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

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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

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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

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Perico

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(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

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80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98Percent sequence identity

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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)

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1 201 401 601 801 1001 1201 1401 1601 1801

Number of sequences sampled

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Richness by sample

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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