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Ecological Genomicsat Konza Prairie
Loretta Johnson, Mike HermanEcological Genomics Institute
KSU
Ecological Genomicsat Konza Prairie
Loretta Johnson, Mike HermanEcological Genomics Institute
KSU
What is Ecological Genomics?What is Ecological Genomics?• integrative field • genetic mechanisms underlying responses
of organisms to their natural environment.• genome-enabled approaches
– functions of single or multiple genes.• biochemical, physiological, morphological,
behavioral responses of adaptive significance
• “finding the genes that matter”.Martin Feder, U of Chicago
Conceptual ModelConceptual Model
ECOSYSTEM RESPONSE
POPULATION ANDCOMMUNITY RESPONSE
ORGANISMAL RESPONSE
GENOTYPICCONTRAINTS
GENE EXPRESSION
PHENOTYPIC EFFECT ONDEVELOPMENT/PHYSIOLOGY
EC
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EC
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GY
GE
NE
TIC
SG
EN
ETI
CS
EC
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EC
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Rationale for Ecological Genomics
• Why is an ecological context necessary?
– Many genomes sequenced, but the function of many genes are not known
– Several studies show completely different gene expression in parallel controlled growth chamber and field studies
• Why is a genetic/genomic context necessary?– Elucidates underlying mechanism for ecological response– Better understanding of phenomena– May provide ability to predict response
MC Ungerer, LC Johnson, M Herman 2007. “Ecological Genomics: Finding the genes that matter in the environment”. Heredity
What are the cross-cutting questions?
• What is the genetic basis for ecological responses to the environment?
• What are the regulatory and genetic pathways involved in organismal responses to their environment?
• What is the ecological context necessary to understand gene expression within organisms?
Approach:Apply the genomic tools available for model organisms to native
organisms growing under field conditions
Advantages • Ecological relevance• Organisms growing under field conditions• Select ecologically dominant organisms
Approach: • to use genome sequences of close relatives of model
organisms to make inferences about non-model organisms
Microarrays and Gene Expression
From:Konstantin V. Krutovskii and David B. Neale (2001) Food and Agriculture Organization of the United Nations, Forest Genetic Resources Working Papers
• 2 growth conditions. e.g. high/low N, H2O
• isolate mRNA →cDNA (complementary DNA)
• hybridize to “chip” with fragments of all the genes.
• readout of gene expressed only in each condition or both conditions.
used to study expression of all genes in a genome at once!
• Common ancestor ~ 10-20 million years.
Applying the genome tools available for corn to big bluestem
Family: Poaceae
Subfamily: Panicoideae
Tribe: Andropogoneae
Genus: Andropogon
Species: Andropogongerardii Vitman
Family : Poaceae
Subfamily: Panicoideae
Tribe: Andropogoneae
Genus: Zea
Species: Zea mays L.
Big Bluestem Corn
Andropogon
• Mass sequencing techniques (454, Solexa)
• Generates 20 million bases in 5 hours
• Produces 200,000 randomized 100bp reads
• Enables new kinds of microbial diversity studies
• Soon many more ecological relevant species can be sequenced
Illustration from www.454.com
New sequencing technology enables
ecological discoveries
Global change
Microbial
Diversity(454 technology)
Ecological
interactions
Functional role of plantendophytes
Genomics of plant-pathogeninteractions
Genomics of plant-herbivoreinteractions
Nematodes ecologicalgenomics
Ecological genomics of big bluestemresponses to simulated climate change
Plant genomics response toirrigation and N
Linking genes and ecosystems: transcriptomeand physiological responses to climate change
Ambient Altered
Physiologicalvariables
Physiologicalvariables
Array BB1rna rna
Physiologicalvariables
Physiologicalvariables
Array IG1rna rna
Andropogon gerardii
Sorghastrum nutans
……..……..……..
……..……..……..
Karen Garrett, Jan Leach, Scot Hulbert, Steve Travers, Zhongwen Tang,Jianfa Bai, Amgad Saleh, Mendy Smith, Alan Knapp and Phil Fay
Goals: 1) Use microarrays to measure transcriptome of native grasses under ambient and
proposed climate change conditions 2) determine candidate genes and functional groups for stress response to climate
change, 3) link physiological phenotype to transcriptome patterns, 4) determine role of individual genes in ecosystem function
Methodology
-Use maize arrays to assess transcriptomeunder variable environmental conditions-Measure physiological variables on same plants- link physiology, expression and environment
• there were statistically significant relationships between gene transcription levels and environmental temperature variables but not water availability variables• functional analysis indicated big bluestem upregulates mostly genes associated with chaperone proteins, and protein biosynthesis in response to stress, while downregulatingtranscription factors
Conclusions
Gene expression responses to delayed precipitation in RaMPs
Travers et al. 2007
Steve Travers
Big bluestem
Nematode Ecological GenomicsKen Jones, Joe Coolon, John Blair, Tim Todd, Michael Herman
Nematode Ecological GenomicsKen Jones, Joe Coolon, John Blair, Tim Todd, Michael Herman
Genetic basis of nematode community responses to environmental change– Nematodes respond quickly and are bioindicators of
environmental disturbance– Define community change at the lowest level using
molecular tools• identify interesting native taxa to study genomic responses• field studies of native soil nematodes
– Determine ecological drivers affecting taxon responses– Identify interesting genes to study
• laboratory-based studies using microarray analysis of a model nematode: C. elegans
– Determine expression levels of genes of interest in native study taxa on Konza prairie
Bact 1 Bact 2
• Lab-based approach using a model organism• Gene discovery using the lab nematode C. elegans• Isolate 3 soil bacteria from Konza soils• Grow C. elegans on Konza bacteria• Use microarrays to discover genes differentially expressed in
response to the different bacterial environments
Are changes in food resources (bacteria) causing the observed changes in the nematode community?
202 unique genes!
Innate immunity MetabolismCuticle/collagenUnknown functionTXN/TLNDevelopmentRibosomeOther
Potential functions:
Are changes in food resources (bacteria) causing the observed changes in the nematode community?
It may be relatedto nematode species ability to respond to pathogens
• Which genes are functionally important? Pathogen-related• Disrupt gene function by mutation• Assess function by measuring fitness of mutants in
identified genes• life table analysis in the lab!
• Predict: removing important gene function will reduce fitness
Environmental and ecological controls on gene expression of root processes in prairie plants
Loretta Johnson & Jyoti Shah, G. K. Surabhi, S. Kumar
Goal: Combine ecology and genomics to better understand root processes under
field conditions -> Identify genes exhibiting altered expression in
response to changing environments (abiotic and biotic stress) e.g., nitrogen, water, grazing
-> Link processes controlling root growth in natural systems to regulation of gene expression in roots
->Take advantage of the genomic resources developed for corn, a close prairie grass relative
• Root processes dominate in tallgrass prairie
• Root productivity exceeds aboveground NPP
• Grasslands contain 15% of the world’s soil carbon, which is primarily derived from root inputs
• Yet, root processes are not well studied
• Candidate genes involved in root proliferation in response to N identified in Arabidopsis
Courtesy photo: Dr Alan Knapp
Why study roots?
PRELIMINARY MICROARRAY RESULTSbig bluestem roots
harvested 2 weeks after N fertilization
11530 features showed hybridization signals (60.05%) to corn features~ 570 genes were significantly hybridized~ 320 showed differential expression
140 genes
Blue=down-regulatedin response to high N
Red= up-regulated in response to high N
186 genes
Control = no N added
High N treatment
2-fold or more up-regulated genes with N
Ribosomal protein
Histones
Growth regulation
DNA binding protein
Plant stress/ Defense
RNA metabolism
Transporter genes Amino acid metabolism
Glycolysis
Lignin biosynthesisLipid metabolism
Glycolysis
Pentose phosphate pathway
Cell wall metabolism
Unknown proteins
Others
Transcription factor Signaling
Root development
:8.7%:6.5%
:3.2%:1.0%
:4.3%:1.0%:4.3%
:3.2%:3.2%
:1.0%:1.0%
3.2%:1.0%
:2.2%:19.5%
:21.7%:4.3%
:7.6%:2.2%
Ribosomal protein
Histones
Growth regulation
DNA binding protein
Plant stress/ Defense
RNA metabolism
Transporter genes Amino acid metabolism
Glycolysis
Lignin biosynthesisLipid metabolism
Glycolysis
Pentose phosphate pathway
Cell wall metabolism
Unknown proteins
Others
Transcription factor Signaling
Root development
:8.7%:6.5%
:3.2%:1.0%
:4.3%:1.0%:4.3%
:3.2%:3.2%
:1.0%:1.0%
3.2%:1.0%
:2.2%:19.5%
:21.7%:4.3%
:7.6%:2.2%
Ribosomal protein Histones Growth harmones DNA binding protein Plant stress/Defense RNA binding protein Transporter genesMembrane associated Nitrogen metabolism Glycolysis Amino acid metabolism Sulfate assimilation Lignin metabolism Respiratory metabolism Unknown proteinsOthers Regulatory proteins (n-metabolism) Signaling Transcription factor
:16.7%:2.1%:2.1%:3.6%:2.9%:2.1%
:1.4%:0.7%
:0.7%:0.7%
:0.7%:0.7%
:2.1%:0.7%
:22.6%:15.3%
:3.6:4.3%
:5.8%
Ribosomal protein Histones Growth harmones DNA binding protein Plant stress/Defense RNA binding protein Transporter genesMembrane associated Nitrogen metabolism Glycolysis Amino acid metabolism Sulfate assimilation Lignin metabolism Respiratory metabolism Unknown proteinsOthers Regulatory proteins (n-metabolism) Signaling Transcription factor
:16.7%:2.1%:2.1%:3.6%:2.9%:2.1%
:1.4%:0.7%
:0.7%:0.7%
:0.7%:0.7%
:2.1%:0.7%
:22.6%:15.3%
:3.6:4.3%
:5.8%
2-fold or more down regulated genes
Global change
Microbial
Diversity(454 technology)
Ecological
interactions
Functional role of plantendophytes
Genomics of plant-pathogeninteractions
Genomics of plant-herbivoreinteractions
Nematodes ecologicalgenomics
Ecological genomics of big bluestemresponses to simulated climate change
Plant genomics response toirrigation and N
Erin Frank
Phytohormone responses to disease and drought stress in Andropogon gerardii
Average SA Concentration by Leaf Section
0200400600800
1000120014001600
Drought Drought +Rust
Watered Watered +Rust
ng/g
Fre
sh T
issu
e
BaseTip
Drought stress doubles disease severity in this context
Erin Frank is comparing gene expression, phytohormone, and polar lipid responses to drought and pathogen stress
Functional role of root endophytes:Arabidopsis microarray dissection of root
endophyte mutualism(Ari Jumponnen)
• Periconia endophytes were isolated from Konza LTER
• Colonize Arabidopsis thaliana
• Improve A. thaliana growth
• Induce defense pathways
• Affymetrix ATH1 arrays to resolve improved growth
Contr
ol
Perico
niasp.
Perico
niamacr
ospino
sa
Shoo
t m
ass
(mg)
100
200
a
c
bA. Periconia in A. thaliana roots
B. Paired mock- and Periconia-inoculated Arabidopsis
C. Arabidopsis growth responses to Periconia
A B
C
Global change
Microbial
Diversity(454 technology)
Ecological
interactions
Functional role of plantendophytes
Genomics of plant-pathogeninteractions
Genomics of plant-herbivoreinteractions
Nematodes ecologicalgenomics
Ecological genomics of big bluestemresponses to simulated climate change
Plant genomics response toirrigation and N
0
200
400
600
800
1000
1200
1400
1600
1800
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98Percent sequence identity
Alp
ha lo
g-se
ries
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Sign
ifican
ce (
p)
0
200
400
600
800
1000
1200
1400
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
P t id tit
Ric
hnes
s
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50S
igni
fican
ce (
p)
Bacterial community response to nitrogen addition(Ken Jones et al)
Bacterial community response to nitrogen addition(Ken Jones et al)
Richness Diversity
• Tag bacterial 16s rDNA sequences by plot to assess community response to perturbations.
• Percent identity of a group of sequences reflects taxonomic level
– e.g. 92% roughly corresponds to family; 97% to species.
• Nitrogen addition reduces richness and diversity
• Question: Is this the reason for the changes in the nematode community?
Massive parallel sequencing to test RaMPeffects on soil microbial diversity
(Ari Jumponnen)• 1,200-1,800 sequences/sample
saturate eukaryon but not bacterial richness in soil
• The temperature or rainfall manipulations did not affect community summary stats (richness, diversity…)
• Individual responsive OTUs (taxa) could be identified (Trichoglossum, Mycosphaerella)
0
100
200
300
400
500
600
700
800
900
1 201 401 601 801 1001 1201 1401 1601 1801
Number of sequences sampled
Num
ber o
f OTU
s ob
serv
ed
Richness by sample
0
50
100
150
200
250
300
350
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98Percent sequence identity
Ambient H20, Ambient T
Ambient H20, Elevated T
Altered H20, Ambient T
Altered H20, Elevated T
Richness by treatment
P>0.05 for all terms.
RaMPS microbe diversity
Future directions for Ecogenwithin the context of Konza
LTER
• Can organisms adjust in time and space to the unprecedented pace of environmental change?
• What are the genetic/molecular mechanisms underlying ecological response to environmental change?
• How can we use long-term experiments as an opportunity to investigate potential genetic differentiation over decades?
• Does genotypic diversity of the dominant plant species have cascading effects to higher trophiclevels?
For more information, visit our website at www.ksu.edu/ecogen