Applying agricultural biotechnology tools and capabilities to enhance food security and nutrition from local food crops to stimulate

sustainable income opportunities for smallholders to reduce poverty

FAO Symposium February 16th & 17th 2016

Howard-Yana Shapiro, PhDChief Agricultural Officer, Mars, Incorporated

Fellow Mars Advanced Research InstituteSenior Fellow, The University of California Davis

Science Advisor MITDistinguished Fellow The World Agroforestry Centre

BREEDING PROGRAMS NEED TO INTEGRATE GENOMICS AND BIOTECHNOLOGY WITH THE CONVENTIONAL BREEDING CYCLE

RELEASE OF CULTIVARS

NOVEL TRANSGENES

NOVEL

GERMPLASM INPUT

FIELD TRIALSCROSSES

SELECTION

+ MARKERS (CAUSAL GENES)

TRANSFORMATION

INCREASING VARIATION

DECREASING VARIATION

Adapted from C.M. Rick

BREEDING PROGRAMS MUST ADAPT TO THE REALITY OFDISRUPTIVE TECHNOLOGIES AND UNPRECEDENTED AMOUNTS OF MARKER, SEQUENCE, PHENOTYPIC,

& META INFORMATION

Imminent Disruptive Technologies for Plant Improvement

DNA Sequencing• Even higher throughput (Illumina)• Even longer reads (PacBio)• Even simpler instruments (Oxford Nanopore)

Unprecedented amounts of data: “Big Data”Inexpensive High Throughput GenotypingHost-Induced Gene Silencing (HIGS)Genome Editing, CRISPR/Cas9Microbiome and Endophyte Exploitation

MinION: disposable DNA sequencer

Disruptive Uncommon Collaboration for Plant Improvement

The AFRICAN ORPHAN CROPS CONSORTIUM & The African Plant Breeding Academy

An Uncommon Collaboration

HOW CAN WE ACHIEVE DURABLE RESISTANCE?

Strategies to enhance durability of resistance

Low tech, low risk (ephemeral?):Pyramid/stack multiple major resistance genes.Heterogeneous deployment in space and time.

Medium tech (more durable?):Utilize durable, minor? genes.Use knowledge of pathogen variability to inform

strategies for resistance gene deployment. New opportunities from novel technologies for

rapid, comprehensive genotyping.High tech (very durable?):

Different evolutionary hurdles (RNAi/HIGS/CRISPR-Cas9).

Proof of concept: Bremia lactucae, lettuce downy mildew (related to Phytophthora spp.)Govindarajulu et al. (2014). Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Pl. Biotech. J. DOI: 10.1111/pbi.1230.

Primary targets: Puccinia striiformis, wheat stripe rust and P. graminis, wheat stem rust (UG99)

Host Induced Gene Silencing of Fungal Pathogens

EXAMPLES OF HOST-INDUCED GENE SILENCING IN MULTIPLE PATHOSYSTEMS

Monocot and dicot hosts. Diverse range of pathogens and pests.Both biotrophic and necrotrophic modes of nutrition.

Weiberg & Jin 2015 Curr. Opin. Biotech.Dutta et al. 2015. Frontiers Microbiol.

Host Pathogen Disease InteractionLettuce Bremia lactucae Downy mildew BiotrophicCorn Diabrotica virgifera Western corn rootworm BiotrophicArabidopsis Heterodera schachtii Sugar beet cyst nematode BiotrophicSoybean Heterodera glycines Cyst nematode BiotrophicTomato Meloidogyne javanica Root knot nematode BiotrophicLettuce Tryphysaria spp Parasitic plant BiotrophicWheat Puccinia striiformis Rust BiotrophicBarely Erysiphe graminis Powdery mildew BiotrophicBanana Fusarium oxysporum Wilt HemibiotrophicBarley F. graminearum Head blight HemibiotrophicTobacco F. verticillioides Ear rot in maize NecrotrophicTomato Botrytis cinerea Soft rot Necrotrophic

Transformed/genome edited varieties

Host Induced Gene Silencing (HIGS) has now been demonstrated in diverse pathosystems. HIGS is therefore likely to be effective against the Aspergillus spp. that produce aflatoxins in multiple crop species.Could provide high levels of resistance to Aspergillus parasiticus and A. flavus and greatly reduce levels of aflatoxin contamination.Could target Aspergillus fungus and/or toxin production. Because the RNAi trigger sequences are several hundred bases long and target vital genes, it will take a major change in the pathogen to overcome it. Consequently resistance based on HIGS is likely to be durable.Because the active component of HIGS is RNA rather than protein, there may be reduced regulatory concerns. Would maintain resistance across a range of farmer practices and environmental conditions.

AFLATOXINS

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Aflatoxins are naturally occurring mycotoxins that are produced by by many species of Aspergillus, a fungus, the most notable being Aspegillus flavus and Aspergillus paraciticus.Found in cassava, cacao, maize, peanuts, millet, rice, sorghum, sunflower seeds trees nuts and many spices.“The prevalence and level of human exposure to aflatoxins on a global scale have been reviewed, and the resulting conclusion was that approximately 4.5 billion persons living in developing countries are chronically exposed to largely uncontrolled amounts of the toxin.

Seems to exacerbate many of the health challenges in the developing world, especially in children.Childhood nutrition and development interfered with.Studies show that aflatoxin interferes with vitamins A and D, iron, selenium, and zinc uptake. Reduces rate of growth and neurological developmentCauses Stunting

Aflatoxin is regulated in the USA by the FDA with an action level of 20 parts per billion (ppb) in most foods and feed

How much is 20 ppb?

1 ppb = 1 part aflatoxin per billion parts grain substrate, or = 0.000000001 (10-9) grams aflatoxin per gram of grain substrate or = 1 nanogram (ng) per gram.

1 ppb is the equivalent of 1 second in 32 years.

1 kernel of maize can have 50,000 ppb of aflatoxin; 40 of these kernels could contaminate a bushel of corn above the 20ppb FDA action level.

Similar, parallel approaches could be implemented formaize, peanut, rice et al. to target multiple diseases and aflatoxin

reduction simultaneously using HIGS and conventional resistance genes

Similar, parallel approaches could be implemented for reduction of other mycotoxins e.g. fumonicins in maize, wheat, et al.

EXPLOITING VARIATION: OPTIONS FOR GENOME EDITING

Ease of creation (specific proteins or RNA)Activity (multigene modifications)Specificity (off-target modifications?)

Deletions (gene knock-outs)Point mutations (allele modification)DNA substitutions (allele replacements)Donor with reporter (indicators)RNA-guided transcriptional modulation

Multiple applicationsfor CRISPR/Cas9-mediated genome editing:

EXPLOITING VARIATION

CRISPR/Cas9-Mediated Genome EditingA disruptive (breeding) technologyMolecular aspects advancing very rapidlyGene knock-outs easyBoth copies of a gene often knocked outMultiple genes (-> 10) can be targeted simultaneouslyAllele replacements and gene insertions much more difficultDemonstrated in numerous crops: rice, sorghum, wheat, cotton, lettuce, corn, orange, ……Improvements in delivery neededTissue culture mutagenicGermline modification without tissue culture advantageous

Future Prospect: Resistance Gene StackingUse CRISPR/Cas9 to integrate known genes within an existing cluster: multiple R genes effective against all known pathotypes and multiple viral, bacterial, fungal, oomycete diseases as well as insect pests and nematodesComplement with HIGS genes against multiple pathogensInherited as single Mendelian unit (recombination repressed)Herbicide resistance gene (e.g. ALS) as selectable marker for locusNeeds cloned genes for resistance to each diseaseGene stack expanded as more resistance genes become availableGenes replaced when overcome by changes in the pathogens

Dm# HIGS# Dm## ALS Xar## Vr## Dm### HIGS##

Crispr (or CRISPR) stands for “clustered regularly interspaced short palindromic repeats” – palindromic meaning DNA sequences that read the same way in either direction

Crispr was Science’s 2015 “Breakthrough of the Year,” but it had been a runner-up in two previous years: “In short, it is only slightly hyperbolic to say that if scientists can dream of a genetic manipulation, Crispr can now make it happen…. For better or worse, we all live in Crispr’s world.

A Few Comments

Dr. Doudna organized a meeting in Napa in January 2015 on guidelines for use of the technique. “Shortly after the meeting, we published a prospective article in Science that urged the global scientific community to refrain from using any genome-editing tools to modify human embryos for clinical applications at this time,” Dr. Doudna wrote in Nature. “We also recommended that public meetings be convened to educate non-scientists and to enable further discussion about how research and applications of genome engineering might be pursued responsibly.” “It was clear that governments, regulators and others were unaware of the breakneck pace of genome-editing research,” she added. “Who besides the scientists using the technique would be able to lead an open conversation about its repercussions?”

Jennifer Doudna, “My Whirlwind year with CRISPR,” Nature, 24/31 December, 2015

While there is confusion in the public mind about what constitutes a genetically modified organism (GMO), it is generally accepted to be an organism into which genetic material from another species as been introduced. Crispr-Cas9 avoids this transgenic approach in many of its applications. Thus governments, companies and the public have not begun to decide how it should be regulated or even defined.