Page 4 - Tomasello_Borra_etal2009

Tomasello, Di Maida, Calvo, Pirrotta, Borra & Procaccini
extracted adapting the standard protocol of the nucleo-
spin plant kit (Macherey–Nagel) to a new automated pro-
cedure. The new methodology involved automated
genomic DNA extraction, PCR setup reactions and frag-
ments analysis plates assembly and was developed for the
Biomek FX automated platform (Beckman Coulter, Ful-
lerton, CA) equipped with ORCA robotic arm, PCR
machine and plates carousel. The Biomek Software allows
for user-defined variables to be included in the method.
Using our settings, a 96-well plate can be processed in
approximately 50 min and up to 12 plates can be pro-
cessed, unattended, in less than 530 min (see Borra et al.
2007 for details). In general, silica-gel-dried tissue samples genotypes according to the given number of loci and
were homogenized using a mixer mills and transferred to
a 96-well plate, where the first extraction buffer, contain-
ing chaotropic salts, denaturing agents and detergents,
was immediately added, as indicated in the kit manual.
Using a vacuum manifold, working on the whole plate,
lysis mixtures were cleared by filtration (300 s at
20 InHg) to remove polysaccharides, contaminations and
residual cellular debris. Using a gripper tool and a 96-tip
multi-pipetting head, the clear supernatant was mixed
with binding buffer and ethanol to create conditions for
optimal binding to the silica membrane and loaded on
the 96-well binding plate. After washing with two differ-
ent buffers, DNA was eluted and stored in low salt buffer
for downstream applications. PCR reactions were set up,
in automation, with 0.5–1 ll DNA; 0.2–0.5 (pmolÆll)1)
each oligo; Taq (0.075 uÆll)1) in a total volume of 20 ll.
A subset of reactions was randomly selected and gel veri-
fied (gel electrophoresis on a 1% agarose gel; TBE 0.5X
stained with ethidium bromide). PCR reactions were
combined in multiplex and fragment analysis sample
plates were assembled in automation, with: 0.4–1.0 ll
PCR samples; 0.4 ll size standard and formamide up to a
total volume of 20 ll (Migliaccio et al. 2005). Fragments
analysis plates were finally analysed on a Beckman coulter
CEQ 2000XL DNA analyser.
Individual multilocus genotypes were assessed by
means of 13 microsatellite loci, of which 12 were nuclear
and one chloroplastic (Poc-trn) (Procaccini & Waycott
1998; Alberto et al. 2003). The analysis of the electropho-
retic profiles was carried out with the software BECK-
MAN CEQ 2000 ver. 3.0. Allelic assignment has been
refined in relation to existing datasets (Arnaud-Haond
et al. 2007b; Serra et al. 2007). Samples which repeatedly
failed to amplify or to give electrophoretic peaks of con-
sistent size were excluded from the analysis. Final statisti-
cal analyses were performed on the total of 218 samples.
Statistical analysis
To establish whether ramets with the same genotypes
were identical by descent (by clonal growth) or identical
Marine Ecology 30 (2009) 288–300 ª 2009 Blackwell Verlag GmbH
Seagrass meadows at the extreme of environmental tolerance
by chance, the psex value was calculated with the program
GENCLONE 1.0 (Arnaud-Haond & Belkhir 2007). Fur-
thermore, we refined our allele scoring to test for the
existence of somatic mutations or scoring errors, re-calcu-
lating psex values for all genotype pairs differing by only
one allele after deleting the variable locus (Arnaud-Haond
et al. 2007a,b). A threshold value of 0.05, below which
identical genotypes were considered truly clonemates, was
selected.
The Ng value (Parks & Werth 1993; Ruggiero et al.
2005) was utilized to establish the theoretical number of
alleles. Ng was calculated as:
Ng ¼ PiL¼1½aiðai þ 1ފ=2 ð1Þ
where L is the number of loci and ai is the number of
alleles at locus i (Reusch et al. 1998).
Genotypic richness for each meadow has been calcu-
lated through the R value (G–1 ⁄ N–1), where G is the
number of the multilocus lineages (MLL) obtained after
re-scoring the database for somatic mutations or scoring
errors, and N is the number of samples. R is equal to one
if all the individuals identified are genetically distinct.
Both R and the allelic richness (A) were calculated for the
complete dataset and after standardization of sample sizes
to N = 16 (equivalent to the smallest number of ramets
from the Plateau population) and 1000 random resam-
pling of sample units using GENCLONE 1.0 (Arnaud-Ha-
ond & Belkhir 2007). Only complete multilocus
genotypes with no missing data for the 13 loci have been
utilized for the calculations above.
Genetic variability has also been estimated by means of
the allelic frequency for each locus, calculated with soft-
ware GENALEX ver. 6.0 (Peakall & Smouse 2006), and
through the observed (Hobs) and expected heterozygosity
(Hexp) calculated using the software GENETIX ver. 4.1
(Belkhir et al. 1996–2002). The Weir & Cockerham
(1984) inbreeding coefficient (Fis) was calculated with the
software FSTAT ver. 2.9.3.2 (Goudet 1995). Linkage dis-
equilibrium tests for all pairs of loci within each popula-
tion were performed with the software ARLEQUIN
(Excoffier et al. 2005).
The Loiselle spatial autocorrelation index (Loiselle et al.
1995) has been utilized to test for the existence of spatial
autocorrelation within sampling sites. The coefficient of
determination (r2) and slope (b) were reported. Analysis
was performed with the software SPAGeDI (Hardy & Ve-
kemans 2002).
Gene flow among populations was estimated using
Wright’s Fst index (1951) and assignment tests. Assign-
ment tests allow the possible assignment of a genotype to
a population different from that of origin. This test was
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