Partial DNA sequence of two PCR fragments obtained using Laminaria digitata microsatellite loci in L essonia nigrescens . The sequence for both species is shown. The complete sequence for A: Ld2/167 locus, containing a tri-nucleotide motif and B: Ld2/520 locus, containg the new primer sequence (underlined sequence Ln 520 F1) and a di-nucleotide motif can be observed in the respective genebank accessions (GenBank AF547165, AY183663, respectively). ∗ : Indicates homology in base sequence between the two species. Bold bases are microsatellite motives, or primer sites. 

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... a species of Laminaria from the Atlantic coasts of Europe, L. digitata (Billot et al., 1998) , were tested in Lessonia nigrescens from the Pacific coasts of Chile. Banding patterns of amplified loci were tried to be improved when they were multilocus so that polymorphism could be tested and compared with those obtained using 30 RAPD-loci, the only other molecular marker available in the same species (Martınez et al., 2003). In this study these markers, although being dominant, were informative enough to detect genetic erosion in this species due to massive mortality caused by strong ENSO events, a phenomenon yet to be compared to co-dominant markers, once these are described. Primers of polymorphic microsatellites, originally described for Laminaria digitata from France (Table 1), were synthesized (Gibco, PE-Applied Biosystems, USA) and tested in three Lessonia nigrescens individuals from Las Cruces (33 30 S), Chile, under the conditions indicated by Billot et al. (1998). DNA was extracted as indicated in Martınez et al. (2003). Amplified bands, of similar molecular weight to those expected for Laminaria digitata (ca. 520 bp for locus Ld2/520 and ca. 167 bp for locus Ld2/167) were iso- lated from low-melting agarose gels, purified with QI- Aquick PCR purification kit (Qiagen) and sequenced in forward and reverse directions in a 310 ABI Perkin- Elmer Automatic Sequencer. Sequence runs were carried out in a 47 × 50 μ m capillary, which was filled automatically with 5 μ L of the performance optimized polymer POP6 TM for sequence analysis (POP4 TM for fragment analysis). Conditions using the Big Dye terminator v.3 were voltage of 12 V, 50 C and run times of 7500 s. The new sequences were deposited in the Gene Bank under accession numbers indicated below. One type of improvement was obtained by using Touchdown PCR (Don et al., 1991) as follows: 10 cycles (94 ◦ C, 70 ◦ C–1 ◦ C descending to 60 ◦ C, 72 ◦ C), then 15 normal cycles at 60 ◦ C, as in Billot et al. (1998). In order to test non-specific reactions, PCR reactions were run using, alternatively, one single primer for each locus. Alternatively, PCR improvement was obtained from sequence data of cleaned PCR products, used to synthesize new primers, more specific to Lessonia nigrescens . For each successfully improved PCR, the reverse primer was ordered NED-labeled (PE Applied Biosystems, USA). Rox-500 size standard (PE, Applied Biosystems, USA) was added to the PCR products and mix was automatically injected in a capillary filled with the POP4 TM polymer of a 310 ABI Perkin-Elmer Automatic Sequencer. The polymer was automatically changed at each new run. Electrophoresis was carried out at 15 kV, 50 ◦ C for 36 min in 1 × ABI310-Automatic Analyzer buffer TM . After PCR improvement of selected loci, band sizes were identified and polymorphism was tested in a random sample of 26 individuals ( = 52 alleles). They were collected from the intertidal in each of five sampling sites on the Chilean coast, between 20 ◦ S and 42 ◦ S (four to six individuals from each site). These sites were the same ones described in Martınez et al. (2003) and they are located from North to South of Chile, on the coasts of Iquique (20 ◦ S, n = 4), Coquimbo (30 ◦ S, n = 5), Las Cruces (33 ◦ S, n = 6), Valdivia (40 ◦ S, n = 5) and Chiloé Island (41 ◦ S, n = 6). Nei’s genetic distances were estimated and represented in a NJ-tree, using the package TFPGA-v1.3, (Miller, 1997). The resulting tree was compared with the one obtained using 4 RAPD primers (C2, C8, C11, L5 from Genset, San Diego, CA, USA, Table 2), which enabled the detection of polymorphism in 30 bands amplified for 10 to 20 individuals sampled in the same localities (Martınez et al., 2003). Expected heterozygosity ( H e ) as estimated from multiplying and adding single allele frequencies ( H = 2 pq + 2 rq + . . . 2 nq ), was compared to the observed heterozygosity ( H o ) the number of observed heterozygotes divided by the total number of individuals. From the 15 primers designed for Laminaria digitata 14 amplified in Lessonia nigrescens . However, in 11 of them there were obtained multilocus banding patterns when using unmodified L. digitata protocols (Figure 1, and summary in Table 3). Monolocus patterns (single bands) were observed only in locus Ld2/148 but its 1000 bp-size was much longer than the expected size range (168–207 bp). Two loci not tested in L. digitata gave only smears (Figure 1, Table 3). Only one locus, Ld2/469-2, did not amplify at all. When 25 forward or reverse primers were tested in PCRs individually, without their respective reverse/forward primer, 14 also gave some amplification product (11 did not amplify) and 12 PCR products gave again multilocus banding profiles (Figure 2). Due to their appropriate size range, two Lessonia nigrescens multilocus loci were chosen for PCR improvement. Sequencing of them allowed a better characterization of loci Ld2/520 (GenBank AY183663) and Ld2/167 (GenBank AF547165) (Figure 3). Although the PCR fragments could not be sequenced completely the data indicated greater conservation of di-nucleotide than tri-nucleotide motives between both brown algal species. The mutation pattern in the two sequences studied seemed to be of a polar type, and less conserved in the sequence flanking the tri-nucleotide motif. Touch down PCR could improve the banding profile for locus Ld2/520 (Figure 4). After PCR improvement through new primer design the estimation of allele frequency for locus Ld2/167 in a random sample of 26 individuals of Lessonia nigrescens showed a similar number of alleles for both species (Table 3). However there was a greater range of allele sizes in Lessonia nigrescens than in Laminaria digitata populations (Table 3). The overall observed heterozygosity ( H = 0 . 384) was much smaller than the expected one ( H e 0 686) and in the two northern samples the H o ‘s were even much smaller than in the rest of the more southern localities (Table 4). Based on these single locus results, two northern Chilean populations (i.e., Iquique/Coquimbo) revealed to be well separated from the rest of populations towards ...