![International Atomic Energy AgencyFood and Agriculture Organization of the United Nations
Some other collaborative projects
• Tomato TILLING by Sequencing (Univ. Hyderabad)
• NIRS,
Sample Name Position Variant TypeCall FrequencyDepth Genome Unique in pools Comments
c5 727 SNP T->[T/A] 0.02 33647 sharm001 727 non-EMS three pools
d4 727 SNP T->[T/A] 0.02 6719 sharm001 727 non-EMS three pools
r15 727 SNP T->[T/A] 0.01 3640 sharm001 727 non-EMS three pools
c10 797 Indel AT/A- 0.01 19479 sharm001 797
c13 1884 SNP C->[C/T] 0.02 22346 sharm001 1884 EMS two pools
r9 1884 SNP C->[C/T] 0.01 93472 sharm001 1884 EMS two pools
c2 2120 SNP A->[A/G] 0.01 53766 sharm001 2120
c2 2181 SNP C->[C/T] 0.02 61617 sharm001 2181 EMS type three pools
d4 2181 SNP C->[C/T] 0.02 8959 sharm001 2181 EMS type three pools
r8 2181 SNP C->[C/T] 0.02 12526 sharm001 2181 EMS type three pools](https://image.slidesharecdn.com/2015-161022014112/85/2015-Bradley-j-Till-Forward-and-reverse-genetics-for-functional-genomics-and-breeding-37-320.jpg)
2015. Bradley j. Till. Forward and reverse genetics for functional genomics and breeding
- 1. Forward and reverse genetics for functional genomics and breeding Bradley J. Till Joint FAO/IAEA Programme Plant Breeding and Genetics Laboratory April 24th, 2015 [email protected]
- 2. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Food Security: “A situation that exists when all people, at all times, have physical, social, and economic access to sufficient, safe, and nutritious food that meets their dietary needs and food preferences for an active and healthy life”. FAO, 2002 • ~ 1 in 8 people in the world suffer from chronic undernourishment • Pressures are expected to increase (climate, population, etc.) • Many approaches for increasing food security, including plant breeding The Challenge of Sustainable Food Security
- 3. Images from Wikipedia commonsHeslop-Harrison J.S. and Schwarzacher T., 2007 Variation within a species is a major resource for the crop breeder http://r4dreview.org/2010/03/exploiting-the-diversity-of-african-yam-bean/ http://ipmworld.umn.edu/chapters/eigenbr.htm http://en.wikipedia.org/wiki/Crop_diversity#mediaviewer/File:Corncobs_edit1.jpg
- 4. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations • Mutations are changes in the DNA sequence of a cell's genome caused by radiation, viruses, transposons, mutagenic chemicals, or errors that occur during meiosis or DNA replication • Mutations are naturally occurring, or can be induced • Induced mutations broaden diversity Mutations provide genetic variation for plant breeding
- 5. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Inducing mutations in plants Image under public domain, National Institutes of Health Lewis John Stadler: Genetic effect of X-rays in maize, wheat, and barley (1920s) Mutation Breeding: The process of treating plant cells with mutagens to facilitate crop breeding •Seed, pollen, cell culture, plant tissues •Chemical or Physical (ionizing radiation) mutagenesis •Orders of magnitude faster than natural mutation •First released variety: “Vorsteland” Tobacco with improved quality in 1934 in Indonesia
- 6. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Physical 87% Chemical 13% Registered Mutant Crop Varieties Plant Species : 224 Total: 3218 http://mvgs.iaea.org/Jankowicz-Cieslak & Till, 2015 in press
- 7. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Examples of mutant varieties VIETNAM • Mutant varieties since the 1980s (>55 total) • Economic impact of over 2 billion USD • Mutant rice growing in 30% of Mekong delta (salinity tolerant) • Award winning quality • Rice, soybean, maize, ornamentals • Over 8 million farmers have profited (income increase by 30%) • 2014 FAO/IAEA Breeding Award Data from: Agricultural Genetics Institute (VAAS), Department of Agriculture and rural development, Institute of Agricultural Science for Southern Vietnam
- 8. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations PERU • Barley & Amaranth • High Andes (>3500 m) • Six-fold yield increase • Adding >6 million USD annually • 2014 FAO/IAEA Breeding Award Photo courtesy Dr. Luz Gomez Pando Examples of mutant varieties
- 9. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Examples of mutant varieties INDIA • Bhabha Atomic Research Centre (Mumbai) • 42 crop varieties (groundnut, mustard, soybean, mung bean, pigeon pea, urdbean, cowpea) • Groundnut varieties cover over 2,970,000 ha. • 2014 FAO/IAEA Breeding Award
- 10. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Mutation Breeding Attributes • tested, proven, robust • ubiquitously applicable • no IP, not regulated • cost effective • increasing local economies
- 11. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations THE PLANT BREEDING AND GENETICS LABORATORY •Training (>30/year) •Services (Mutagenesis) •Adaptive R&D/Tech Transfer • In direct support of TC projects • “upstream” efforts to develop, evaluate and test new approaches (CRPs) • “Smart” methods for developing countries (Low-cost, non-toxic, do-it-yourself)
- 12. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations THE PLANT BREEDING AND GENETICS LABORATORY •Training (>30/year) •Services (Mutagenesis) •Adaptive R&D/Tech Transfer • In direct support of TC projects • “upstream” efforts to develop, evaluate and test new approaches (CRPs) • “Smart” methods for developing countries (Low-cost, non-toxic, do-it-yourself)
- 13. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations THE PLANT BREEDING AND GENETICS LABORATORY •Services (Mutagenesis) • International treaty on plant genetic resources for food and agriculture & SMTA • Dosage optimization • Seed & veg. prop • ~ 40 requests/year Mirta Matijevic
- 14. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations THE PLANT BREEDING AND GENETICS LABORATORY Adaptive R&D/Tech Transfer • In direct support of TC projects • “upstream” efforts to develop, evaluate and test new approaches (CRPs) • methods for labs with limited infrastructure (Low-cost, non-toxic, do-it-yourself)
- 15. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Mutation disocvery & optimization Traditional “forward” genetics Mutagenesis Phenotyping Reverse genetics Mutagenesis Find mutations in genes Phenotypic validation Clone gene causing trait Varietal release ?
- 16. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations PCR seed bank Mutation discovery heat, cool Mutagenesis XSequencing TILLING Targeting Induced Local Lesions IN Genomes McCallum, et. al., 2000
- 17. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Advantages of TILLING • Yields a spectrum of point alleles and small indels • Mutations are irreversible • Efficient for small genes • Can focus on region of interest • Applicable to almost any organism • Only phenotype selected plants (10s versus 1000s) • Non-transgenic
- 18. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Examples of TILLING Projects • Individual projects • Public services Organism Mutagen Spectrum Density (per kb) Arabidopsis EMS >99% GC-AT 1/200 Barley EMS ~70% GC-AT 1/140 1/1000 Maize EMS >99% GC-AT 1/500 Pea EMS >99% GC-AT ~1/700 Rice EMS, Az- MNU, Gamma Mixture ~1/130 – 1/6000 Sorghum EMS >90%GC-AT ~1/500 Soybean EMS, MNU 75-90% GC- AT 1/140 – 1/500 Tomato EMS ~90% GC-AT 1/300-1/700 Wheat EMS >99% GC-AT 1/20-1/40 * Many different groups
- 19. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Mutation density and polyploidy1mutationperXkb 0 100 200 300 400 500 600 •Density and spectrum allow estimation of ideal population size to recover desired alleles Data from chemical mutagenesis B. J. Till et al., Genome Res 13, 524 (2003). B. J. Till et al., BMC Plant Biol 4, 12 (2004). A. J. Slade et al., Nat Biotechnol 23, 75 (2005). B. J. Till et al., BMC Plant Biol 7, 19 (2007). J. L. Cooper et al., Genetics 180, 661 (2008). J. L. Cooper et al., BMC Plant Biol 8, 9 (2008). Jankowicz-Cieslak et al., Plant Biotech Journal 12 (2012)
- 20. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Examples of TILLING for trait improvement Crop Trait Target genes Reference Wheat Waxy Starch, High amylose GBSSI, SBEIIa Slade et al, 2005, 2012 Pea Plant height gibberellin 3β- hydrolase Tirques et al, 2007 Potato Improved Starch GBSSI Muth et al, 2008 Tomato Fruit color Phytoene synthase 1 Gady et al, 2011 Sorghum Altered hydrogen cyanide potential CYP79A1 Blomstedt, et al, 2012
- 21. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Why so few vegetatively propagated food crop varieties? Banana & Cassava: Staples for 1 billion Primarily veg prop. Years of breeding support In total only 5 released mutant varieties (14% of population, only 0.16% of database)
- 22. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Tissue mutagenesis & TILLING for banana Joanna Jankowicz-Cieslak • High density of induced point mutations (1/57kb) • Rapid dissolution of chimeric sectors Jankowicz-Cieslak et al, Dec 2012
- 23. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Most mutant varieties by forward genetics Mutagenesis Phenotyping Clone gene causing trait Varietal release ? • Many crops only self-fertilized • Almost no information on types of mutations and genes involved in traits • It is very difficult to identify gene/mutation in large genome crops • Knowledge of mutation and gene for functional genomics, markers, etc.
- 24. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Example of TC project support : The spread of wheat stem rust race Ug99 • First report of virulence for Sr31 1999 (Pretorius et al. 2000 Plant Dis. 84:203) • Stem rust did not appear in Uganda during the following years • Presence in Kenya reported in 2002 although Kenyan data indicate that it may have existed since 1999 • Detected in Ethiopia in 2003 • Most leading cultivars and breeding lines became susceptible to Ug99
- 25. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations An interregional approach for enhancing resistance to stem rust • IAEA interregional project started in 2009, through the department of Technical Cooperation (INT/5/150) • 21 countries and 4 international institutions (CIMMYT, FAO, IAEA, ICARDA) participate in this project • University of Eldoret, Kenya, used as the screening hot spot • Goal: to generate novel genetic variation among wheat and barley varieties using mutagenesis to improve resistance
- 26. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations An example from Kenyan materials • Mutation induction was done using gamma rays from Cobalt source (60co) in Seibersdorf Laboratories Vienna, Austria • Plants grown and tested in Kenya • Training provided at Seibersdorf • Two new varieties released in 2014 Ego Amos
- 27. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations What next? Lab supporting cloning mutated genes causative for resistance Mapping (~5 years) Ug99 partners: Jorge Dubcovsky, Ksenia Krasileva, UC Davis, Hermann Buerstmayr, BOKU, Austria Miriam Kinyua, Amos Ego, U. Eldoret, Kenya, Tom Fetch, Agri-food Canada, Rob Park, U. Sydney 1. Seed from Kenya 2. Genotyping by Sequencing 4. Exome capture for mutation discovery 3. Disease resistance validation – Canada Australia Cloning by sequencing
- 28. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Exome is a fraction of the genome
- 29. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Reducing sequencing loads- (CRP D24012) Figure : Isabelle Henry and Luca Comai, UC Davis Genome Center
- 30. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Pilot exome capture for discovery of rare mutations (sorghum and cassava) Gamma M2 N6 Gamma EMS M1V3 17 selected for sequencing (1genotype) 15 plants from 2 Sudanese genotypes selected for sequencing M1V3 Self Souleymane Bado Owen Huynh Joanna Jankowicz-Cieslak
- 31. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Recovery of indels in mutagenized material 25 kb bin analysis Collaborators: Isabelle Henry and Luca Comai, UC Davis Genome Center
- 32. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Name Sample(s) Chr/Scaffold Start End Length (kb) Type In/del A 75b_1 & 75b_2 Chromosome1 39125000 39525000 400 Het Deletion B 75b_1 & 75b_2 chromosome 1 44825001 45525000 699.999 ? Insertion C 75b_1 & 75b_2 super12 3725001 4300000 574.999 Het Deletion D 300a_2 Chromosome 2 42950001 43090000 139.999 Het Deletion E 300a_1 chromosome_10 42340001 42600000 259.999 ? Deletion F 300a_2 Chromosome 2 38975001 39225000 249.999 Het Deletion G 75_b_1 chromosome_8 32800001 32910000 109.999 ? Insertion Preliminary summary of sorghum indel analysis • Both insertions and deletions recovered • Indels found in 4/5 analysed mutants in one Sorghum line (But not the other) • Smaller bin analysis suggests some false negatives • More individuals would help with detection
- 33. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Transferring technologies to developing countries Issues to consider for molecular biology: •Varying infrstructure •Toxic waste storage and disposal •Lack of constant power makes -80C freezers problematic •Can be difficult to get liquid nitrogen •Kits for sample analysis are expensive, and you need to practice to get good at molecular methods •Many applications require a hiqh quality genomic DNA from leaf tissue
- 34. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Low-cost methods suitable for most labs Bernhard Hofinger Owen Huynh WEED CELERY L W1 W2 W3 W4 C1 C2 C3 C4 U L 1x 1.7x 10x 20x 1x 2.2x 13x 26x • High quality DNA at 1/10th the cost • No toxic chemicals • Protocols and kits for MSs • > 80 scientists from >30 MSs trained since 2013 Joanna Jankowicz-Cieslak Adapted method for Tef (Ethiopia) & Sugarbeet (Poland)
- 35. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Low-cost methods suitable for most labs 1st National Workshop on TILLING in Crop Plants – University of Hyderabad, India April 13-24 – Prof. Ramesh Sharma Genomic DNA Activity of extracted nucleases
- 36. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Technology transfer- Low-cost methods for Mauritius Babita Dussoruth • Low cost Ecotilling to characterize local germplasm • Low cost methods for DNA extraction: Banana, barley, cassava, wheat, rice, tomato, breadfruit, chilli, sorghum, mango, litchi, peach, Ceylan olive, coffee, roses, anthurium, jackfruit, chrysanthemum, etc. EcoTILLING in Cochliobolus sativus – Mohammed Jouhar - SYRIA AAB ABB
- 37. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Some other collaborative projects • Tomato TILLING by Sequencing (Univ. Hyderabad) • NIRS, Sample Name Position Variant TypeCall FrequencyDepth Genome Unique in pools Comments c5 727 SNP T->[T/A] 0.02 33647 sharm001 727 non-EMS three pools d4 727 SNP T->[T/A] 0.02 6719 sharm001 727 non-EMS three pools r15 727 SNP T->[T/A] 0.01 3640 sharm001 727 non-EMS three pools c10 797 Indel AT/A- 0.01 19479 sharm001 797 c13 1884 SNP C->[C/T] 0.02 22346 sharm001 1884 EMS two pools r9 1884 SNP C->[C/T] 0.01 93472 sharm001 1884 EMS two pools c2 2120 SNP A->[A/G] 0.01 53766 sharm001 2120 c2 2181 SNP C->[C/T] 0.02 61617 sharm001 2181 EMS type three pools d4 2181 SNP C->[C/T] 0.02 8959 sharm001 2181 EMS type three pools r8 2181 SNP C->[C/T] 0.02 12526 sharm001 2181 EMS type three pools
- 38. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Some other collaborative projects • NIRS, Proteomics, Metabolomics of mutant rice (BOKU, U. Vienna) • Citrus greening disease & TILLING (University of Florida) • Grass pea TILLING (JIC, RevGenUK) • Banana disease Tropical Race 4 resistance (TBRI, Bioversity, Stellenbosch Univ, DuRoi) • Epigenomics of adaptation in barley (GMI, Vienna)
- 39. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Summary • Global factors such as population growth and climate change have a negative impact on food security • Nucleotide variation is the major source of variability for the plant breeder • Mutations can be induced in organisms at a much faster rate than occur naturally • Mutation breeding has been widely successful in developed and developing countries, but more needs to be done • Improving traditional approaches and innovative methods
- 40. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Acknowledgments DH Screening IPK Gatersleben, Germany Jochen Kumlehn, Andrea Mueller, Ingrid Otto Kutno, Poland Kamila Kozak-Stankiewicz Ethiopia U Likyelesh Gugsa Citrus University of Florida, USA Janice Zale Banana Stellenbosch University, South Africa Altus Viljoen Du Roi Laboratory, South Africa, Anne Davson Low-Cost methods AREU Mauritius Babita Dussoruth, Binita Saraye University of Nebraska, USA Rob Laport AEC, Syria Mohammad Jouhar COLLABORATORS Ug99 Miriam G. Kinyua Ego K. Amos Tom Fetch Rob Park Cloning Jorge Dubcovsky (Davis) Ksenia Krasileva (Sainsbury) Genotyping and mapping BOKU, Tulln, Austria Buerstmayr lab Tomato- TILLING by Sequencing University of Hyderabad Group of Ramesh Sharma Exome Sequencing UC Davis, USA Luca Comai, Isabelle Henry Sorghum & Low Cost ARC, Sudan Mayada Beshir Plant Breeding and Genetics Laboratory Stephan Nielen Joanna Jankowicz-Cieslak Souleymane Bado Abdelbagi Mukhtar Ali Ghanim Mirta Matijevic Farzaneh Taassob Shirazi Guenter Berthold Andreas Draganitsch Plant Breeding and Genetics Section Pierre Lagoda Fatma Sarsu Ljupcho Jankuloski Past Members Owen Huynh Chikelu Mba Brian Forster Bernhard Hofinger Krzysztof Kolloch Malgorzata Kupc And many more…
- 41. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations THANK YOU!
- 42. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Seedling and adult plant phenotyping • Seedling infection types • Adult plant infection response --------Resistant-------- ---Susceptible--- 0 0; ; 1 2- 2+ 3 3+ 4 *13 new resistant lines identified
- 43. International Atomic Energy AgencyFood and Agriculture Organization of the United Nations Natural (Spontaneous) mutation Rate is about 1 mutation every billion base pairs In humans that means that we have about 6 mutations in each of our cells However, most mutations are functionally silent & only those mutations in gametes (egg & sperm/pollen) are passed onto offspring… Plant breeders use natural mutations that have accumulated over many thousands of years But, slow rate of spontaneous mutation is a major limitation