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International Journal of Aquaculture 2012
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6
(1.7%) repeats. Ninety (76.3%) sequences contained
more than one microsatellite. But most of these
sequence contained clustered and complex repeated
structures, only fifty-six sequences had sufficient
flanking regions for primer designing.
1.2 Amplification and polymorphism
Thirteen primer pairs showed reliable and reproducible
amplification in 32 wild individuals. Sequences of
these 13 microsatellites were submitted to GenBank
(accession numbers FJ594998 – FJ595016). Four of the
13 primer pairs produced no more than 2 alleles per
individual and were considered valid single-locus
microsatellite markers (Table 1). The 4 single-locus
microsatellite loci were polymorphic with allele
numbers ranging from 5 to 15 per locus (mean, 11±4.55).
The expected and observed heterozygosity ranged
from 0.60 to 0.92 (mean, 0.79±0.14) and from 0.52 to
0.90 (mean, 0.67±0.24), respectively (Table 1). No
linkage disequilibrium was detected among all the loci
at
p
>0.05 (after Bonferroni correction). One locus
(
HUBA07
) showed significant (
p
<0.05 after Bonferroni
correction) deviation from Hardy–Weinberg equilibrium.
HUBA10
showed signs of null alleles as indicated by
Micro-checker (1 000 randomizations) (Table 1),
suggesting that the presence of null alleles is the main
cause for the deviation from HWE at these loci. The
four microsatellite markers developed here should be
useful for population genetics studies in this species.
Table 1 Primer sequences and characteristics of 4
Babylonia areolata
single-locus microsatellite markers
Locus
GenBank
acc. no.
Repeat motif
§
Primer sequences (5'-3')
Ta
(
℃
)
MgCl
2
(mM)
n a as
H
E
H
O
P
HWE
F: tgtgacatgaacaagggacttc
HUBA01
FJ594998 (TG)
n
R: aggaaactcagagcattcgtg
62
1.2
30 10 302-340 0.92 0.88 0.056
F: aaaagtgcttctcgtgcattg
HUBA07
†
FJ595004 (AG)
N
(TG)
N
R: aacttgtctctttcggggaac
60
1.2
27 15 219-309 0.60 0.36 0.006
F: atctccgtcgtcttgtgattg
HUBA10
†
FJ595007 (TG)
N
(AG
n
R: tgctggtattcagtcctgtcc
58
2.0
31 5 380-450 0.87 0.60 0.023
F: tacgacgtgtttgacgtgttg
HUBA18
FJ595015 (TC)
N
(TTC)
N
(GTG)
N
R: tgacctcacgcaagaaaagag
60
1.5
29 14 390-430 0.76 0.84 0.025
Note: The number of individual analyzed (n), number of alleles (a), allele size range in base pairs (as), observed (
H
O
) and expected
(
H
E
) heterozygosities are presented for each locus. Ta is the annealing temperature and
P
HWE
is the probability of Hardy–Weinberg
equilibrium.
§
Repeat motif: N, pure; n, interrupted; Values in bold represent significant probability estimates after correction for
Bonferroni multiple tests (initial α< 0.05/5 = 0.01);
†
Loci showing evidence for null alleles suggested by Micro-Checker
1.3 Duplicated loci
The remaining nine (69%, 9 of 13) duplicated loci
amplified more than 2 alleles in most individual
(Table 2).
The extra alleles can not be eliminated through
increasing amplification stringency (lower Mg
2+
concentration and higher annealing temperature),
suggesting that they were not caused by nonspecific
amplification, but by amplification of duplicated loci.
Four (30.8%, 4 of 13 ) of them amplified 2 to 4
parallel loci per individual between 100 and 550 bp,
which were clearly linked in tandem as they were
separated in well-defined patterns. For example,
HUBA02
amplified unambiguous non-overlapping
two parallel loci linked obviously, one to two
fragments that differed by 8n bp per locus, with a
locus-to-locus distance of about 145 bp (Figure 1).
This can be explained by the model of [forward
primer] - 145 bp- [forward primer] -267 bp - (8 bp) n-
[reverse primer]. This finding implies that one of the
primers sequence may be located within a satellite
near the microsatellite loci so that
there is significant
tandem duplication of microsatellite loci and/or their
flanking sequences in
B. areolata
genome.
2 Discussion
Those 4 duplicated loci amplified parallel loci
separated in well-defined pattern observed in this
study were similar to those microsatellite loci that
were linked in tandem with duplicated structures, e.g.
satellites or mini-satellite sequences found in Atlantic
surfclam,
Spisula solidissima
(6.7%, 2 of 30) (Wang et
al., 2009b), white-lipped pearl oyster,
Pinctada