Molecular Methods in Ecology & Evolution€¦ · 2014-03-18 1 Molecular Methods in Ecology & Evolution Helena Westerdahl, March 18th 2014 RNA dCTP cpDNA dNTP mtDNA Molecular Methods

2014-03-18

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Molecular Methods in Ecology & Evolution

Helena Westerdahl, March 18th 2014

RNA

dCTP

dNTPcpDNA

mtDNA

Molecular Methods in Ecology & Evolution

Helena Westerdahl, February 4th 2014

What is the evolutionary history and relationships among members of a group of species?

How genetically diverse are populations comprising a single species across a broad environmental gradient?

How many males have fathered the chicks in a clutch, and which are these males?

Why do some individuals suffer from a specific infection while others do not?

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How many species of Mouse lemurs are there on Madagascar?Mouse lemurs - the world’s smallest living primates. In 1992, there were two named species - Now more than 15 speciesMitochondria – Now nuclear DNAResults indicate that Microcebus is one of the world’s most speciose genera of primate.

Nuclear & Mitochondrial DNA

16,000 bp

haploid genome

no recombination

maternal inheritance

high mutation rate (10-8)

3,000,000,000 bp

diploide genome

recombination

Mendelian inheritance

low mutation rate (10-9)

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Genome ‘categorization’

Nuclear DNA

Single copy Repetitive

Tandemly repeated

Coding10% Non-coding 90%

Microsatellite

1-6

Minisatellite

10-60

Satellite

> 100

Methods to study genetic variation

dominant co-dominant

AFLP

RAPD

RFLP

Same length Different lengths

SSCP microsatellites

DGGE minisatellites

RSCA insertion/deletion

SNP

Cloning & seq.

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AFLP

RAPD

RFLP



DGGE minisatellites


SNP

Cloning & seq.

Methods to study genetic variation- Same length

PCR productsof the same length

1 2 3

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• DGGE• SSCP• Cloning and

DNA sequencing• SNP• RSCA


1 2 3


DGGE Denaturing Gradient Gel Electrophoresis

A=TG=C

denaturant30%

60%DGGE 1 PCR fragment=1 bandSSCP 1 PCR fragment=2 bands

homozygote 1 bandheterozygote 2 bands

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DGGE Denaturing Gradient Gel Electrophoresis

a b c d e f g h i j k l m n o

1

2

3

4

5

67

8

3 MHC alleles 4 MHC alleles

SSCPSingleStrand ConformationPolymorphisms

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Cloning and DNA sequencing

PCR product is ligated

into vectors (plasmids)

A single vector inserted

into a bacteria (E.coli)

Bacteria grow over night

Colonies with inserts are

amplified using PCR

These PCR-products

are sequenced

ATGTCCAGT…

ATGTCAAGT…

ATGTCCAGT…

ATGTCAAGT…

One PCR per clone

Sanger sequencing

A T G CA* T* G* C*

A*AT*ATG*ATGA*one primer

PCR fragments

cocktail

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• DGGE• SSCP• Cloning and

DNA sequencing• SNP• RSCA


1 2 3


SNP, single nucleotide polymorphisms

T or C

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SNP screening using

*Q-PCR with probes

* Chip-based SNPs

PCR amplification ofalleles a & b

ab

Hybridization of PCR fragments to a fluorescent -labelled reference sequence (r*)

r*

a

Heteroduplexes are formed, but only the fluorescent will be visualized in the sequencing robot

allelea,r*

b,r*

r*,r*

not detecteda,ba,aetc.

Output from the sequencing robot

Step 1Step 2

Step 3

Step 4

ref.seq. a b

ref.seq.

The hybridization is done against 4 -7 reference sequences in total and heritability analyses show which alleles that corresponds to which peak

RSCA Reference Strand mediated Conformation Analyses

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DGGE minisatellites


SNP

Cloning & seq.



AFLP

RAPD

RFLP

RFLP Restiction Fragment Length Polymorphisms& Southern Blots

long fragments

short fragments

A B C

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kb

9.4

6.6

4.4

2.32.0

1.0

1 2 3

Pheasant

kb

9.4

6.6

4.4

2.32.0

1.0

1 2 3

Pheasant Great reed warbler

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Male ZZ Female ZW

Random Amplified Polymorphic DNA RAPD- Molecular Sexing

single primerPCR

200

500

1000

2000bp

Agarose gel

RFLP + RAPD= AFLP

E-primer

M-primer

T

C

Pre-amplification

E-primer

M-primer

TCA

CAC

Selective amplification

• Two different restriction enzymes chop the DNA in roughly 1 million pieces

• Synthetically made adaptors are attached to the fragments

• A selected subset of the DNAfragments between the

adaptors are amplified with PCR in two steps

• PCR fragments separated by size

Amplified Fragment Length Polymorphisms

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5’-GAATTC-3’3’-CTTAAG-3’

EcoRI

5’-TTAA-3’3’-AATT-5’

MseI / TruI

Restriction enzymes

CTCGTAGACTGCGTACC-AATTCNNNNNT-TACTCAGGACTCATCATCTGACGCATGGTTAA-GNNNNNAAT-GAGTCCTGAGTAGCAG

E-adaptor M-adaptor

AFLP gels

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DGGE minisatellites


SNP

Cloning & seq.



AFLP

RAPD

RFLP

..ctcagtctggtagggatcggcgtcacacacacacacacatcggatttagccgtatcggtttaaatgctgg……….

..gagtcagaccatccctagccgcagtgtgtgtgtgtgtgtagcctaaatcggcatagccaaatttacgacc

gggatcggc

Primer1

cctaaatcgg

Primer2

Cell nucleus

PCR products of different lengthsMicrosatellites, Simple sequence repeats (SSR), Short tandem repeats (STR)

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Mol Ecol Resour. 2013 Dec 17. doi: 10.1111/1755-0998.12213. [Epub ahead of print]Characterization of the house sparrow (Passer domesticus) transcriptome:

a resource for molecular ecology and immunogenetics.Ekblom R, Wennekes P, Horsburgh GJ, Burke T.

How can microsatellites be found?

Table 3. Number and type of microsatellite repeats identified from the house sparrow transcriptome.

Type Min. repeat number Total number of repeatsNumber of repeats with primer sequence

Di- 10 151 19

Tri- 8 90 18

Tetra- 4 464 226

Penta- 4 197 52

Hexa- 4 66 12



968 microsatellite repeat sequences in the house sparrow transcriptome. As tri- and hexanucleotide repeats (nonreading frame interrupting) are the least common types, a majority of these are expected to be situated in untranslated regions of the expressed genes.

.


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Table 3. Number and type of microsatellite repeats identified from the house sparrow transcriptome.

Type Min. repeat number Total number of repeatsNumber of repeats with primer sequence

Di- 10 151 19

Tri- 8 90 18

Tetra- 4 464 226

Penta- 4 197 52

Hexa- 4 66 12



968 microsatellite repeat sequences in the house sparrow transcriptome. As tri- and hexanucleotide repeats (nonreading frame interrupting) are the least common types, a majority of these are expected to be situated in untranslated regions of the expressed genes.

We also identified 3177 SNPs. Note that there is a risk that the described flanking sequences of some of these SNPs span an exon/intron boundary. Consequently, SNP genotyping efficiency using gDNA might be reduced.


Genotyping microsatellites

”stutter”or

”shadow”bands

70

90

94

1 2 3 4 5 6 7 8 9 10 11 12

Heterozygote for 94 & 96

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Summary Microsatellites

• Markers isolated from the target species, or borrowed from other species

• Mutation rate orders of magnitude higher than nucleotide substitions

• Back-mutations frequent – homoplacies

• Excellent for parentage, heterozygosity and assigment test



DGGE minisatellites


SNP

Cloning & seq.



AFLP

RAPD

RFLP

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PCR Temperature profileReagentsTemplate

DetectionLabellingPrimersTerminators

GelRedEthidium bromideSyber gold

MatrixAgaroseAcrylamidPolymer

Interpretation PCR errorscontamination

What is the evolutionary history and relationships among members of a group of species?

How genetically diverse are populations comprising a single species across a broad environmental gradient?

How many males have fathered the chicks in a clutch, and which are these males?

Why do some individuals suffer from a specific infection while others do not?

How many species of mouse lemurs are there on Madagascar?

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Summary of Methods

MtDNA AFLP Microsatellites

Paternity analyses NO (+) +++

Inbreedning NO + +++

Population structure ++ +++ ++

Cryptic species +++ +++ +

Species Phylogenies +++ ++ +

Documents

Molecular Methods in Ecology & Evolution€¦ · 2014-03-18 1 Molecular Methods in Ecology & Evolution Helena Westerdahl, March 18th 2014 RNA dCTP cpDNA dNTP mtDNA Molecular Methods