Exploiting Wheat’s Distant Relatives

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Presentation delivered by Dr. Ian King (University of Nottingham, UK) at Borlaug Summit on Wheat for Food Security. March 25 - 28, 2014, Ciudad Obregon, Mexico.
http://www.borlaug100.org

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Exploiting Wheat’s Distant Relatives

  1. 1. Exploiting Wheat’s Distant Relatives Ian and Julie King
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. Nottingham London IanJulie
  7. 7. University Park Jubilee Campus Kings Meadow Campus Sutton Bonington Campus
  8. 8. University of Nottingham Malaysia Campus University of Nottingham China Campus
  9. 9. 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
  10. 10. (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
  11. 11. (Sears 1981) 1) The identification of introgressions is difficult and time consuming Why has introgression not been used more widely?
  12. 12. 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
  13. 13. 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
  14. 14. (Sears 1955 and 1981) 2) Introgressions were frequently very large and carried deleterious genes
  15. 15. (Sears 1955 and 1981) 2) Introgressions were frequently very large and carried deleterious genes Need Genetic Markers
  16. 16. (Sears 1981) Need for high throughput techniques to identify and characterize introgressions
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. • 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
  21. 21. Thinopyrum bessarabicum Highly salt tolerant (Forster; King) Thinopyrum elongatum Yield, biomass, photosynthetic capacity, salt tolerance
  22. 22. Aegilops speltoides Disease and insect resistant
  23. 23. Aegilops umbellulata Resistance to leaf rust (saved the US wheat crop late 50s early 60s), bread making quality – (Sears)
  24. 24. Triticum urartu Wheat A genome donor photosynthetic capacity
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. 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)
  29. 29. Shrivelled grain culture – dissection
  30. 30. Shrivelled grain culture – growing plants
  31. 31. Ancestral/wheat crossing – F1 hybrids Backcrossing – BC1, BC2 – Recombinants Colchicine treated F1 hybrids - Amphidiploids Amphidiploid seed multiplication Marker development , sterile culture of embryo/grain
  32. 32. Gustafson Glasshouse
  33. 33. Gustafson Glasshouse MK2 Crossing all year round
  34. 34. 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
  35. 35. • 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:
  36. 36. Chinese Spring (F) Aegilops speltoides (M) X F1 BC1 BC2 X Paragon X Paragon
  37. 37. 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
  38. 38. 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
  39. 39. 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
  40. 40. Triticum aestivum x Triticum urartu BC1 hybrids 1A 2A 3A 5A 6A 7A BC1 1 BC1 2
  41. 41. • 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
  42. 42. WISP Axiom® 820k array • 96 format 2 PEG design • Includes SNPs among elite lines • Plus SNPs between elites and landraces, and non- wheat relatives
  43. 43. 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
  44. 44. Sample screening • Elite varieties • Landraces • Wheat relatives • Synthetics • Mapping populations • Deletion lines
  45. 45. 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
  46. 46. 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
  47. 47. 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
  48. 48. 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
  49. 49. - 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
  50. 50. – 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
  51. 51. 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
  52. 52. 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
  53. 53. Multiplication of amphidiploids
  54. 54. 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
  55. 55. 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
  56. 56. 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
  57. 57. In order to obtain the maximum value from the introgression material developed they need to be screened for a wide range of traits
  58. 58. • 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
  59. 59. 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
  60. 60. Flowering morphology
  61. 61. 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
  62. 62. 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.
  63. 63. Plant Breeding Institute 66
  64. 64. 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
  65. 65. 5214 kb 949 kb Very high throughput, 20k plants per year feasible for one person…. Root phenotyping (very high throughput)
  66. 66. Root phenotyping (very high throughput)
  67. 67. Winter Wheat var: Glasgow
  68. 68. 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
  69. 69. 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
  70. 70. An Automated Root Phenotyping Platform
  71. 71. Discovering new biological mechanisms Lateral Root Hydropatterning
  72. 72. CIMMYT Screening of the germplasm produced at Nottingham will take place at CIMMYT (Mexico) Matthew Reynolds, Hans Braun, David Bonnett
  73. 73. UK/Brazil BBSRC Nottingham/Plant Genomics and Breeding Center Partnering Award Julie King Ian King Antonio Costa de Oliveira
  74. 74. 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
  75. 75. 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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