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SEPG  1565 - Date Awarded 1998

The characterisation of microsatellite loci in two polar sea urchin species

Dr. Christiane Biermann

Present address:
Friday Harbor Laboratories,
620 University Road
Friday Harbor,
WA 98250-9299, USA
email: biermann@fhl.washington.edu

Address during SEPG, 1998:

Molecular Ecology and Fisheries Genetics Laboratory
University of Hull,
Hull HU6 7RX

A substantial part of population genetic work is the  identification of suitable molecular markers. Although some protein-coding genes are known in regular sea urchins, noncoding regions, e.g. short simple repeats, which permit finer resolution for ecological genetic questions, have not been isolated before. My analyses of (larval-mediated) gene flow in circumantarctic sea urchins proposed for this SEPG has not been completed yet; therefore I here describe the characterisation of sea urchin microsatellite loci.

Microsatellites are tandem repeats of very short DNA sequence motifs (1-5 base pairs) and are generally selectively neutral. Because of their high mutation rate they offer fine-scale resolution for genetic fingerprinting, from paternity assignment up to studies of population structure.

In order to isolate specific loci from an unknown genome, random pieces of genomic DNA are cloned into bacteria, which are then probed with (e.g. radioactively) labelled pieces of DNA that represent the target sequence. I hybridised the sea urchin DNA library with a mixture of nucleotide probes representing repeats of the motifs AC, AG, AAC, AAG, ATC, CAG, GAC, GCC, GTG, TAC, AAAC, AAAG, AAAT, CGTA, GACA, GACT, GATA, GCAT, GCGA, and GTAT. The inserted piece of DNA from positive (hybridising) bacterial colonies was then isolated and sequenced.

The genomic library can be enriched to increase the representation of target loci. I chose RAPD- (random amplified polymorphic DNA-) enrichment, in which the library is constructed from PCR-products obtained by amplifying genomic DNA with a single, short primer. The rationale for this is that a region will only amplify if it is flanked by inverted repeats (matching the RAPD-primer); and inverted repeats frequently bracket a simple repeat region.  I constructed RAPD-enriched libraries from eleven ligations for one Sterechinus (Antarctic), and for two populations of Strongylocentrotus droebachiensis (circumarctic). My screening and isolation successes are shown in the following table:

Microsatellite loci do not appear to be very abundant in either of the two sea urchin species, and, if found, are rarely long and usually interrupted. Longer, more perfect repeats without interruptions are expected to show more length variation; the variability of these largely imperfect sea urchin loci has to be assessed empirically by testing a number of individuals for each locus. The last category in the table above lists the longest/least interrupted loci whose flanking regions permit the design of specific primers. Their repeat motifs are listed in the following table:

This is one of the first investigations of echinoderm microsatellites. One other laboratory has supposedly isolated sea urchin variable repeat loci, but this is unpublished. It appears that microsatellites are not very frequent or long in echinoids. This is surprising as I have found a lot of variability, especially in a repeat-region, in the nuclear "bindin" gene, and because the species examined have large census population sizes. Of course the loci I found, although not long and perfect, may still show variability at a scale useful to elucidate gene flow patterns around the Antarctic continent.

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