2015 hm clause

transcript

Controlling Plant Traits with Environment, Genetics and Genomics

Kevin M. Folta

Professor and ChairHorticultural Sciences Department

University of Florida

Kevin M. Folta, Professor and Chair, Kevin M. Folta, Professor and Chair, Horticultural Sciences Department, University of FloridaHorticultural Sciences Department, University of Florida

Environmental, Genetic and

Genomic Manipulation of High Value Plant

Traits

THE THREE THINGS I DO…

SCIENCE SCIENCE COMMUNICATIONCOMMUNICATION

SPONSOREDSPONSOREDRESEARCHRESEARCH

DEPARTMENTDEPARTMENTCHAIRMANCHAIRMAN

• Functional genomicsFunctional genomics• Gene discoveryGene discovery• Marker developmentMarker development• Light signalingLight signaling• Light effects on high-Light effects on high-

value plant /fruit value plant /fruit traitstraits

• Promoting public Promoting public understanding of understanding of biotechnologybiotechnology

• Training scientists to Training scientists to discuss biotech with discuss biotech with concerned concerned audiences. audiences.

• Breeding / new varietiesBreeding / new varieties• Crop physiology and Crop physiology and

productionproduction• Molecular geneticsMolecular genetics• GenomicsGenomics• Organic and Organic and

sustainable productionsustainable production• Weed scienceWeed science• Plant nutrition, water Plant nutrition, water

useuse• Space biologySpace biology• Cell and developmental Cell and developmental

biologybiology• Postharvest physiologyPostharvest physiology

Environmental modification of plant traits using narrow bandwidth illumination

Novel genomics approaches to identifying genes affecting fruit flavors

Identifying new regulators of plant metabolism

Genetics EnvironmentGxE

Breeding, induced mutations, selection, GMO

Not so much control. Spacing, colored mulches, fertilizer… EMO!

1. Photomorphogenesis and plant development

2. Testing the hypothesis – Pre-harvest control of plant traits

3. Testing the hypothesis – Post-harvest control of plant product quality

4. Strawberry flavors and novel methods of marker discovery

PhytochromesPhytochromesCryptochromesCryptochromes

PhototropinsPhototropins

LOV-domain proteinsLOV-domain proteins

Green Sensor? Green Sensor?

The Light SensoriumThe Light Sensorium

UVR8UVR8

If this is so great, why don’t we do it now?

Application

Arabidopsis thaliana

Broccoli Mustards Kales

Why Microgreens are a Great System!!

Experiments are fast (a few days)

Experiments are simple (put in light, measure)

Data show that plants are not Arabidopsis

Information creates new IP and information for growers\

These are a high-value specialty crop and there is significant interest in new colors and flavors.

New concepts in basic plant science

“Microgreens”

Colorful or flavorful seedlings

High value, $60-$80 / lb

Huge demand

Painting Seedlings with Light

Far-red blue-red

Plasticity in Beet Sprouts

Blue-far red

red-far red

Broccoli Sprouts

white red FR red FR red blue blue blue FR

Light Quality

Relative anthocyanin

Quantity and Quality of Glucosinolates

Glucosinolate (µmol g-1)

Light condition

Dark White Far-Red Red Blue

Aliphatic

4MSOB1.72 ±

1.68 ± 0.12

a2.48 ±

1.88 ± 0.35

ab 1.77 ± 0.05 a

5MSOP0.17 ±

0.12 ± 0.02

a0.28 ±

0.17 ± 0.01

a 0.17 ± 0.08 a

40HI3M0.58 ±

0.17 ± 0.01

b0.27 ±

0.15 ± 0.02

b 0.29 ± 0.10ab

4MTB0.21 ±

0.01ab

0.25 ± 0.02

a0.27 ±

0.04ab

0.16 ± 0.02

b 0.37 ± 0.23ab

unknown0.12 ±

0.26 ± 0.02

0.20 ± 0.03

0.28 ± 0.03

bc 0.34 ± 0.05 c

Total aliphatic

2.80 ± 0.26

2.48 ± 0.15

a3.50 ±

2.64 ± 0.17

ab 2.94 ± 0.18ab

Indole

I3M0.18 ±

0.06 ± 0.01

b0.11 ±

0.11 ± 0.03

abc 0.10 ± 0.01 c

4MTI3M0.02 ±

0.01 ± 0.00

0.01 ± 0.01

0.02 ± 0.01

ac 0.01 ± 0.00 b

1MTI3M0.12 ±

0.02 ± 0.00

b0.03 ±

0.01bc

0.04 ± 0.00

ac 0.02 ± 0.00 b

Total indole0.32 ±

0.09 ± 0.01

b0.15 ±

0.17 ± 0.03

c 0.13 ± 0.01 c

Total3.12 ±

0.29ab

2.57 ± 0.15

a3.65 ±

2.81 ± 0.14

ab 3.07 ± 0.18ab

Carvalho and Folta, 2014

Specialty lettuces

Can we breed for the trait of phenotypic plasticity?

One genotype, many phenotypes

w/ Thomas Colquhoun, Dave Clark and their students

Controlling Sensory Quality

Experimental Plan

Flowers, Fruit, Herbs in experimental conditions

Analyze by GC/MS

Grassy- green

Apple/pineapple

Fruity, banana

Fruity, sweet

Manipulation of the light spectrum allows us to predictably steer plant physiology, development and metabolite accumulation

Microgreens are a high-value system to begin to investigate the limits of light effects on seedling traits

Specific wavelengths are now being investigated for the abiltiy to change post-harvest quality.

Strawberries and FlavorsStrawberries and Flavors

Applications of Genetics and GenomicsApplications of Genetics and Genomics

Coupling Gene to Function in Strawberry (Fragaria spp.)

Kevin M. Folta Professsor and Chair

Horticultural Sciences DepartmentUniversity of FloridaGainesville, FL USA

kfolta@ufl.edu

Jeremy Pillet, Alan Chambers, Iraida Amaya, Hao-Wei Yu, Vance Whitaker, Jin-He Bai, Anne Plotto, Michael

Schwieterman, Thomas Colquhoun

Vance Whitaker

Anne Plotto

Vance Whitaker

Alan Chambers Jeremy Pillet Thomas Colquhoun& Mike Schwieterman

What is Strawberry? Small, herbaceous perennial in the Rosaceae

family. Exists at several ploidy levels, wild are typically

diploid, cultivated varieties are octoploid Diploid draft genome in 2010 Allo/autopolyploid commercial varieties Breeding for yield, size, flowering habits and

postharvest quality has de-prioritized flavors

Diploid Strawberry- the Arabidopsis of the Specialty Crops!

Arabidopsis thaliana F. vesca

Genome size 163 Mb 240 Mb

Perenniality No Yes

Plant Size/Stature Grow 2.5 inch pot 4 inch pots

Genome sequenced Yes Yes

EST Resources Much, but incomplete Next Gen based, > massive

Reproduction Flowers Flowers or runners

Stable Transformation Floral Dip Agro, particle bombardment

Transient Expression System In fruit.

Cycling (seed to seed) 8 weeks 12 -16 weeks

Genetic tools Many! Limited T-DNA, activation tag, EMS, TILLING

Relevance to crops Some MUCH!

Strawberries and FlavorsStrawberries and Flavors

Applications of Genetics and GenomicsApplications of Genetics and Genomics

There is room to improve strawberry fruit quality

•Flavors•Post-harvest attributes•Nutriceuticals

•Can speed with marker-assisted breeding

•How do we leverage genomics tools?

“Consumer-Assisted Selection”

Consumer sensory panels and psychophysics evaluation

Analytical chemistry

Resequencing and allele discovery

Marker development,selection and validation

Gamma Decalactone – imparts peachy flavors

Methyl Anthranilate – “sticky” grape

Gamma Decalactone – imparts peachy flavors

Methyl Anthranilate – “sticky” grape

Present in about 50% of germplasmInherited as a single dominant locus

Not present in existing varieties

Found in diploid strawberry and some older heirloom varieties

Linking Traits to Genes Identification of two flavor-associated genes

through analysis of bulk-segregant RNAseq

Identification of flavonoid-pathway modulating genes using transcription correlation network analysis.

Mara des BoisElyana

γ-DecalactoneMethyl anthranilate

RNA-seq ResultsGenes with transcript evidence

>12,000

Chambers et al,. 2014 BMC Genomics

RNAseq Results

Very small number of transcripts tightly associated with the presence/absence of gamma decalactone.

One was an omega-6-desaturase

Hoffman et al., 2006

Transient Expression /

Suppression via Agroinfiltration

Stable Transformation

Octoploid

Diploid

Functional Validation Systems

CTRL 1 CTRL 2 CTRL 3 hpFAD1 1 hpFAD1 2 hpFAD1 3 hpFAD1 4

FAD1 expression

CTRL 1 CTRL 2 CTRL 3 hpFAD2 hpFAD3 hpFAD4 hpFAD5

Gamma Decalactone content

Silencing of FAD1 leads to decreased

gamma decalactone

accumulation

I. Amaya, J. Pillet, et al. unpub.

Plants Possessing the FaFAD1 Gene Can Produce Gamma DThe defect is due to a large deletion on LG 3

A PCR-based MarkerThe Gene is Not Detected in

Non-Gamma-D Genotypes

Methyl Antranilate ProductionCandidates

MA found in only several non-production varieties

Repeated grouping around MA presence/absence

Methyl transferase

One Transcript is Always Expressed AmongMA Producers

Methyltransferase+ methyl donor

Anthranilic acid Methyl anthranilate

A Transcript Common in All Fruits that Produce MA Encodes a Methyltransferase

Mara C5 Mara C6 Mara C7 S4 1 S4 2 S4 5 S4 6

Methyltransferase expression

200000

400000

600000

800000

1000000

Mara C5 Mara C6 Mara C7 S4 1 S4 2 S4 5 S4 6

Methyl Anthranilate content

Transient Silencing of the Methyltransferase leads to lower levels of MA in ripe fruits

Transcript Abundance

Methyltransferase Expressed in E.coli, then Fed Anthranilate

Peach notes from Gamma Decalactone require the presence of FAD1

A methyltransferase is necessary, but not sufficient, for production of MA in cultivated varieties tested. Marker in development.

Transcript Network Correlation Analysis

“Guilt by association”

How can we mine existing datasets to identify novel regulators of known pathways?

Flavonoid Pathway

Has a central role in flavors, colors and nutriceutical compounds.

Well understood.

Transcripts in the pathway are known to be co-regualted

Transcripts for core flavonoid pathway genes are co-expressed

Hoffman et al., 2006

Transient Expression /

Suppression via Agroinfiltration

Stable Transformation

Octoploid

Diploid

Functional Validation Systems

TCP11 Overexpression Affects Nodes of Flavonoid Pathway

PCL1-like Overexpression Affects Nodes of Flavonoid Pathway

Repression of a SCARECROW-LIKE transcription factor decreases expression throughout the pathway.

Validation will continue in stable transgenics

Conclusions

Bulk-segregant analysis of transcriptome data can be used to identify candidate genes for flavors.

New regulators of well-known pathways can be identified through analysis of transcript correlation networks.

Transcriptomes from small populations can be powerful if samples are carefully chosen, and can be mined for new findings.

Grateful to:

kfolta.blogspot.com@kevinfolta

kevinfolta@gmail.com

"There is a path to truth and sincerity that you must guard and defend“

-- Teruyuki Okazaki

“It is our mission to stand up for the truth that science gives us.”

Dr. Jack PayneSVP UF/IFAS

The shoulders we stand upon

Those that support our science (and often unpopular outreach)

Ideas that inspire my outreach

The agencies that fund us.