Isolation of Microsatellite Loci

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Isolation of Microsatellite Loci

• For the lizard species Sceloporus grammicus
  (Squamata, Phyrnosomatidae)

P.H.Degnan, Biology Department, Providence
College, May 4th 2001
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Purpose

• Develop a library of microsatellites for
  S.grammicus to help further the understanding of
  behavioral, ecological chromosomal and
  evolutionary questions that influence the
  maintenance of the Tulancingo hybrid zone.

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Characterization of the S. grammicus complex

• First characterized by Weigmann (1828).
• Phrynosomatid lizard that ranges from southern
  Texas and throughout Mexico at elevations from
  ?2400m-3200m (?7000-10000ft).
• The lizard are polygynous, viviparous and
  sexually dimorphic.

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Characterization of the S. grammicus complex

• Species complex exhibits multiple Robertsonian
  rearrangements due mainly to chromosomal
  fissions.
• This has lead to the identification of 8
  distinct chromosomal races that have 2n from 32
  to 46 chromosomes.
• Recent evidence leads to the conclusion that the
  cytotypes represent incipient species derived
  from as ancestral 2n32

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Chromosomes of Sceloporus grammicus 2n32
(Standard)
Pr. 1
Pr. 2
Pr. 3
Pr. 5
Pr. 4
Pr. 6
Macrochromosomes
1
2
3
7
4
5
8
6
Y
X2
Microchromosomes
X1
sex chromosomes
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Characterization of the S. grammicus complex

• Chromosomal polytypy occurs more often in
  mammalian species but is rare amongst
  non-mammalian vertebrates.
• Despite the varying chromosomal races there are
  regions where these cytotypes have shared
  territories resulting in the capacity for
  hybridization.
• The Tulancingo Hybrid zone is a region that
  involves hybridization between F5 and FM2
  cytotypes

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Tulancingo Transect
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Characterization of the S. grammicus complex

• Studies have shown that F5 cytotype show a
  significant preference for oak forests and the
  FM2 have a preference for more xeric
  environments.
• Matings between the paternal cytotypes results in
  hybrids (F1) which have shown reduced levels of
  fitness, but backcrosses help stabilize
  chromosomal hybrids.

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Characterization of the S. grammicus complex

• Past studies have utilized meristic characters,
  mtDNA, and allozymes as tools for evolutionary
  and population questions.
• Now employ microsatellites as a tool for these
  questions as well behavioral, biochemical, and
  paternal studies

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What are Microsatellites?

• Microsatellites are Mendelian, codominant
  selectively neutral, highly polymorphic genetic
  markers.
• These regions of DNA exhibit short repetitive
  nucleotide sequence motifs. (Mono, di, tri,
  tetra, penta and hexanucleotide repeats)

AAT Motif
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What are Microsatellites?

• Their highly repetitious nature leads to the
  biological mutability of these regions. In the
  absence of selective pressures, these mutations
  cause the formation of distinct alleles that can
  be used for detailed population structures and
  related studies.
• Length polymorphisms amplified with primers on
  either side of the repeat region in a polymerase
  chain reaction (PCR).

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Steps in isolation of ?sats

• Construction of a partial genomic library
• Genomic DNA extraction
• Plasmid ligation
• Insertion into competent cells
• Screen Colonies with ?satellites probes
• Plasmid isolation/purification
• Sequence genomic DNA inserts
• Design of Primers

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Genomic DNA extraction

• Frozen tissue sample (-80oC) dry ground in mortar
  and pestle, then incubated at 55oC in STE buffer,
  10mg/ml Proteinase K, and 20 SDS.
• Phenol/Chloroform extractions
• DNA precipitated by adding 2M NaCl and100 EtOH
• Pellet spun down and dried before resuspension in
  dH2O
• Sample run on an 0.7 Agarose gel

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Genomic DNA extraction

• DNA digested with restriction enzyme SAU3A
• Product run on 0.7 gel and through use of PCR
  ladder, fragments from 200-500bp were excised.
• DNA purified from gel

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Plasmid Ligation

• DNA ligated into multiple priming site of pZeroTM
  -2 plasmid (Invitrogen)
• Plasmids transfected into competent bacterial
  cells (E. coli)
• Cells plated on LB-Kanamycin (0.03g/L) plates and
  grown for 24hrs at 37oC

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Plasmid Ligation
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Screening of colonies

• Nylon transfer membranes placed on top of plates
• Membrane removed and adherent cells denatured,
  neutralized and baked at 65oC for 2 hrs to allow
  hybridization of the DNA.
• Membranes hybridized with radiolabeled
  oligonucleotide probes (?-33P) in 5x SSC, 0.5
  SDS and 5x Denhardts solution.
• After hybridization the membranes were washed
  before being developed on X-ray film.

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Screening of colonies
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Isolation of positives and regrow
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Isolation of positives and regrow

• Positives selected from hotspots on
  autoradiographs.
• Return to original plates scrape up cells with
  sterile toothpick and transfer to 5ml culture
  media (SOC media).
• Cultures grown for 24hrs at 37oC in shaking H2O
  bath
• 2ml of cells purified for plasmids using Quick
  and Dirty protocol or QAIprep spin tubes.
• Run a gel to double check presence of plasmid and
  relative concentration of DNA.

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Plasmid preparation

• Quick and Dirty vs. QAIprep

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Sequence plasmid insert

• Purified Plasmid sent to Brown University
  sequencing facility.
• Sample contained 0.5?g of plasmid dsDNA and
  3.2pmol T7 primer.
• Samples run on ABI Prism 377 DNA Sequencer.

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Sequence plasmid insert
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Sequence plasmid insert
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Design Primers

• Primers designed using MacVectorTM 6.5.3
• Primer design based on
• Primer size (18-20bp)
• Product size (100-300bp)
• Annealing Temp (55-75oC)
• Percentage GC content
• Self-Annealing
• Forward and reverse primers that suit these
  requirements were then ordered from Integrated
  DNA Technologies

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PCR Protocol
DNA polymerase
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Isolated microsatellites
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Sg Microsatellites
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Sg 202CAG
Sg 205CAG
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Future Analysis

• Assessment of local mating strategies
• Are Females multiply mated?
• How do genotypes of F1 effect mate selection?
• Can F1 males act as dominant (?) males?
• Do subordinate (satellite) males have capability
  to mate?
• Are there loci that are cytotype specific?
• Are there any hormonal /biochemical differences
  between cytotypes?

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Acknowledgements

• Tom Earl 01 Ruth Lambert
  99

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Acknowledgements
Brenden LeFebvre 01
Mike MacPhee 02
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Acknowledgements
Amanda Townsend 01
Jennifer Boie 01