Download URCAS presentation

Microsatellite Identification in the Thick-billed Parrot
(Rhynchopsitta pachyrhyncha)
By:
Daniel Acosta
Howard Hughes Medical Institute-NMSU Research
Scholar
Dr. Wright’s Lab
Department of Biology
Thick-billed Parrot
•
International Union for
Conservation of Nature (IUCN
2007)
•
World Parrot Trust
•
Historic range: Southwestern
United States and Northern
Mexico (Snyder et. al., 1999)
•
Habitat degradation and
fragmentation from logging
Range
Genetic Variation
•
High degree of genetic variation to reduce
the impact of founder effect, which may
lead to genetic differentiation
•
To adapt to a changing environment and to
avoid reduced reproductive fitness.
•
Importance to any translocation.
Microsatellites
•
Microsatellites are simple sequence repeats (1-6) base pairs long: e.g.
GTGTGTGTGTGT or ACGACGACGACGACGACGACG found in the
genome of both prokaryotic and eukaryotic organisms
•
Found in coding and non-coding regions
•
They have a high mutation rate and high variability in natural populations.
•
High degree of polymorphism
•
All of these characteristics makes microsatellites a class of genetic marker
that is highly useful to assess genetic variation.
Methods: Isolation of Microsatellites
DNA Extraction
Boil clone in TE buffer
PCR
Genetic Library
T3/T7
(Kongrit et. al., 2008) T3/T7/GT10
(Zane et. al. 2002)
Methods: Primer Design
Primer Design
Sequencing
CCGAGTAGGACAGAGCCTTGGGTGGCATGGTTTAGTGGG
AGGTGTCCCTGCCCACGGCATGGGGTTTGGAACTAGATG
ATCTTAAGGTCCTTTACAGCCCTAACTGTTCTATGATTCTAT
TGGGTCTCAAGGGTTTGTGTGTGTGTGTGTGTGTGTGTG
TGTGTGTGTGTTCATTTTCCTCTTCAGGGTGGA
ATAAGAGCCTTGAATTACAACATTAAACCTTTTAAATGG
Test for amplification in
thick-billed parrot DNA
Optimize Primers
Polymorphism
Polymorphism
•
Having genetic diversity
•
Proportion of loci polymorphic: Number of polymorphic loci / total number of
loci sampled
•
We can also calculate allelic diversity: If we have sampled 6 loci and the
diversity is as follows (1, 3, 3, 2, 2, 3) Allelic diversity=(1+3+3+2+2+1) / 6 =
2
•
These calculations tell us a great deal about the genetic variation of the
target population
Progress and Future Goals
•
Up to date: 3 primers we
designed and 4 designed for other
species.
•
These primers have been
optimized for [Mg] and annealing
temperature.
•
We now propose to use these
primers to assess the genetic
variation not only of the wild
population, but of the captive
population.
•
Compare wild population to
captive population
Acknowledgements
•
•
•
•
•
•
•
Howard Hughes Medical Institute-NMSU Research Program
Dr. Timothy Wright
Ph. D Student Erin Schirtzinger
Ph. D Student Swati Mukherjee
Ph. D Student Alejandro Salinas
Nadine Lamberski @ San Diego Zoo
Kari L. Schmidt. @ American Museum of Natural History
References
IUCN 2007. Rhynchopsitta pachyrhyncha.
<http://www.iucnredlist.org/search/details.php/19715/all> (March 26, 2
008 2007).
Kongrit, C. et. at. (2008). Isolation and characterization of dinucleotide
microsatellite loci in the Asian elephant (Elephas maximus). Molecular
Ecology 8, 175-177
Snyder, N. F. R., E. C. Enkerlin-Hoeflich, and M. A. Cruz-Neto. 1999. Thickbilled Parrot (Rhynchopsitta pachyrhyncha) The Birds of North America 24
Zane, L., Bargelloni, L., & Patarnello, T. (2002). Strategies for microsatellite
isolation: a review. Molecular Ecology 11, 1-16g
Picture Sources
•
•
•
•
•
•
http://www.avianweb.com/images/birds/parrots/thickbilledparrots/thickbilled.jpg
(map)
http://content.cdlib.org/xtf/data/13030/xw/ft0f59n6xw/figures/ft0f59n6xw_000
01.gif
http://www.aviary.org/~aviary/images/thick-billed%20parrots.jpg
http://www.dkimages.com/discover/previews/928/55058803.JPG
http://www.sabo.org/images/tbpanest.jpg
http://www.expeditionswest.com/adventures/2004/sierra_madre/JourneyOn
eSatevo/images/DSCF3627.jpg
Questions??