Brief description
Tissue samples were collected from newly born pups from 15 individual colonies between 1989 and 2000, to look at variable nuclear microsatellite markers to allow the investigation of genetic population structure and sex-biased dispersal.Lineage
Maintenance and Update Frequency: notPlanned
Statement: - Sample collection -
Three separate sampling events were conducted between 1989 and 2000, and a total of 15 individual colonies were sampled. Five colonies (Beagle Island, North Fisherman Island, Buller Island, Six Mile Island and Kangaroo Island) were sampled on all three occasions. Breeding colonies were visited towards the end of the breeding season, which was calculated by extrapolation from previously published breeding data (Gales et al. 1994). Estimates of pup production for the sampled colonies are taken from Chapter 2 of the thesis.
Tissue samples were taken from newly born pups of a single breeding season to ensure the sampling of individuals from their birth colony, and to minimise the sampling of related individuals. Females produce only one pup per season and so each pup represents a single maternal line. Live pups were targeted as the primary source, although freshly dead pups were sampled where available. Exceptions to this occurred on Dangerous Reef and The Pages, where for a number of reasons only dead pups were sampled. Pups were captured by hand, and a small piece of tissue removed from the tip of the middle digit of the right hind flipper. Tissue was preserved in a solution of 95% EtOH and 100mM EDTA to help stabilise nucleic acids. Total genomic DNA was extracted as in Gemmell & Akiyama (1994), and thirteen microsatellite loci designed for other pinniped species were screened to determine the degree of cross species suitability and polymorphism of markers.
Statement: - Screening of microsatellite loci -
DNA extraction techniques were described in Chapter 3 of the thesis. Approximately 20-50ng of template DNA was added to the PCR reaction mixture, which was derived from Gemmell et al. (1997) using direct incorporation of a radio-labelled nucleotide. Individual PCR reaction consisted of 1ul X10 reaction buffer, 2.5mM MgCl2, 0.25 uM of both forward and reverse primers, 0.25U Taq, 5mM of three dNTP's (ATP, GTP, TTP) and 0.5mM of CTP, 0.01ul of (alpha-labelled) P-32 CTP, 60mM TMAC (Tetramethyl ammounium chloride), 1% formamide and dH2O to a final volume of 10ul.
PCR products were separated on 6% polyacrylamide gels, and allele sizes determined by comparison with a known DNA sequence. Gels were run for approximately 3-4 hours at 100W, or until products were sufficiently separated, based on estimates of fragment size. The microsatellite loci used were designed for a number of other species of pinniped, and known to have cross species utility (Gemmell et al. 1997). Gels were dried and exposed to autoradiography film (KodakTM X-OMAT) for 2-4 days. Films were then developed and alleles scored by size in relation to a known sequence of an M13 plasmid.
Notes
PurposeTo use this genetic information to formulate discrete population boundaries to assist in the effective management and conservation of the Australian Sea Lion.
Created: 21 08 2007
Data time period: 1989 to 2000
text: westlimit=112.9; southlimit=-36.4; eastlimit=138.7; northlimit=-27.7
Subjects
41 131005 |
BIOSPHERE |
EARTH SCIENCE |
ECOLOGICAL DYNAMICS |
Genetic variation |
Microsatellite markers |
Neophoca cinerea |
Oceans | Marine Biology | Marine Mammals |
Population Dynamics |
Population genetics |
SPECIES/POPULATION INTERACTIONS |
oceans |
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Other Information
(PhD thesis)
Identifiers
- global : 6932a7b0-4f8e-11dc-87ba-00188b4c0af8
