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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/