| Date post: | 07-Aug-2015 |
| Category: |
Science |
| Upload: | university-of-florida |
| View: | 74 times |
| Download: | 4 times |
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
E
G
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
ON
OFF
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
white
Far-red blue-red
dark
red
Plasticity in Beet Sprouts
blue
Blue-far red
red-far red
Broccoli Sprouts
0
2
4
6
8
10
12
14
16
18
white red FR red FR red blue blue blue FR
Light Quality
An
tho
cya
nin
ng
/g F
.W.
Relative anthocyanin
Quantity and Quality of Glucosinolates
Glucosinolate (µmol g-1)
Light condition
Dark White Far-Red Red Blue
Aliphatic
4MSOB1.72 ±
0.15a
1.68 ± 0.12
a2.48 ±
0.16b
1.88 ± 0.35
ab 1.77 ± 0.05 a
5MSOP0.17 ±
0.01a
0.12 ± 0.02
a0.28 ±
0.16a
0.17 ± 0.01
a 0.17 ± 0.08 a
40HI3M0.58 ±
0.09a
0.17 ± 0.01
b0.27 ±
0.05b
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 ±
0.01a
0.26 ± 0.02
bc
0.20 ± 0.03
ab
0.28 ± 0.03
bc 0.34 ± 0.05 c
Total aliphatic
2.80 ± 0.26
ab
2.48 ± 0.15
a3.50 ±
0.37b
2.64 ± 0.17
ab 2.94 ± 0.18ab
Indole
I3M0.18 ±
0.01a
0.06 ± 0.01
b0.11 ±
0.01c
0.11 ± 0.03
abc 0.10 ± 0.01 c
4MTI3M0.02 ±
0.00a
0.01 ± 0.00
bc
0.01 ± 0.01
ab
0.02 ± 0.01
ac 0.01 ± 0.00 b
1MTI3M0.12 ±
0.03a
0.02 ± 0.00
b0.03 ±
0.01bc
0.04 ± 0.00
ac 0.02 ± 0.00 b
Total indole0.32 ±
0.04a
0.09 ± 0.01
b0.15 ±
0.02c
0.17 ± 0.03
c 0.13 ± 0.01 c
Total3.12 ±
0.29ab
2.57 ± 0.15
a3.65 ±
0.40b
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
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
>1,0
00
>1,2
00
>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
0
10
20
30
40
50
60
70
80
90
CTRL 1 CTRL 2 CTRL 3 hpFAD1 1 hpFAD1 2 hpFAD1 3 hpFAD1 4
FAD1 expression
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
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
0
20
40
60
80
100
120
140
160
180F_
Mar
a
F_El
yana
F_00
6
F_02
4
F_03
7
F_04
2
F_05
1
F_08
9
F_09
1
F_09
3
F_09
8
F_10
3
F_15
2
F_19
3
F_20
3
F_20
4
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
0
0,5
1
1,5
2
2,5
3
Mara C5 Mara C6 Mara C7 S4 1 S4 2 S4 5 S4 6
Methyltransferase expression
0
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
"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.
