Polymorphic microsatellite markers in a cephalopod: the veined squid Loligo forbesi

dynamics and even species integrity is currently hampered by a lack of variable and dependable markers. The use of morphometric or meristic characters in this group is problematical due to extreme plasticity of body form and growth, making accurate and consistent study difficult (Pierce et al. 1994). The successful application of biochemical or molecular genetic markers in such cases in other groups has unfortunately not been repeated in cephalopods: allozyme and mitochondrial DNA studies have been hampered by extremely low levels of genetic variability in many species (Carvalho et al. 1992; Brierley et al. 1993, 1995; Norman et al. 1994); and development of DNA techniques generally is held back by difficulties of extracting good-quality DNA and lack of sequence and gene order information. The burgeoning application of microsatellite DNA markers, including successes in organisms with low variability detectable by other methods (Hughes & Queller 1993; Jarne et al. 1994), offers a way forward. The current study reports an initial description of highly polymorphic microsatellite loci in a cephalopod, the NE Atlantic squid Loligo forbesi, suitable for use in population studies and the analysis of mating systems in these behaviourally complex animals. Total DNA was extracted, using a modified CTAB method, from arm tips of freshly killed squid. A 300–600 bp partial genomic library was constructed for L. forbesi using ligation of size-selected Sau3AI digested total DNA into pUC18/BamHI vector (Pharmacia), then transfection into DH5α competent cells (Gibco BRL), following standard procedures (Sambrook et al. 1989; Rassmann et al. 1991). Recombinant colonies were probed with an array of repeat motif oligonucleotides (AAT10, AAG8, AAC9, AAAT9, AAAC6, AAAG6, GATA6) and dinucleotide polymers (AC/GT, AG/CT). High-stringency positives were PCR amplified and cycle sequenced (ABI kit) using universal pUC18 primers, run on an ABI automated sequencer. Primers were designed, using the programme OLIGO 4.1 (National Biosciences Inc., Plymouth, MN, USA), from unique sequence flanking a range of the microsatellite repeat types found (di-, triand tetra-motifs; perfect and imperfect arrays): 10 pairs giving the best results are presented in Table 1. GATA repeat arrays, although relatively common, were not used either because of the Sau3AI cut site falling at the end of repeat or due to complex multiple repeat structures. PCR reactions were performed under the following conditions: 120 s at 94 °C, then 30 cycles of 60 s at 92 °C, 60 s at the specific annealing temperature (see Table 1) and 10 s at 72 °C, using an MJ Research PTC-100 thermal cycler. Subsequent screening of population samples demonstrated that use of a ‘Touchdown’ protocol (see Don et al. 1991), employing an initial set of 10 cycles dropping from 5 °C above the annealing temperature in 0.5 °C increments, gives cleaner products from some older samples. Reaction mixes contained 20 ng template DNA, 1.5–2.5 mM MgCl2, 0.2 mM each nucleotide, 5 pmol of each primer (forward primer end-labelled with 32P γATP), 0.2 U Taq polymerase (Boehringer Mannheim) with the manufacturer’s supplied 1× buffer, in a final reaction volume of 10 μl. Amplified products were resolved on 6% denaturing polyacrylamide gels (after Sambrook et al. 1989), product sizes being determined by comparison to an M13mp8 DNA sequence standard. Results of optimization and screening of variability, using DNA CTAB-extracted from ethanol preserved mantle tissues of 25 individuals randomly selected from across the range of L. forbesi in the NE Atlantic, for the 10 loci are presented in Table 1. All primer pairs amplified products of the predicted size. Standard pedigree analysis is almost impossible for this species, due to the sensitivity of individuals to captivity, but genotyping from four broods within egg strings from single females did not indicate departure from Mendelian allelic segregation expectations (unpublished data). Levels of variability detected are high, number of alleles ranging from 7–15 per locus and percentage heterozygosity between 50 and 91%. Initial screening of larger population samples for several of these PRIMER NOTE