2014-03-18 1 Molecular Methods in Ecology & Evolution Helena Westerdahl, March 18 th 2014 RNA dCTP dNTP cpDNA mtDNA Molecular Methods in Ecology & Evolution Helena Westerdahl, February 4 th 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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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
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2014-03-18
1
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?
2014-03-18
2
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.
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
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.
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.
.
How can microsatellites be found?
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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
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.
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.
How can microsatellites be found?
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
Same length Different lengths
SSCP microsatellites
DGGE minisatellites
RSCA insertion/deletion
SNP
Cloning & seq.
Methods to study genetic variation
dominant co-dominant
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?