltoides – 22 genotypes sent to Bristol for primer validation and genotyping. All 22 genotypes had segments of Ae. speltoides present. Least number of segments = 5 Highest number of segments = 12 • Need to make more backcrosses to isolate lines with single introgressions • Far more introgressions than expected Ambylopyrum muticum, Triticum urartu, Ae. caudata, Secale cereale, Thinopyrum Intermedium, Thinopyrum ponticum, Thinopyrum elongatum
KASP
Isolate genotypes with a single introgression
Triticum aestivum x Triticum urartu BC1 hybrids
1A 2A 3A 5A 6A 7A
BC1 1
BC1 2
• KASP can be used to screen several 1000’s of plants with circa 56 quickly and relatively cheaply How can you screen with higher resolution and use more of the SNP’s that have been developed?
KASP
Isolate genotypes with a single introgression
WISP Axiom® 820k array
• 96 format 2 PEG design
• Includes SNPs among elite lines
• Plus SNPs between elites and landraces, and non-wheat relatives
WISP Axiom® 820k array
• 96 format 2 PEG design
• Includes SNPs among elite lines
• Plus SNPs between elites and landraces, and non-wheat relatives
Ex. 38, ooo Aegilops speltoides SNPs
Sample screening • Elite varieties • Landraces • Wheat relatives • Synthetics
• Mapping populations
• Deletion lines
820K Array ~ 593,755 validated SNPs
Axiom 384HT Breeders chip ~35K SNPs Mapped, Codominant Even coverage Good PIC score
Axiom 384HT Progenitors ~35K SNPs
Axiom 384HT Others… ~35K SNPs
Being manufactured now. Available to breeders For spring 2014 Public and IP free!
Spring 2014
High Resolution Identification of introgressions
A
B
D
Wheat ph1/ph1
X
Wild relative (R)
ph1
X A
B
D
Wheat Ph1/Ph1
High throughput screening of 1000’s of BC1 and
subsequent backcross progeny to identify
recombinants
The technology is now available
to exploit the distant relatives of
wheat
Axiom® 35K array Identify introgressions etc + KASP- used in later generations
A
B
D
Wheat ph1/ph1
X
Wild relative (R)
ph1
X A
B
D
Wheat Ph1/Ph1
High throughput screening of 1000’s of BC1 and
subsequent backcross progeny to identify
recombinants
The technology is now available
to exploit the distant relatives of
wheat
Axiom® 35K array Identify introgressions etc + KASP- used in later generations
Step change in identification
and characterization of Introgressions
Thinopyrum intermedium 440016 X Chinese Spring Mutant P208/533
Thinopyrum elongatum 401007 X Chinese Spring Mutant 84
Aegilops mutica 2130004 X Chinese Spring Euploid 94
Secale cereale 428373 X Chinese Spring Mutant 84
Thinopyrum bessarabicum 531712 X Chinese Spring Mutant 84
2. Wheat/ancestral introgression - Amphidiploids
- Develop a series of wheat/ancestral amphiploids from different species and accessions
(retaining the ancestral parents).
Wheat
X
Wild relative
Amphidiploids in a Paragon background
(Trait analysis – heat, salt, disease resistance etc)
Chromosome double
Season 1
Self Season 2
Multiplication
Season 3/4
Trait analysis,
Phenotyping platform
Ph1/Ph1
Ph1/Ph1
Ph1/Ph1 Ph1/Ph1
2. Wheat/ancestral introgression - Amphidiploids
– Colchicine – (Colchicum autumnale – autumn crocus)
inhibits microtubule polymerization
by binding to tubulin - spindle poison
= inhibiting chromosome segregation during meiosis
– Caffeine - (Coffea arabica – coffee)
inhibits plant cell cytokinesis
after nuclear division transverse cell walls fail to form – binucleate cells
- sister nuclei fuse or enter mitosis together and become polyploid
• Wheat/distant relative F1 hybrids at 4 tiller stage
• Split plants in half, trim roots and shoots
• Treatment overnight with a solution of 0.1% Colchicine, 2% DMSO, Tween-20; or 3 g/L caffeine
• Washing off traces, potting, cool temperature at start (15˚C)
• Tag ears that shed pollen – fertility still low, sometimes only later tillers affected (8th or later)
• Very careful threshing
Chromosome doubling
Colchicine treated F1 hybrid plants.
F1 hybrid shedding pollen.
Aegilops speltoides x Chinese Spring Eup
F1 hybrid setting seed.
Aegilops mutica x Chinese Spring Eup
Amphidiploid grains produced from 20 different distant relative species,
32 different accessions Distant relative species Wheat F1
hybrid
M0
grain
M1
grain
Aegilops caudata 2090001 Paragon 2 13 G
Aegilops caudata 2090001 Pavon 76 1 10 G
Aegilops caudata 2090002 Highbury 3 10 G
Aegilops comosa var. comosa
276970
Paragon 1 4* G
Aegilops mutica 2130004 CS Eup 94 2 7 216
Aegilops mutica 2130008 CS Eup 94 1 1 32
Aegilops mutica 2130012 Highbury 1 6 166
Aegilops mutica 2130012 CS Eup 94 3 5 167
Aegilops mutica 2130012 Pavon 76 2 3 30
Aegilops mutica 2130012 Paragon 1 4 89
Aegilops peregrina 604183 Capelli 1 1* G
Aegilops sharonensis 584411 Pavon 76 1 1 G
Aegilops speltoides 2140007 Pavon 76 1 1 32
Aegilops speltoides 2140008 Highbury 3 18 75
Aegilops speltoides 2140008 CS Eup 94 3 9 420
Aegilops speltoides 2140020 CS Eup 94 1 3 4
Aegilops speltoides 393495 Highbury 2 13 G
Aegilops speltoides 449340 Pavon 76 1 2 19
Aegilops umbellulata 542377 Pavon 76 1 1 37
Aegilops umbellulata 554410 CS Eup 94 2 8 94
Secale anatolicum P208/141 CS Eup 94 2 10 619
Secale anatolicum P208/141 Pavon 76 1 1 G
Secale anatolicum P208/142 CS Eup 94 17 201 12699
Secale anatolicum P208/142 Highbury 1 36 G
Distant relative species Wheat F1
hybrid
M0
grain
M1
grain
Secale anatolicum P208/142 Highbury 2 8* G
Secale anatolicum P208/142 CS Eup 94 2 26 G
Secale anatolicum P208/142 CS Eup 94 1 2* G
Secale iranicum P208/11 Pavon 76 1 2 G
Secale iranicum P208/151 CS Eup 94 1 1 G
Secale montanum P208/369 Capelli 1 1* G
Secale montanum P208/369 Pavon 76 1 1 G
Secale segetale P208/143 CS Eup 94 2 2 G
Secale vavilovii 573649 CS Eup 94 1 5 G
Secale vavilovii 573649 Pavon 76 1 2 G
Thinopyrum bessarabicum 531712 CS Mut P208/535 3 27 175
Thinopyrum elongatum 401007 CS Mut 84 1 3 G
Thinopyrum elongatum 401007 CS Mut P208/511 8 20 84
Thinopyrum elongatum 401007 CS Mut P208/533 1 2 1
Thinopyrum elongatum 401007 Paragon 6 20 59
Thinopyrum elongatum 401007 Paragon Mut 2 2 34 113
Thinopyrum elongatum 401007 Paragon Mut 11 1 13 1
Thinopyrum elongatum 401007 Paragon Mut 12 1 4 38
Thinopyrum intermedium 440016 Highbury 1 2 G
Thinopyrum ponticum 636523 Paragon 1 1 G
Thinopyrum ponticum VIR2 CS Mut P208/535 1 1 G
Thynopyrum bessarabicum 531711 CS Eup 94 1 1 G
Thynopyrum turcicum P208/201 CS Eup 94 1 28 G
Triticum timopheevii P95-99-1-1 Highbury 1 3 41
Triticum turgidum 94748 Highbury 1 11* G
Triticum urartu W Highbury 1 2 7 G - growing (in glasshouse/ in vernalization) * - from caffeine treated plants
Multiplication of amphidiploids
Secale anatolicum
x Chinese Spring Euploid 94
(Amp 1/6)
56 chromosomes
Multicolour GISH A genome – yellow; 14 chromosomes B genome – purple; 14 chromosomes D genome – red; 14 chromosomes Rye genome –green; 14 chromosomes
Multicolour GISH A genome – yellow-blue; 14 chromosomes B genome – purple; 14 chromosomes D genome – red; 12 chromosomes Ae. mutica genome –green; 13 chromosomes
Aegilops mutica x Chinese Spring Euploid 94
(Amp 28/1)
53 chromosomes
Multicolour GISH A genome – green; 11 chromosomes B genome + Ae. speltoides genome – purple; 28 (14 + 14) chromosomes D genome – red; 12 chromosomes
Aegilops speltoides x Pavon 76
(Amp 43/1)
51 chromosomes
In order to obtain the maximum value from the introgression material developed they
need to be screened for a wide range of traits
• Salt – India
• Heat/drought tolerance – Sydney, India, CIMMYT
• Water and nutrient use efficiency – Nottingham, Sydney, CIMMYT
• Mineral content (Boron/Aluminium) - Nottingham
• Roots – Nottingham, RRES
• Photosynthetic capacity/chloroplast cell structure/biomass – Nottingham, RRES, CIMMYT
• Disease/Insect Resistance – RRES, Sydney, India, CIMMYT
• Biofuel (ethanol) – Nottingham
Phenotyping
Development of an international phenotyping platform - to determine the potential of the introgressions being generated
Phenotyping
Photosynthesis Found increased photosynthesis in some BC1 plants of Triticum urartu, Aegilops speltoides and Thinopyrum bessarabicum – actually all the species he looked at. The Triticum urartu and Thinopyrum bessarabicum BC1 plants of interest have been included in the genotypes sent to Bristol for genotyping. SCPRID PhD student – Cannan - has continued the work. Found plants of interest among the BC3 Aegilops speltoides. Disease resistance Fusarium head blight, JIC; Take – all, RRES Biofuels
Flowering morphology
New collaboration: The University of Nottingham £2.2M The University of Sydney Directorate of Wheat Research, India Agharkar Research Institute, India CIMMYT
New amphidiploids to be screened on numerous sites in UK, Australia and India for wide range of phenotypic characteristics: Water use efficiency Nitrogen use efficiency Mineral uptake Photosynthetic capacity Rust resistance
India and Australia
SCPRID Programme
Screening novel wheat germplasm in SCPRID (UoN/DWR/ARI)
Four Indian students recruited to start in academic year 2013-14 • Urmila Devi (introgression work) • Jaswant Singh (tolerance to hostile soils / root phenotyping) • Ajit Nehe (nitrogen-use efficiency) • Kannan Chinnathambi (photosynthetic efficiency)
The three‘physiology’ students based in India until June 2014 (DWR/ARI) Jaswant will start work immediately with DWR supervisors to establish field-trial plots: - 6 sites of poor quality soils - 1 standard site with 2x N-levels Ajit and Kannan return to India in Oct./Nov. 2013 following initial training 2013/14: Indian genotypes (n=40, 4 replicate plots), root and shoot traits measured 2014/15: Amphidiploid material and Indian genotypes (n=40) 2015/16: Amphidiploid material (n=40) 2016/17: Amphidiploid material (n=40) Jaswant, Ajit and Kannan will spend periods of 2014 and 2015 in UK to develop high-throughput assays for root and shoot phenotyping of all ancestral lines, amphidiploids and introgression series. This will inform choice of material for bulking and field-trials.
Plant Breeding Institute
66
Root phenotyping (very high throughput)
Item unit cost (£ excl. VAT) Unit per tank Cost per tank (£ excl. VAT)
Frames and panels 152.55 152.55
Water reservoirs (custom drip trays) 32.80 9 295.20
19" X 24" Anchor paper (inc. cutting) 0.212 192 40.70
240 mm x 300 mm black polythene 0.0114 192 2.19
Q Connect foldback clip 19 mm 0.0072 192 1.38
Hoagland’s solution (16.3 g) 17.20 2.88 g 2.72
CAPITAL COSTS: £447.75 per tank
+camera and stand
RECURRING ITEMS: £0.25 /individual
STAFF: £0.70 /individual
GROWTH-ROOM: £0.50 /individual
Sowing: <5 person hours per tank, <£0.50 per individual at £18 h-1
Imaging: <2 person hours per tank, <£0.20 per individual at £18 h-1
Growth room costs: £100 per tank per run = £0.50 per individual, assume £8k per year at £200 per week for 40 weeks
Current cost = ~£1.45 per individual, excl. capital depreciation
5214 kb 949 kb
Very high throughput, 20k plants per year feasible for one person….
Root phenotyping (very high throughput)
Root phenotyping (very high throughput)
Winter Wheat var: Glasgow
Glasgow Ae. buncialis Ae. uniaristata T. dicocchodies Ae. variablis
Ae. genticulata Ae. markgafii Ae. columnaris T. urartu Ae. peregrina
Glasgow Ae. biuncialis Ae. uniaristata T. dicoccoidies Ae. variablis
Ae. genticulata Ae. markgafii Ae. columnaris T. urartu Ae. peregrina
Hounsfield CT Facility An X-ray Computed Tomography Facility for Rhizosphere Research
Key Features
• 3 CT Scanners working from 0.5 µm to 5 mm resolution
• Accommodating samples up to 25 cm diameter & 1 m length
• Rapid scanning within 10 minutes
• Automated sampling system enabling 4-D visualisation
• Automated root imaging via RooTrak
• New Building opening Feb 2014
Malcolm Bennett
An Automated Root Phenotyping Platform
Discovering new biological mechanisms
Lateral Root Hydropatterning
CIMMYT
Screening of the germplasm produced at Nottingham will take place at
CIMMYT (Mexico)
Matthew Reynolds, Hans Braun, David Bonnett
UK/Brazil
BBSRC Nottingham/Plant Genomics and Breeding Center Partnering Award
Julie King Ian King
Antonio Costa de Oliveira
http://wheatisp.org/
“In the next 50 years, we will need to harvest as much wheat as has been produced since the beginning of agriculture, some 10,000 years ago."
The BBSRC wheat breeding programme is divided into 4 pillars (Landraces, Synthetics, Alien Introgression, Elite Wheats) and 2 themes (Phenotyping and Genotyping). These are represented by the 6 circles below; each is clickable and takes you to the website of the respective area).
Free of IP
Ian & Julie King Csilla Nemeth Surbhi Mehra Caiyun Yang Paul Kasprzak Duncan Scholefield Stella Edwards Stephen Ashling PhD Students: Jonathan Atkinson Urmila Dogra Paul Waldron Jason Raynor Lauren Baker Jack Heath New Postdoctoral Researchers Andras Cseh - Hungary Glacy Silva - Brazil Research Fellow BBSRC/Nottingham Research Fellow BBSRC/Nottingham Postdoc position ERC
King’s Group
http://www.wheatisp.org/
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