Molecular Ecology Notes (2002), 2, 242–244doi:10.1046/j.1471-8278 .2002.00212.x
© 2002 Blackwell Science Ltd
Blackwell Science, Ltd
Isolation and characterization of 20 polymorphic
microsatellite loci for Scaptodrosophila hibisci
ALEX C. C. WILSON,*‡ PAUL SUNNUCKS*§ and J. S. F. BARKER†
*Department of Biological Sciences, Macquarie University, NSW 2109, Australia, †School of Rural Science and Natural Resources,
University of New England, Armidale, NSW 2351, Australia
Scaptodrosophila hibisci is an endemic Australian Drosophilidae that breeds in the flowers
of native Hibiscus. Here we report the isolation and amplification of 20 polymorphic
microsatellite loci. We cloned these microsatellites because loci developed for Drosophila
melanogaster failed to amplify in S. hibisci. Null alleles were detected at six loci, and five
were X-linked. Two of the primer pairs amplified an unlinked ‘bonus’ locus. One locus
containing juxtaposed microsatellite loci was suitable for designing an additional set of
primers. Mean number of alleles per locus was 10, mean HO and HE per locus were 0.532
and 0.636, respectively.
Keywords: Drosophilidae, juxtaposed loci, null alleles, segregation distortion, sex linkage, X-linkage
Received 6 December 2001; revision received 17 January 2002; accepted 31 January 2002
Scaptodrosophila hibisci is an endemic Australian member
of the family Drosophilidae that breeds in the flowers of
native Hibiscus species. Compared to the genus Drosophila,
very little is known of the biology and genetics of the
Scaptodrosophila species. Here we report the isolation and
amplification of 20 polymorphic microsatellite loci for S.
hibisci. Loci developed for Drosophila melanogaster failed to
amplify in S. hibisci (P. England personal communication).
DNA was extracted from 45 S. hibisci males using a salting-
out protocol (Sunnucks & Hales 1996). Microsatellites were
cloned and screened largely following Taylor et al. (1994).
Template DNA for polymerase chain reaction (PCR)
screening was extracted from single flies by salting-out
(Sunnucks & Hales 1996), and resuspended in 20 µL of
1 × TE (1 mm EDTA, 10 mm Tris base pH 7.5). Microsatel-
lite PCR was carried out using an MJ Research PTC 200
thermocycler in 10 µL reactions containing approximately
50 ng of DNA, 0.5 U of Taq polymerase (Promega), 50 mm
KCl, 10 mm Tris-HCl (pH 9.0), 0.1% Triton X-100, 2 mm
MgCl2, 200 µm of dGTP, dCTP and dTTP, 20 µm dATP,
10 pmol of each primer and 0.05 µL of [α33P]-dATP at
10 mCi/mL. PCR cycling conditions (‘touchdown’ pro-
grams) were as follows: initial denaturation 94 °C 2 mins,
followed by one cycle of v °C 30 s, 72 °C 45 s, 94 °C 15 s; one
cycle of w °C 30 s, 72 °C 45 s, 94 °C 15 s; one cycle of x °C
30 s, 72 °C 45 s, 94 °C 15 s; one cycle of y °C 30 s, 72 °C 45 s,
94 °C 15 s; 30 cycles of z °C 30 s, 72 °C 45 s, 94 °C 15 s; final
extension 72 °C for 2 mins. For PCR program PMS1: v = 62,
w = 61, x = 59, y = 57 and z = 55; PMS2: v = 55, w = 53, x =
51, y = 49 and z = 47; PMS3: v = 53, w = 51, x = 49, y = 47 and
z = 45. PCR program PMS4 is the same as PMS3 except that
35 cycles at the final annealing temperature (z) were per-
formed. For PCR program AMS1 the first four extension
cycles (temps v-y) were each cycled twice through and the
z temperature cycle repeated 28 times. For AMS1: v = 65, w
= 64, x = 63, y = 61 and z = 60.
Approximately 14 000 recombinant colonies were
screened for (GA)n and (CA)n microsatellite repeats.
Sequences were obtained from 45 positive colonies and
primers were designed for 22 loci. Four of the loci for which
primers were designed were abandoned because they
amplified poorly or not at all. Only four of the loci con-
tained pure dinucleotide repeats (Sh34d, Sh74X, Sh89X,
Sh94), three contained compound repeats (Sh8ii, Sh36c,
Sh72X) and 10 impure or interrupted repeats (Table 1).
Nine loci were (CA)n microsatellites and seven (GA)n.
Although not probed for, one pure mononucleotide repeat
(Sh29c) and one interrupted (AT)n repeat (Sh48) were
Correspondence: Alex Wilson. ‡Present address: Center for Popu-
lation Biology, University of California, Davis, CA 95616 USA.
Fax: + 1530 7521449; E-mail: firstname.lastname@example.org
§Present address: Department of Genetics, La Trobe University,
Bundoora, VIC 3086, Australia
PRIMER NOTE 243
© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 242–244
Table 1 Twenty polymorphic microsatellite loci isolated from Scaptodrosophila hibisci, with primer sequences and characteristics of each locus. Repeat structure is derived from sequence
of original cloned allele.
(Accession #) Repeat
(bp) Primer 5′ → 3′
XLi X-linked locus. *Null allele(s) positively identified at the locus by the presence of null homozygous individuals in population samples. †Null allele identified in segregation analyses
(Wilson unpublished data). HO is observed heterozygosity and HE is unbiased expected heterozygosity (Nei 1978) — both averaged over the two populations. See text for PCR program
244 PRIMER NOTE Download full-text
© 2002 Blackwell Science Ltd, Molecular Ecology Notes, 2, 242–244
isolated. Two of the primer pairs, Sh88 and Sh90, amplified
a locus additional to the one for which the primers were
designed. These we call Sh88+ and Sh90+. The repeat types
of these additional loci (Sh88+ and Sh90+) are currently
unknown. Both additional loci are apparently unlinked to
the cloned loci with which they coamplify.
Two microsatellite clones were isolated that each con-
tained two microsatellite motifs separated by a region of
unique sequence large enough to design an additional set
of primers to amplify the two microsatellite containing
regions separately (juxtaposed microsatellite loci: Estoup
et al. 2000). One of these was difficult to amplify and was
dropped. The other locus (Sh8) contained two microsatel-
lites, Sh8i and Sh8ii. These juxtaposed loci, however,
showed no significant linkage disequilibrium in any of
nine populations (Barker, unpublished data).
Allelic diversity and average observed and expected
heterozygosity were computed across 21 males and 21
females from each of two populations — Balgownie,
NSW (34°23.149′-S, 150°52.022′-E), and Beatrice Creek,
Queensland (20°50.576′-S, 148°40.760′-E). The number of
alleles per locus ranged from two to 24 over the 84 ind-
ividuals typed from the two populations (Table 1). The mean
number of alleles per locus was 9.65. Observed hetero-
zygosity ranged from 0.024 to 0.834, and expected hetero-
zygosity from 0.023 to 0.888 (Table 1).
Of the 20 loci, null alleles were detected at six, and five
were X-linked (Sh9X, Sh72X, Sh74X, Sh78X and Sh89X;
Table 1), i.e. the locus was always homozygous in males (in
fact hemizygous) and heterozygous or homozygous in
females. Sex-linkage is expected to influence rates of micro-
satellite evolution (Hedrick & Parker 1997), as sex-linked
loci have a smaller effective population size than auto-
somal loci. Studies in D. melanogaster have attempted to
characterize the location and concentration of microsatel-
lites across the genome (Pardue et al. 1987; Lowenhaupt
et al. 1989; Schug et al. 1998; Bachtrog et al. 1999). In D. melano-
gaster, Bachtrog et al. (1999) report a total microsatellite
density on the X chromosome more than twice as high as
on autosomes. This is in stark contrast to the situation in
another dipteran, Bactrocera tryoni, in which none of 16 loci
tested was X-linked (Kinnear et al. 1998; M. Frommer per-
sonal communication). Quantitative information on X-
linkage is generally difficult to derive from the literature,
but we note that across a wide range of taxa, very few sex-
linked microsatellites have been reported.
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