Microsatellite DNA Variation of the Gametophyte Clones Isolated from Introduced Laminaria japonica (Phaeophyta) and L. longissima of China and Varieties Derived from them

Development of Laminaria microsatellite DNA marker

About 30% of colonies of the library contained inserts with microsatellite DNA motifs at a middle position. 167 colonies were sequenced commercially with the automatic sequencer ABI3730 (Sangon, Shanghai, China). Of about 138 sequences obtained 65 most desirables (microsatellite DNA located at a middle position and the flanking regions of both sides were long enough) were used to design the primers as the first trial. Unfortunately, only 18 pairs of primers detected polymorphism in the gametophyte clones isolated from either L. japonica (Group 1) or L. longissima (Group 2) (Tables 1 and 2). In the total sample, the number of alleles per locus ranged from two to seven with an average of 3.9, and the Nei's gene diversity ranged from 0.369 (SH8 and H180) to 0.753 (H120) with an average of 0.554 (Table 3). It was also found that Laminaria microsatellite DNA usually was short and tended to be imperfect (Table 2). Very interestingly, 13 loci were found to be independent of each other in Group 1 in linkage disequilibrium testing, while only one locus was found to be independent in Group 2.

Microsatellite DNA variation

As listed in Table 3, the allelic diversity (mean number of alleles) of Group 1 was significantly higher than that of Group 2 (2.9 vs. 1.8, P < 0.05). Similarly, the Nei's gene diversity of Group 1 was also significantly higher than that of Group 2 (0.414 vs. 0.161, P < 0.05). Both allelic diversity and Nei's gene diversity of Group 3 were lower than those of Group 1. Both allelic diversity and Nei's gene diversity of Group 4 were lower than those of Group 1 but higher than those of Group 2. In addition, significant differences of either allelic diversity or Nei's gene diversity or both were observed between female (Population 1) and male (Population 2) gametophyte clones in each group. Significant genetic differentiation was detected between groups and between populations in each group (P < 0.05) (Table 4). The variation among groups accounted for 39.95%, while that among populations accounted for 21.65% of the total (Table 5).

The differentiation was also reflected by Nei's genetic distances (Table 6). The distances between Group 1 and Group 2 and between Group 2 and Group 3 reached 1.142 and 1.036, respectively, while that between Group 1 and Group 3 was only 0.278. It was very interesting to note that the genetic distance between Group 1 and Group 4 was obviously longer than that between Group 3 and Group 4 (0.686 vs. 0.291). The genetic distances between groups and between populations in each group were more clearly reflected in Figure 1, where female and male clones in each group were clustered first, then Group 1 and Group 3 clustered, and finally Group 3 and Group 4 clustered.

The relationship of the four groups was also clearly demonstrated in the assignment test (Table 7). About 33.3% of the gametophyte clones of Group 1 were assigned to Group 3, but not to Group 4. In contrast, 14.3% of the gametophyte clones of Group 2 were assigned to Group 4, but not Group 3. No clones of both Group 3 and Group 4 were assigned back to Group 1 or Group 2. In addition, recent population size reduction (bottleneck effect) was detected in all groups.

Gametophyte clones

In total, 90 Laminaria gametophyte clones were used in the present study. The number of clones across four groups and two sexes are listed in Table 1. These clones were conserved indoor by Shandong Oriental Ocean Sci-tech Co. Ltd. (Yantai, China), which represented the variation of introduced L. japonica and L. longissima of China and the varieties derived from them. The gametophyte clones were harvested by filtering through a sieve cloth and were stored in 70% ethanol – 15 mM ethylene diamine-tetraacetic acid buffer (EDTA) (pH 8.0) and at −20 °C.

DNA isolation

DNA was isolated from about 100 mg of gametophyte following the protocol described previously (Patwary and Van Der Meer 1994; Wang et al. 2005b; Varela-Alvarez et al. 2006) with minor modifications. In brief, the gametophyte was ground into powder in liquid nitrogen, transferred into 3 mL extraction buffer (2% CTAB, 1.4 mol/L NaCl, 20 mmol/L EDTA, pH 8.0, 100 mmol/L Tris-HCl, pH 8.0 and 2%β-mercaptoethanol), mixed well and incubated at 65 °C for 1 h. The emulsion was mixed with 0.33 volume of 5 M potassium acetate (KAc) (pH 7.5), placed on ice for 30 min and centrifuged at 16 000g for 20 min. The supernatant was extracted with phenol and chloroform (24:1, v:v). The DNA was precipitated with 0.6 volume of isopropanol. The DNA was re-suspended in 300 μL extraction buffer and extracted again with phenol and chloroform after adding 100 μL KAc (pH 7.5). The DNA was precipitated with two volumes of absolute alcohol after adding 0.10 volume of NaAc (pH 5.2), washed with 70% ethanol, air-dried briefly and dissolved in tris(hydroxymethyl)aminomethane ethylene diamine-tetraacetic acid (TE) buffer.

Development of microsatellite DNA markers

A microsatellite DNA containing a short genomic DNA fragment enriched library of L. japonica was constructed following the protocol of Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) (Vos et al. 1995; Zane et al. 2002). Streptavidin coated on magnetic beads conjugated with the biotin covalently linked to (AC)₁₂ oligonucleotide, which then annealed with single strand microsatellite DNA containing DNA fragments. Microsatellite DNA containing DNA fragments were obtained by separating glass beads from the buffer after hybridization and releasing them from the beads by denaturation. The DNA strand was re-amplified, inserted into a pMD18-T vector (Takara, Dalian, China) and electroporated into Escherichia coli DH5α. The colonies were each subjected to three independent polymerase chain reaction (PCR) screenings in order to check whether microsatellite DNA were located at the middle of the insert. The first PCR reaction was carried out using two universal sequencing primers on the vector. The second reaction was carried out using (AC)₁₂ oligonucleotide and forward sequencing primer, and the third reaction was carried out using (AC)₁₂ oligonucleotide and the reverse sequencing primer. A colony was desirable for sequencing only if the PCR product that was amplified by both sequencing primers was longer than that amplified by either (AC)₁₂ oligonucleotide and the forward sequencing primer or (AC)₁₂ oligonucleotide and the reverse sequencing primer. Those recombinants with desirable inserts were commercially sequenced with the automatic sequencer ABI3730 (Sangon). The sequences with relatively longer microsatellite DNA at the middle and flanking sequences at both sides were used to design primers using Primer Premier 5.0 software (http://www.premierbiosoft.com/primerdesign/).

Genotyping of gametophyte clones

Gametophyte clones were genotyped with the primers designed. PCR reaction was carried out in a 25-μL volume containing one reaction buffer, 2.0 mM MgCl₂, 200 μM dNTP (each), 200 μM primers (each direction) and 1 U Taq DNA polymerase. The thermal cycling condition was as follows: pre-denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at the temperature appropriate for every pair of primers (Table 1) and extension at 72 °C for 1 min and an extra extension at 72 °C for 10 min. PCR product was separated on 6% denaturing acrylamide gel and visualized by silver staining.

Data processing

The number of alleles and Nei's gene diversity of female and male gametophytes (populations) isolated from four different sources of material (groups) and Nei's unbiased genetic distances between them, were calculated using POPGENE version 1.44 (Yeh et al. 2000). Linkage disequilibrium was tested with GENEPOP version 3.4 (Raymond and Rousset 1995) with the significance level of all multiple tests compared with the sequential Bonferroni adjustments (Rice 1989). Significant deviation from linkage disequilibrium was acceptable when the possibility of a multiple test was less than the possibility adjusted with the sequential Bonferroni method. A neighbor-joining tree of eight populations was constructed using MEGA3 (Kumar et al. 2004) based on Nei's unbiased genetic distances. Various F-statistics were calculated using FSTAT 2.9.3 (Goudet 1995) with their significances tested by permutation, of which, F_ST was the measure of genetic differentiation, which was less biased when the sample size was small (Gaggiotti et al. 1999). The proportion of genetic differentiations among groups and among populations was estimated using hierarchical analysis of molecular variance (AMOVA) implemented in ARLEQUIN 2.000 (Schneider et al. 2000). Recent effective population size reduction (bottleneck effect) was determined using BOTTLENECK software (Cornuet and Luikart 1996). The probability that an individual's genotype would be found in any of the four groups was calculated using the assignment test implemented in STRUCTURE 2.1 (Pritchard et al. 2000).