Microsatellite loci isolated from the Caribbean coral, Montastraea annularis

Information concerning the genetics of reef-building coral species plays a critical role in assessing coral reef health and provides baseline data from which management authorities can plan the design of marine sanctuaries. Speculation regarding anthozoan genomes has asserted that little repetitive DNA is present and there is, consequently, a scarcity of microsatellites contained therein. Classic studies of genome composition suggest that the cnidarian genome contains about 3% highly repetitive DNA, whereas mammalian species may have as much as 10% (Britten & Davidson 1971). Observations from our single-copy library construction (Severance et al. 2004) indicated that highly repetitive DNA accounts for approximately 6% of cloned genomic DNA in Montastraea annularis. Difficulties in isolating microsatellite DNA from Scleractinia may, therefore, be species specific and directly related to overall genome size and complexity differences among Scleractinian taxa (Marquez et al. 2002). Here, we report the isolation of seven microsatellite loci of the Caribbean coral reef dominant, M. annularis, from microsatellite-enriched nuclear DNA libraries.

Microsatellite loci were cloned from azooxanthellate M. annularis gametes using an enrichment protocol that combines approaches described by Kandpal et al. (1994), Fischer & Bachmann (1998), Edwards et al. (1996), Kijas et al. (1994) and University of Florida Microsatellite Workshop protocols. Total cell DNA (tDNA) (3 µg) was digested with Sau3AI in a 100-µL reaction. Digested tDNA (100 ng) was ligated to 1 µg of reverse-complementary oligonucleotide linkers containing BglII restriction sites (underlined; BglIIA, 5′-GATCGTCCAGATCTAAGCAAGAGCACA-3′ and BglIIB, 5′-CTCTTGCTTAGATCTGGAC-3′) using T₄ DNA ligase in a 20-µL reaction. A 1 µL volume of the linker-tDNA ligation was amplified in a 100 µL polymerase chain reaction (PCR) containing 2.5 mm MgCl₂, 1 × Reaction Buffer (Promega), 5 µg bovine serum albumin, 1 µm of BglIIB oligonucleotide linker, 200 µm dNTPs and 1.25 U Taq DNA polymerase (Promega) and thermal cycled under the following conditions (Omn-e Hybaid): 95 °C for 3 min followed by 30 cycles of 1 min at 95 °C, 1 min at 60 °C and 2 min at 72 °C and a final 72 °C extension for 7 min. A 20 µL volume of the PCR product was denatured at 98 °C for 10 min, placed on ice for 2 min and hybridized at 48 °C overnight with 20 ng each of 3′ biotin-labelled oligonucleotides 5′-(TC)₁₅TATAAGATA-3′, 5′-(AC)₁₅TATAAGATA-3′ and 5′-(CAA)₁₀TATAAGATA-3′ in a final total volume of 100 µL. Hybridized products were captured using Streptavidin MagneSpheres (Promega) and eluted in a 100-µL volume. The eluant was hybridized using a second round (denaturing, oligo-hybridizing, streptavidin capture and elution) to minimize nonspecific binding. The eluant (10 µL) was amplified in a 100-µL PCR reaction using the same reagent concentrations and thermal cycling parameters as described above. The PCR product (25 µL) was digested with BglII in a 100-µL volume and ligated to BamHI-digested dephosphorylated Bluescript-SK-plasmid (Stratagene). Ligations were transformed with Sure-shot cells (Invitrogen) and plated on antibiotic media. Single clones were PCR amplified using 12.5 pmol each of M13 primers with the same PCR conditions as described above. The PCR products were sequenced using Perkin-Elmer ET Terminator protocols and size sorted on an ABI Prism 310 DNA sequencer (Applied Biosystems, Inc.).

From the clone microsatellite flanking sequences, primers were designed using primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/primer3www.cgi). Of 23 loci isolated, seven were amplifiable from field samples of M. annularis and primers corresponding to these loci were labelled with a fluorescent 5′-tag. Some 124 samples from four geographically distinct regions were genotyped and the estimated number of alleles, mean observed and expected heterozygosities were calculated using arlequin (Schneider et al. 2000) (Table 1). Under similar amplification conditions (Table 1), these loci readily amplified 150 samples of the sibling species, M. faveolata, suggesting the applicability of these markers not only to population genetic structure analyses but also for the resolution of species boundaries within this taxonomic complex.