Applying agricultural biotechnology tools and capabilities to enhance food security and nutrition from local food crops to stimulate sustainable income opportunities for small holder farmers to reduce poverty presentation by "Howard-Yana Shapiro, Mars Incorporated, Dranesville and University of California Davis, Davis, United States of America"

1. 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, PhD
Chief Agricultural Officer, Mars, Incorporated
Fellow Mars Advanced Research Institute
Senior Fellow, The University of California Davis
Science Advisor MIT
Distinguished Fellow The World Agroforestry Centre

2. 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 OF
DISRUPTIVE TECHNOLOGIES AND UNPRECEDENTED
AMOUNTS OF MARKER, SEQUENCE, PHENOTYPIC,
& META INFORMATION

3. 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 Genotyping
Host-Induced Gene Silencing (HIGS)
Genome Editing, CRISPR/Cas9
Microbiome and Endophyte Exploitation
MinION: disposable
DNA sequencer

4. Disruptive Uncommon Collaboration for
Plant Improvement
The AFRICAN ORPHAN CROPS CONSORTIUM &
The African Plant Breeding Academy

5. An Uncommon Collaboration

6. 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).

7. 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

8. 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 Interaction
Lettuce Bremia lactucae Downy mildew Biotrophic
Corn Diabrotica virgifera Western corn rootworm Biotrophic
Arabidopsis Heterodera schachtii Sugar beet cyst nematode Biotrophic
Soybean Heterodera glycines Cyst nematode Biotrophic
Tomato Meloidogyne javanica Root knot nematode Biotrophic
Lettuce Tryphysaria spp Parasitic plant Biotrophic
Wheat Puccinia striiformis Rust Biotrophic
Barely Erysiphe graminis Powdery mildew Biotrophic
Banana Fusarium oxysporum Wilt Hemibiotrophic
Barley F. graminearum Head blight Hemibiotrophic
Tobacco F. verticillioides Ear rot in maize Necrotrophic
Tomato Botrytis cinerea Soft rot Necrotrophic

9. 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.

10. AFLATOXINS
10

11. 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.

12. 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
development
Causes Stunting

13. 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?

14. 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.

15. Similar, parallel approaches could be implemented for
maize, 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.

16. EXPLOITING VARIATION: OPTIONS FOR GENOME EDITING
Ease of creation
(specific proteins or RNA)
Activity
(multigene modifications)
Specificity
(off-target modifications?)

17. Deletions (gene knock-outs)
Point mutations (allele modification)
DNA substitutions (allele replacements)
Donor with reporter (indicators)
RNA-guided transcriptional modulation
Multiple applications
for CRISPR/Cas9-mediated
genome editing:
EXPLOITING VARIATION

18. CRISPR/Cas9-Mediated Genome Editing
A disruptive (breeding) technology
Molecular aspects advancing very rapidly
Gene knock-outs easy
Both copies of a gene often knocked out
Multiple genes (-> 10) can be targeted simultaneously
Allele replacements and gene insertions much more difficult
Demonstrated in numerous crops: rice, sorghum, wheat, cotton,
lettuce, corn, orange, ……
Improvements in delivery needed
Tissue culture mutagenic
Germline modification without tissue culture advantageous

19. Future Prospect: Resistance Gene Stacking
Use 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 nematodes
Complement with HIGS genes against multiple pathogens
Inherited as single Mendelian unit (recombination repressed)
Herbicide resistance gene (e.g. ALS) as selectable marker for locus
Needs cloned genes for resistance to each disease
Gene stack expanded as more resistance genes become available
Genes replaced when overcome by changes in the pathogens
Dm# HIGS# Dm## ALS Xar## Vr## Dm### HIGS##

20. 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

21. 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

22. 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.