DNA analysis: the last frontier for biologists Lesson 1 of MEFF Spring 2007 Taught by Dr. Matteo Garbelotto.

DNA analysis: the last frontier for biologists

• Lesson 1 of MEFF Spring 2007

• Taught by Dr. Matteo Garbelotto

Forest pathology and mycology:Forest pathology and mycology:the P.I.the P.I.

•THE LAB

•IMPORTANCE OF FORESTS

Aesthetic valueAesthetic value

Production of timber and fuelProduction of timber and fuel

Terrestrial ecosystems containing Terrestrial ecosystems containing maximum biodiversitymaximum biodiversity

Habitat for millionsHabitat for millions

Huge role in geological protection Huge role in geological protection from erosion, floods, etc.from erosion, floods, etc.

One of the most significant sinks for carbon

FORESTS ARE ESSENTIAL FORESTS ARE ESSENTIAL FOR LIFE ON THE PLANETFOR LIFE ON THE PLANET

FUNGI ARE ESSENTIAL FOR FORESTS

WHAT ARE FUNGI ?WHAT ARE FUNGI ?

• Individuals belonging to the kingdom fungi• They are actually more closely related to

animals than plants (cell wall contains chitin, do not photosynthesize)

• The feed by extrogenous digestion (food base digested outside their cells and then simple elements absorbed )

• Indefinite growth: size can range from unicellular organisms) to the largest beings in the world

Largest living organisms in the Largest living organisms in the world are fungiworld are fungi

Aerial view of a single fungal Aerial view of a single fungal individual growing in the soil individual growing in the soil

and killing plantsand killing plants

Tens of acres occupied by a single individual, older than Tens of acres occupied by a single individual, older than

5000 years!!!! (Northern Michigan)5000 years!!!! (Northern Michigan)

Thanks to their web-like indefinite Thanks to their web-like indefinite growth in soil and plant substrates growth in soil and plant substrates and their way of digesting nutrients and their way of digesting nutrients fungi play a critical role in recycling fungi play a critical role in recycling

nutrients which can then be nutrients which can then be reutilized by plants reutilized by plants

•Fungi like this one will actually decay the woody matter and physically free space for new generations of trees, besides recycling the nutrients

The weblike structure of fungi, usually The weblike structure of fungi, usually immersed in the soil or in plant matter is immersed in the soil or in plant matter is involved in an essential symbiosis that involved in an essential symbiosis that

greatly enhances the ability of plants to growgreatly enhances the ability of plants to grow

•pianta

•fungo

The visible part of root tips of The visible part of root tips of most trees is actually a mantle most trees is actually a mantle of fungal hyphae fused with the of fungal hyphae fused with the

plant tissueplant tissue

What is the deal of this mutualism?

• Fungus absorbs nutrients for plants• Plant gives fungus carbohydrates it produces via

photosynthesis

There are thousands of mycorrhzial fungal There are thousands of mycorrhzial fungal species, and only at times do they produce the species, and only at times do they produce the

classical fruit body (e.g.mushrooms) above classical fruit body (e.g.mushrooms) above groundground

•In absence of fruit body: how can we identify them?

DNA can be extracted from any part of an DNA can be extracted from any part of an organism, like the web-like hyphae emanating organism, like the web-like hyphae emanating

from this root tipfrom this root tip

•DNA sequence identified these threads as Tricholoma matsutake

Tricholoma matsutakeTricholoma matsutake grows in Eurasia and is grows in Eurasia and is prized enormously by the Japanese who pay prized enormously by the Japanese who pay

over 1000 dollars a poundover 1000 dollars a pound

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Using DNA data we were able to prove Using DNA data we were able to prove Tricholoma matsutakeTricholoma matsutake also grows in the also grows in the

eastern US and in Mexicoeastern US and in Mexico

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The mexican fungus is sold in Japan and the The mexican fungus is sold in Japan and the revenues make up to 30% of the economy of revenues make up to 30% of the economy of

some mountain communitiessome mountain communities

What about planting pines What about planting pines where these fungi, essential for where these fungi, essential for

the growth of pines, do not the growth of pines, do not exist?exist?

In the soil under pines we found In the soil under pines we found DNA sequences of an imported DNA sequences of an imported

fungusfungus

Fungi and fungus-like Fungi and fungus-like organisms are responsible for organisms are responsible for

90% of plant diseases90% of plant diseases

•Affecting a portion of a plant

•Affecting low• numbers of trees

•Epidemics (exotic diseases)

•Native diseases play a fundamental role of eliminating overcrowding and eliminating the weakest individuals, but exotic diseases can negatively change ecosystems by wiping out entire populations

Like the conquistadores brought Like the conquistadores brought diseases that were lethal to diseases that were lethal to those who had never been those who had never been

exposed to them, so do exotic exposed to them, so do exotic diseases cause true devastation diseases cause true devastation in plant communities because of in plant communities because of

lack of coevolution between lack of coevolution between hosts and microbeshosts and microbesQuickTime™ and a

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Fungal pathogens are true Fungal pathogens are true invadersinvaders

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are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

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•Invasions are more frequent before frequency and intensity of movement of goods and people are greatly on the rise

Forest pathogens can never be Forest pathogens can never be eradicatederadicated

How can people transport How can people transport pathogenspathogens

• By transporting plants and plant partsBy transporting plants and plant parts– Crops, and seedsCrops, and seeds– Raw foodRaw food– Ornamental plantsOrnamental plants

Untreated lumberUntreated lumber

SoilSoil

Insects vectoring fungiInsects vectoring fungi

HOW to identify species and HOW to identify species and possibly provenance of possibly provenance of

pathogenspathogens

• Microscopic organisms with few Microscopic organisms with few differentiating featuresdifferentiating features

• Often cannot be culturedOften cannot be cultured

: USING DNA SEQUENCES: USING DNA SEQUENCES

•Species I Species II(more virulent)

•Europa

•Nord America

A

B

C

D

ABCD are four sequences ABCD are four sequences uniquely found in the four types uniquely found in the four types

of pathogof pathogensens

• If association of sequence and pathogen is unequivocal, sequence can be used for diagnostics

• If you were and official in the US which sequence (I.e. pathogen) should you be most concerned abou….t

What is DNA?What is DNA?

• The code of life, four different bases are arranfed in triplets of different combinations, each triplet codes for an amminoacid. Aminoacids are assembled to form proteins

•DNA is a tight double helix and is arranged in physical sticks called chromosomes

Ecah chromosome containsEcah chromosome contains

Portions that are garbage they do not code for a protein, these evolve more rapidly, and are more likley to differentiate organisms

Portions that code for proteins, the more essential is the protein, the less the DNA will be allowed to mutate, less useful to differentiate organisms

Let’s read the DNA sequence of Let’s read the DNA sequence of a variable portion of a a variable portion of a

chromosomechromosome

•AGT GAA CCA G

Mutation-evolution

• AGA GAA CCA G

Further mutation-evolution

•AGA GAG CCA G

Let’s build an evolutionary or family tree Let’s build an evolutionary or family tree using these sequencesusing these sequences

•AGT GAA CCA G

Uncle Scroodge

• AGA GAA CCA G

Donald Duck

•AGA GAG CCA G

Hewey

•EVE

Lewy

Dewey

Who stole the cookies ?Who stole the cookies ?

• AGA GAG CCA G

Who is Who is AGA GAG CCA G

•AGT GAA CCA G

Uncle Scroodge

• AGA GAA CCA G

Donald Duck

•AGA GAG CCA G

Hewey

•EVE

Lewey

Dewey

And if the culprit were And if the culprit were AGA GAT CCA G ?

•AGT GAA CCA G

UNCLE SC

• AGA GAA CCA G

DD

•AGA GAG CCA G

H

•EVE

L

D

If the culprit were If the culprit were AGA GAT CCA G ?

•AGT GAA CCA G

US

• AGA GAA CCA G

DD

•AGA GAG CCA G

H

•EVA

L

D

•Maybe•But which one?

•AGT GAA CCA G

US

• AGA GAA CCA G

DD

•AGA GAG CCA G

H

•EVE

•AGA GAT CCA G

D

•AGA GAC CCA G

L

And if the culprit were And if the culprit were GGC AAA TCT G ?

•AGT GAA CCA G

US

• AGA GAA CCA G

DD

•AGA GAG CCA G

H

•EVE

•AGA GAT CCA G

D

•AGA GAC CCA G

L

If the culprit were If the culprit were GGC AAA TCT G ?

• With the available database it would be impossible to identify the culprit

• By enlarging the database we may fiund the match in somebody else not present in the current database. E.g. Mickey mouse

The last frontiers in ScienceThe last frontiers in Science11

• To sequence an entire genome to understand how it functions

We sequenced to genomes of We sequenced to genomes of related fungus-like organisms related fungus-like organisms

(Science)(Science)• Discovered that they produce hundreds of

molecules to relate with plants

• The kingdom called Straminopila has seen the fusion of photosynthetic and non-photosynthetic organisms

The new frontiers of ScienceThe new frontiers of Science22

• To sequence one gene, one locus, for hundreds of species to increase our databases regarding communities and entire ecosystems

Sequencing of groups of Sequencing of groups of organismsorganisms

• Sequencing all of the organisms on the island of Moorea

• Sequencing all the species in an herbarium