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Mutation Breeding in AgricultureDr. Bradley J. Till

1The screen versions of these slides have full details of copyright and acknowledgements

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Mutation Breeding in Agriculture

Dr. Bradley J. TillJoint FAO/IAEA Programme

Plant Breeding and Genetics Laboratory

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Outline

• Background on plant breeding

• Foundations of mutation breeding and examples

• Reverse genetics for functional genomics and breeding

• Considerations for vegetatively propagated crops

• New technologies for mutation assisted breeding

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~ 10,000 years of plant breeding

Plant Breeding: The process of altering the genetics of plants to create desirable traits

Photo: John Doebley, http://teosinte.wisc.edu/images.html

• Involves selection or creation of genetic variation & phenotypic evaluation of plants

• Selective pressure by humans (seed shattering, grain size, plant height, etc.)



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Why is plant breeding needed?

• Historically: Domestication and breeding are the foundations for modern society

• Presently: Increasing pressures on global food security

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 billion go hungry every day

• Many approaches for increasing food security, including plant breeding

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• 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

Mutations provide genetic variation for plant breeding

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Induced mutations to increase biodiversity

• Hermann Muller: Novel variation can be induced by treatment of cells with ionizing radiation (1920s, Nobel prize in physiology or medicine in 1946)

From The Physical Basis Of Heredity, Thomas Hunt Morgan. Philadelphia: J.B. Lippincott Company 1919



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Inducing mutations in plants

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

• Direct or indirect usage (elite variety, introgression, radiation-induced translocations)

• First released variety: “Vorsteland” Tobacco with improved quality in 1934 in Indonesia

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Why induced mutations for breeding?

• Create new variation not found in nature

• Generate novel alleles orders of magnitude faster than occur spontaneously

• Generate variation in organisms where traditional introgression is impeded (e.g., linkage drag, or asexual propagation)

• Broadly applicable

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Over 3200 registered mutant varieties

http://mvgs.iaea.org/



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Physical 87%

Chemical 13%

Registered Mutant Crop Varieties (2012)

Cereals 1591

Flowers 645

Legumes 493

Oil crops 111

Others 378

• Total: 3218• Plant species: 224

http://mvgs.iaea.org/

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How does mutation breeding work?

Example of seed mutagenesis

Mutation inductionM1 generation

chimeric Phenotyping Multi location trialsVarietal release

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Optimizing mutagenesis in seed propagated crops

0Gy 100Gy 200Gy 300Gy 400Gy 500Gy

0

20

40

60

80

100

120

Doses (Gy)100 200 300 400 5000 600S

eedl

ing

heig

ht a

s pe

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tage

of c

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Types of mutagens commonly used

• Chemical: Alkylating agents (EMS, MNU), Sodium azide, other chemicals

– Treatment: Soak plant material in a specific concentration of chemical for specific time

• Physical: Ionizing radiation (Gamma rays, X-rays, fast neutron, ion beam), Ultraviolet

– Treatment: Expose plant material to source for calculated time to reach desired dosage; Treatment can be acute, chronic, or split dosage

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Spectrum/ types of mutations

• Point mutations

• Insertions

• Deletions

• Duplications

• Translocations

• Inversion

Chemical

Physical

Translocation

Duplication

Insertion

Deletion Inversion

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Effects of mutations on genes

• Missense

• Nonsense

• Read through

• Silent

• Truncation

• Regulatory/dosage effect

Point mutations (chemical and physical)

Chromosome alteration (physical)



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Effect of mutations on gene function

• Recessive loss of function

• Dominant gain of function

• Dominant negative

• No effect

• Lethality

• Reversion

* The mutagen and dosage can be modified to control the type and frequency of mutations

Somatic tulip mutation: Source: User Dmccabe, Wikimedia commons

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Examples of mutant varieties

Barley: Golden promise

Slide courtesy Brian Forster

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Examples of mutant varieties (2)

Slide courtesy Brian Forster

Mutant varietyMutant gene

Mutagen/ cross with

mutantCountry Value

or areaBasis of value assessment

Golden Promise (1967) ari-e.GP

Gamma rays UK -Scotland

US$ 417 million

Crop value during 1977-2001

Diamant (1965) and derived varietiessdw1

X-rays / crosses

with Diamant

Numerous European countries

2.86 million

ha

Total area already planted in 1972



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Examples of mutant varieties (3)

• Gamma irradiation

• “Green Revolution” genes

• 15% yield advantage

• Used to create 20 varieties

Photo in public domain: USDA ARS

Semi-dwarf rice “Calrose 76”

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Examples of mutant varieties: Developing countries

• Sudan, Tissue Culture Laboratory, Agricultural Research Corporation (ARC)

• Released 2007

• Supported by IAEA

• 30% yield advantage over local varieties

Banana: Al-Beely

Photo: Dr. Mohamed Ahmed Ali

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Examples of mutant varieties: Developing countries (2)

Rice: “VND” varieties

VND95-20

• Salinity tolerance

• Short duration

• ~30% of Mekong delta

• Supported by IAEA



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Barley: La Molina, Centenario

• High altitude varieties

• Peru, high Andes (>3500 m)

• Six-fold yield increase

• IAEA support

Photo courtesy Dr. Luz Gomez Pando

Examples of mutant varieties: Developing countries (3)

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Mutation breeding attributes

• Tested, proven, robust

• Ubiquitously applicable

• Not hazardous, not regulated

• Cost effective

• Increasing local economies

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Taking advantage of knowledge of genes & gene function

http://www.genomesonline.org, October 29, 2012



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Forward and reverse genetics

Traditional “forward” genetics

Reverse genetics

Phenotyping Clone gene causing trait

Find mutations in genes Phenotypic validationMutagenesis

Mutagenesis

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Targeting induced local lesions in genomes

• Reverse-genetics with induced point mutations and small indels

• Random and high-density mutagenesis allows recovery of mutations in all genes in a small population

• Target genes and gene regions of interest

• DNA and seed library can be used for years & made available to many labs

• Applicable to most organisms, non-transgenic

“TILLING” : McCallum, et al., 2000

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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

* References provided at end of presentation



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Mutation density and polyploidy

• Density and spectrum allow estimation of ideal population size to recover desired alleles

1 m

utat

ion

per X

kb

Data from chemical mutagenesis

* References provided at end of presentation

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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

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• Many important species, including staple food security crops for over 1 billion people in the developing world, but receiving comparatively little investment

• Mitotic propagation (obligate or facultative)

• All plants have capacity for mitotic vegetative/clonal reproduction via totipotent stem cells

• Arose through competitive evolutionary advantage, and/or human selection

• Induced mutations to increase genetic diversity and generate novel phenotypes

Vegetatively propagated crops & mutagenesis



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Musa: Banana and plantain

• 87% locally consumed, 120 countries

• Basic food crop for ~ 400 million people

• Wide genetic diversity

• Diploid and polyploid, edible triploids sterile/parthenocarpic (obligate vegetative propagation)

• Vulnerable to fungal disease/ drought

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Tissue mutagenesis for banana

Recovery

DNAMutation discovery

• 4000 meristems isolated

• 1% EMS for 3 hours

• ~80% survival

• Clonal propagation to M1V6 = 1.5 years

Meristem isolation

M0V1 M0V1

Wild-type AAA

M1V1 M1V1

EMS

Meristem isolation & bisection

Repeat6 X

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33IRDye 700 IRDye800

Mutations identified by TILLING using EMD

For 768 samples:

Target Size (bp) # mutationsACETRANS 1415 6

AMTHLTR 1410 2

DNAJ 1395 1

ELF3 1416 9FTSJMT 1369 3

GHF17 1402 1

MALSYN 1402 3

NPH3 1427 2

PAAL2 1422 3

PUF 1472 2

RNDR 1500 1

Total 33



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Spectrum of recovered alleles

• What about density?

• What is the effective population size?

• 100% GC->AT

• 36% silent, 49% missense

• 15% truncation (exp = 4.6%)

1.000.27

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Recovery

Model 1: Slow dissolution of chimeras

Mutagenesis of isolated meristems

• % of siblings with same mutation allows estimation when chimeras dissolved

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Model 2: Diplontic selection and rapid dissolution of chimeras

Recovery

• 100% inheritance in siblings

Mutagenesis of isolated meristems (2)



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Evaluation of inheritance of mutations in siblings

• Mutation density estimation

(13430*(96+(96*0.09))/33)*1.5

= ~1 mutation / 57 kb

• Data supports model of rapid

chimera dissolution

Gene target Allele Line number

Number identified

Number screened

ACETRANS C215T MT1_5 6 6C227T MT90_83 4 4C653T MT47_33 10 10

G1127A MT49_43 10 10C1312T MT73_6 9 9

AMTHR C717T MT80_53 73 74G1007A MT90_83 5 5

FTSJMT G284A MT57_33 10 10*C623T MT82_73 8 13*C623T MT90_23 3 11*C623T MT94_33 2 18C986T MT82_33 9 27

MALSYN C182T MT99_83 2 3G685A MT89_53 4 4G1309A MT81_63 7 7

RNDR G824A MT80_53 73 74

Total 235 285

% 82.5

* Corrected 91.4

Jankowicz-Cieslak et al., Plant Biotechnology Journal 10: 1056-1066

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Developing a diploid banana TILLING population

Mutagenesis

+/- +/- +/- +/-

Cell suspension (Calcutta4, AA, Sigatoka resistant, & hybrids)

In vitro culture, >1000 heterozygous plants

Test for mutation density (EMD or NGS)Recover desired alleles

LowHigh

Field propagation and self-fertilization only candidates(low fecundity overcome through clonal propagation)

What is the frequency of meiotic defects?

Jorge Lopez (INIVIT) CubaMarty Dickman TAMU

IAEA coordinated research project D 24012

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Gamma irradiation(> deleterious & dominant alleles?)

N3

N4

N5

N6N8

N9 N7

TILLING

Genotyping

Mutagenesis in facultative vegetatively propagated plants: Cassava



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New technologies for mutation breeding

• “Next Generation Sequencing” (mutation density/spectrum, TILLING, mapping/cloning)

• Targeted mutations with engineered nucleases (TALENs, ZFNs, Meganucleases)

• “Phenomics”

Enhancing the efficiency of the induction and screening of mutations

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Summary

• Induced mutations provides genetic variation to improve yield, quality, biotic and abiotic resistance

• Easily transferable to developed and developing nations

• Applied for over 70 years, adding billions to the global economy

• Forward and reverse-genetic approaches can be applied

• New technologies promise to enhance the efficiency of the process

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Plant Breeding and Genetics LaboratoryBrian Forster

Joanna Jankowicz-Cieslak

Owen Huynh

Souleymane Bado

Mirta Matijevic

Bernhard Hofinger

Farzaneh Taassob Shirazi

Gilbert Seballos

Guenter Berthold

Andreas Draganitsch

Past MembersChikelu Mba

Marta Brozynska

Kamila Kozak-Stankeowicz

Danilo Moreno

Joy Nakitandwe

Plant Breeding and Genetics SectionPierre Lagoda

Madeleine Spencer

Stephan Nielen

Fatma Sarsu

Collaborators (Banana and Cassava)Jorge Lopez, INIVIT, CubaMartin Dickman, TAMU, USA

Aime Diamuini, CGEA, D.R. CongoLuca Comai, Isabelle Henry, Henriette O’Geen

UC Davis

AND MANY MORE!

Acknowledgments



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Appendix 1: References and further reading• Ahloowalia, B. S., M. maluszynski and K. nichterlein, 2004 Global impact of mutation-derived varieties.

Euphytica, 135: 187-204

• Barkley, N. A., and M. L. Wang, 2008 Application of TILLING and EcoTILLING as reverse genetic approaches to elucidate the function of genes in plants and animals. Current Genomics, 9: 212-226

• Blomstedt, C. K., R. M. Gleadow, N. O'donnell, P. Naur, K. Jensen et al., 2012 A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production. Plant Biotechnology Journal, 10: 54-66

• Bovina, R., V. Talame, S. Silvio, M. C. Sanguineti, P. Trost et al., 2011 Starch metabolism mutants in barley: A TILLING approach. Plant Genetic Resources-Characterization and Utilization, 9: 170-173

• Chawade, A., P. Sikora, M. Brautigam, M. Larsson, V. Vivekanand et al., 2010 Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes. BMC Plant Biol, 10: 86

• Colbert, T., B. J. Till, R. Tompa, S. Reynolds, M. N. Steine et al., 2001 High-throughput screening for induced point mutations. Plant Physiol ,126: 480-484

• Comai, L., K. Young, B. J. Till, S. H. Reynolds, E. A. Greene et al., 2004 Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J, 37: 778-786

• Cooper, J. L., B. J. Till, R. G. Laport, M. C. Darlow, J. M. Kleffneret al., 2008 TILLING to detect induced mutations in soybean. BMC Plant Biology, 8: 9

• Dahmani-mardas, F., C. Troadec, A. Boualem, S. Leveque, A. A. Alsadon et al., 2010a Engineering melon plants with improved fruit shelf life using the TILLING approach. PLoS One, 5: e15776

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• Dahmani-mardas, F., C. Troadec, A. Boualem, S. Leveque, A. A. Alsadon et al., 2010b Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. Plos One, 5

• Dong, C., J. Dalton-morgan, K. Vincent and P. Sharp, 2009 A Modified TILLING Method for Wheat Breeding. Plant Genome, 2: 39-47

• Elias, R., B. J. Till, C. Mba and B. Al-safadi, 2009 Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Res Notes, 2: 141

• Furbank, R. T., and M. Tester, 2011 Phenomics--technologies to relieve the phenotyping bottleneck. Trends Plant Sci, 16: 635-644

• Gady, A. L., F. W. Hermans, M. H. Van De Wal, E. N. Van Loo, R. G. Visser et al., 2009 Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations. Plant Methods, 5: 13

• Gottwald, S., P. Bauer, T. Komatsuda, U. Lundqvist and N. Stein, 2009 TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1. BMC Res Notes, 2: 258

• Greene, E. A., C. A. Codomo, N. E. Taylor, J. G. Henikoff, B. J. Till et al., 2003 Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics, 164: 731-740

• Hadrich, N., Y. Gibon, C. Schudoma, T. Altmann, J. E. Lunn et al., 2011 Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity. Journal of Plant Physiology, 168: 1395-1405

Appendix 1: References and further reading (2)

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• Himelblau, E., E. J. Gilchrist, K. Buono, C. Bizzell, L. Mentzer et al., 2009 Forward and reverse genetics of rapid-cycling Brassica oleracea. Theor Appl Genet, 118: 953-961

• Hurtado-gonzales, O. P., C. Rippetoe, D. Gobena, L. Finley and K. H. Lamour, 2006 Development of reverse genetic TILLING resources for the oomycetesPhytoahthora capsici and P sojae., Phytopathology, 96: S51-S52

• Jain, S. M., B. Till, P. Suprasannaand N. Roux, 2011 Mutations and Cultivar Development of Banana, pp. 203-217 in Banana Breeding, Progress and Challenges, edited by M. P. A. A. Tenkouano. CRC Press

• Jankowicz-cieslak, J., O. A. Huynh, S. Bado, M. Matijevic and B. J. Till, 2011 Reverse-genetics by TILLING expands through the plant kingdom. Emirates Journal of Food and Agriculture, 23: 290-300

• Jankowicz-cieslak, J., O. A. Huynh, M. Brozynska, J. Nakitandwe and B. J. Till, 2012 Induction, rapid fixation and retention of mutations in vegetatively propagated banana. Plant Biotechnology Journal, 10: 1056-1066

• Kandavelou, K., and S. Chandrasegaran, 2009 Custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes. Methods Mol Biol, 544: 617-636

• Kawaura, K., M. Takaku, T. Imai and Y. Ogihara, 2009 Molecular analysis of the Q gene controlling spike morphology with TILLING lines of common wheat. Genes & Genetic Systems, 84: 476-476

• Lababidi, S., N. Mejlhede, S. K. Rasmussen, G. Backes, W. Al-said et al., 2009 Identification of barley mutants in the cultivar 'Lux' at the Dhn loci through TILLING. Plant Breeding, 128: 332-336




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• Le Signor, C., V. Savois, G. Aubert, J. Verdier, M. Nicolas et al., 2009 Optimizing TILLING populations for reverse genetics in Medicago truncatula. Plant Biotechnology Journal, 7: 430-441

• Martin, B., M. Ramiro, J. M. Martinez-zapater and C. Alonso-blanco, 2009 A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis. BMC Plant Biol, 9: 147

• Mba, C., R. Afza, S. Bado and S. H. Jain, 2010 Induced Mutagenesis in Plants Using Physical and Chemical Agents, pp. 111-130 in Plant Cell Culture: Essential Methods, edited by M. R. Davey and P. Anthony. John Wiley & Sons, Ltd.

• Mba, C. M., R. Afza, J. Jankowcz-cieslak, S. Bado, M. Matijevic et al., 2009 Enhancing Genetic Diversity Through Induced Mutagenesis in Vegetatively Propagated Plants, pp. 293-296 in Induced Plant Mutations in the Genomics Era, edited by Q. Y. Shu. Food and Agriculutre Organization of the United Nations, Rome

• Mccallum, C. M., L. Comai, E. A. Greene and S. Henikoff, 2000a Choosing optimal regions for TILLING. Plant Physiol, 123: 439-442

• Mccallum, C. M., L. Comai, E. A. Greene and S. Henikoff, 2000b Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol, 123: 439-442

• Mussolino, C., and T. Cathomen, 2012 TALE nucleases: tailored genome engineering made easy. Curr Opin Biotechnol, 23: 644-650

• Muth, J., S. Hartje, R. M. Twyman, H. R. Hofferbert, E. Tacke et al., 2008 Precision breeding for novel starch variants in potato. Plant Biotechnol J, 6: 576-584


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• Ng, P. C., and S. Henikoff, 2003 SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 31: 3812-3814

• Okabe, Y., E. Asamizu, T. Saito, C. Matsukura, T. Ariizumi et al., 2011 Tomato TILLING Technology: Development of a Reverse Genetics Tool for the Efficient Isolation of Mutants from Micro-Tom Mutant Libraries. Plant and Cell Physiology 52: 1994-2005

• Ozias-akins, P., M. L. Ramos, P. Faustinelli, Y. Chu, S. Maleki et al., 2009 Spontaneous and induced variability of allergens in commodity crops: Ara h 2 in peanut as a case study, Regulatory Toxicology and Pharmacology54: S37-S40

• Perry, J., A. Brachmann, T. Welham, A. Binder, M. Charpentier et al., 2009 TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements. Plant Physiology 151: 1281-1291

• Porch, T. G., M. W. Blair, P. Lariguet, C. Galeano, C. E. Pankhurst et al., 2009 Generation of a Mutant Population for TILLING Common Bean Genotype BAT 93. Journal of the American Society for Horticultural Science 134: 348-355.

• Raghavan, C., M. E. B. Naredo, H. H. Wang, G. Atienza, B. Liu et al., 2007 Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Molecular Breeding 19: 87-101

• Rogers, C., J. Wen, R. Chen and G. Oldroyd, 2009 Deletion based reverse genetics in Medicago truncatula. Plant Physiol. 151: 1239-1249

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• Sabetta, W., V. Alba, A. Blanco and C. Montemurro, 2011 sunTILL: a TILLING resource for gene function analysis in sunflower. Plant Methods, 7

• Slade, A. J., S. I. Fuerstenberg, D. Loeffler, M. N. Steine and D. Facciotti, 2005 A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol, 23: 75-81

• Slade, A. J., C. Mcguire, D. Loeffler, J. Mullenberg, W. Skinner et al., 2012 Development of high amylose wheat through TILLING. Bmc Plant Biology, 12: 69

• Sreelakshmi, Y., S. Gupta, R. Bodanapu, V. S. Chauhan, M. Hanjabam et al., 2010 NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications. Plant Methods, 6

• Stephenson, P., D. Baker, T. Girin, A. Perez, S. Amoahet al., 2010 A rich TILLING resource for studying gene function in Brassica rapa. BMC Plant Biol, 10: 62

• Suzuki, T., M. Eiguchi, T. Kumamaru, H. Satoh, H. Matsusaka et al., 2008 MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics,279: 213-223

• Till, B. J., C. Burtner, L. Comai and S. Henikoff, 2004a Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res, 32: 2632-2641

• Till, B. J., J. Cooper, T. H. Tai, P. Colowit, E. A. Greene et al., 2007 Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol, 7: 19

• Till, B. J., J. Jankowicz-cieslak, L. Sagi, O. A. Huynh, H. Utsushi et al., 2010 Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling. Theor Appl Genet, 121: 1381-1389




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• Till, B. J., S. H. Reynolds, C. Weil, N. Springer, C. Burtner et al., 2004b Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol, 4: 12

• Till, B. J., T. Zerr, L. Comai and S. Henikoff, 2006 A protocol for TILLING and Ecotilling in plants and animals. Nature Protocols, 1: 2465-2477

• Triques, K., B. Sturbois, S. Gallais, M. Dalmais, S. Chauvin et al., 2007 Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J, 51: 1116-1125

• Tsai, H., T. Howell, R. Nitcher, V. Missirian, B. Watson et al., 2011 Discovery of Rare Mutations in Populations: TILLING by Sequencing. Plant Physiology, 156: 1257-1268

• Uauy, C., F. Paraiso, P. Colasuonno, R. K. Tran, H. Tsai et al., 2009 A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol, 9: 115

• Van harten, A. M. (Editor), 1998 Mutation Breeding. Theory and Practical Applications. Cambridge University Press, Cambridge

• Wang, N., Y. J. Wang, F. Tian, G. J. King, C. Y. Zhang et al., 2008 A functional genomics resource for Brassica napus: development of an EMS mutagenized population and discovery of FAE1 point mutations by TILLING. New Phytologist, 180: 751-765

• Xin, Z., M. L. Wang, N. A. Barkley, G. Burow, C. Franks et al., 2008 Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population. BMC Plant Biol, 8: 103


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