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Received: 5 November 2016 Accepted: 22 November 2016 DOI: 10.1111/jai.13391
S H O R T C O M M U N I C AT I O N
Development and characterization of 21 novel microsatellite markers for the Australian smelt Retropinna semoni (Weber, 1895) M. R.-U. Islam | D. J. Schmidt | J. M. Hughes Australian Rivers Institute, Griffith University, Nathan, QLD, Australia Correspondence Md. Rakeb-Ul Islam, Australian Rivers Institute, Griffith University, Nathan, QLD, Australia. Email:
[email protected]
1 | INTRODUCTION
with the diversity in migratory life histories (Hughes et al., 2014; Woods et al., 2010). In this study, twenty-one new polymorphic mi-
The Australian smelt Retropinna semoni is a common freshwater fish
crosatellite markers were developed and characterized for the study
species widely distributed throughout coastal and inland drainages
of northern lineages of Australian smelt. These markers will be used
of southeastern Australia. They form large shoals in the mid to upper
for analysis of population structure, life history variation and species
water column and inhabit deep slow flowing pools as well as shallow
delimitation.
fast flowing riffle-runs. Adults live in a diverse array of environments including slow flowing low land rivers, small coastal streams, river impoundments, upland river and streams, dune lakes and brackish
2 | MATERIALS AND METHODS
water estuaries (Pusey, Kennard, & Arthington, 2004). Australian smelt are formally recognised as being two described taxa R. se-
Randomly-sheared genomic DNA libraries were prepared for
moni (Weber), and R. tasmanica McCulloch, but recent genetic
Illumina paired-end sequencing using two smelt specimens repre-
analyses have recognized a complex of five or more cryptic species
senting informal lineages designated ‘SEQ’ and ‘CEQ’ (Hammer et al.,
throughout their geographic range based on allozymes, microsat-
2007; Schmidt et al., 2016). Genomic DNA was isolated from tissue
ellites and mitochondrial DNA data (Hammer, Adams, Unmack, &
voucher GUB_433 (=lineage SEQ, Twin Bridges Reserve, Brisbane
Walker, 2007; Hughes, Schmidt, Macdonald, Huey, & Crook, 2014;
River, -27.430457 152.639357) and voucher GUM_433 (=lineage
Schmidt, Islam, & Hughes, 2016). Migratory life history patterns
CEQ, Conondale Bridge, Mary River, -26.727511 152.713604).
vary greatly among these genetic groups, ranging from freshwater
Genomic DNA was extracted from fin tissue using the DNeasy
residents to facultative amphidromy and estuarine-resident pop-
Blood and Tissue kit (Qiagen) following the manufacturer’s direc-
ulations (Crook, Macdonald, & Raadik, 2008; Hughes et al., 2014;
tions. DNA was sheared to an approximate mean length of 400 bp
Woods, Macdonald, Crook, Schmidt, & Hughes, 2010). Information
using the M220 Focused-ultrasonicator (Covaris), and an Illumina
on population structure and life history variation in two northern
MiSeq-compatible sequencing library was prepared. A double index
lineages geographically designated ‘CEQ’ and ‘SEQ’ has received
two-step library preparation was used, with all steps performed in
relatively little attention despite speculation these lineages may
the presence of solid phase reversible immobilization (SPRI) beads
represent cryptic species (Hammer et al., 2007; Page & Hughes,
(iTru protocol: Travis Glenn, pers. comm.; Faircloth & Glenn, 2012).
2010; Schmidt et al., 2016). Microsatellites are powerful DNA mark-
Paired-end sequencing was performed on the Illumina MiSeq
ers to measure genetic variation within and between populations
Benchtop Sequencer at the Griffith University DNA Sequencing
(Muneer et al., 2009). They have been used extensively for studies
Facility, using a 600 cycle MiSeq reagent kit v3. The two libraries
of stock identification and population differentiation (Schmidt et al.,
each generated ~1.2 × 107 paired-end reads. Overlapping paired
2014; Wright & Bentzen, 1994). Microsatellite variation has been
reads were merged with Geneious v9.1.5 software (Kearse et al.,
examined within and between R. semoni lineages in southeastern
2012), using the Flash v1.2.9 plugin (min overlap 20, max overlap
Australia, demonstrating a range of population structures consistent
200; Magoc & Salzberg, 2011). Then 1.6 × 106 merged reads in size
J Appl Ichthyol. 2017;1–5.
wileyonlinelibrary.com/journal/jai © 2017 Blackwell Verlag GmbH | 1
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ISLAM et al.
2
range 200–580 bp were sampled for each library. Reads contain-
in GenBank under accession numbers KY124334 – KY124354
ing microsatellites from the genomic libraries were selected using
(Table 1). Locus names beginning with letters ‘BS’ were isolated
the QDD pipeline version 3.1 (Meglecz et al., 2014). Selected loci
from voucher GUB_433 (=lineage SEQ; Table 1). Locus names be-
contained ≥10 uninterrupted repeats and had minimal target region
ginning with letters ‘MS’ were isolated from voucher GUM_433
complexity indicated by the QDD design category ‘A’. Complete mi-
(=lineage CEQ; Table 1). Of the 21 primer pairs, only 3 loci derived
tochondrial genomes of both voucher specimens were previously
from the CEQ lineage were polymorphic (MS6, MS19 and MS24;
assembled from a sample of the raw paired-end data used here
Table 1). The remaining 18 polymorphic loci were all derived from
(Schmidt et al., 2016). A total of 48 primer pairs (24 pairs from each library) were
the SEQ lineage (Table 1). Characteristics of these loci are described in Table 1. The number of alleles (NA) per locus ranged from 3 to
screened using a sample of eight smelt specimens to check for
20 (mean = 9.29). The observed (HO) and expected (HE) heterozygo-
successful amplification and polymorphism. Four unique 20-mer
sity ranged from 0.111 to 0. 969 (mean = 0.756) and 0.358 to 0.945
oligonucleotide tails were added to 5′ end of the forward primers
(mean = 0.734), respectively. The polymorphic information content
as described by Real, Schmidt, and Hughes (2009). All subsequent
(PIC) of all loci ranged from 0.334 to 0.926 (mean = 0.691), indi-
microsatellite screening was carried out in 10 μl PCR reaction mix-
cating that these markers are highly informative (Botstein, White,
ture consisting of 0.5 μl of genomic DNA, 0.2 mM reverse primer,
Skolnick, & Davis, 1980). No locus pair showed significant linkage
0.05 mM tailed forward primer, 0.2 mM tailed fluorescent tag (either
disequilibrium. However, deviation from Hardy–Weinberg equilib-
FAM, VIC, NED or PET, Applied Biosystems), 1× PCR buffer (Astral
rium (HWE) was found in three loci (BS1, BS15 and MS19) after
Scientific) and 0.02 units of taq polymerase (Astral Scientific). The
Bonferroni correction, and two loci (BS1 and BS2) showed potential
following basic thermocycler settings for the polymerase chain re-
presence of null alleles.
action (PCR) were performed: initial denaturation at 94°C for 4 min, followed by 35 cycles at 94°C for 1 min, 57°C for 30 s, 72°C for 1 min and a final extension at 72°C for 7 min. Fluorescently labelled
4 | DISCUSSION
amplified PCR products were pooled and added to 10 μl of Hi-Di™ formamide with 0.1 μl of GeneScan™ 500 LIZ size standard and run
Genetic diversity of the 21 polymorphic loci in the current study
on to an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems)
was similar to five loci developed previously for R. semoni by Hillyer,
according to the manufacturer’s instructions. Genemapper ver-
Woods, and Hughes (2006), showing a moderate genetic diversity
sion 3.1 software (Applied Biosystems) was used to edit and score
(Hillyer loci: mean NA = 10.8, HO = 0.65 and HE = 0.71 vs new loci:
genotypes. The best loci were selected on the basis of amplification suc-
mean NA = 9.29, HO = 0.756 and HE = 0.734). Microsatellite loci of Hillyer et al. (2006) were developed for R. semoni lineages from
cess rate, the presence or absence of stutter peaks, peak intensity,
the southern extent of the species’ range and have proven use-
the polymorphism of the loci, the number of alleles and hetero-
ful for assessing the population structure at various spatial scales
zygosity. Selected loci were further characterised using a popula-
in southern populations (Hughes et al., 2014; Woods et al., 2010).
tion sample (n = 32) of R. semoni collected from the Brisbane River
However, only two loci (sm-26, sm-80) described by Hillyer et al.
(27°30′16.048″S; 152°55′49.991″E), Queensland, Australia. The
(2006) amplified consistently in northern R. semoni lineages ‘SEQ’
number of alleles (NA), allelic range, observed heterozygosity (HO)
and ‘SEC’, and these showed limited polymorphism (R. Islam pers.
and expected heterozygosity (HE), Hardy-Weinberg equilibrium
obs.). Allelic diversity of the 21 new loci presented here indicates
(HWE) test were calculated using Arlequin version 3.5 (Excoffier &
that they will be valuable for population genetics research on
Lischer, 2010). Linkage disequilibrium (LD) between the loci was
northern lineages. Three loci that showed significant departure
evaluated using GENETIX version 4.05 (Belkhir, Borsa, Chikhi,
from HWE should be used with caution in further population ge-
Raufaste, & Bonhomme, 1996). The potential for null alleles, large
netic analyses. Potential presence of null alleles was found in two
allele dropout and stuttering to interfere with scoring accuracy was
loci, which might be the result of mutations in the primer annealing
tested for each microsatellite locus in each sample using MICRO-
sites (Callen et al., 1993). In conclusion, most of the microsatel-
CHECKER version 2.2.3 (Van Oosterhout, Hutchinson, Wills, &
lite loci that were developed here could be used to investigate the
Shipley, 2004). The polymorphic information content (PIC) for
genetic diversity and population structure of the Australian smelt
each locus was calculated using CERVUS version 3.0.3 (Kalinowski,
fish.
Taper, & Marshall, 2007). AC KNOW L ED G EM ENTS
3 | RESULTS
We would like to thank Leo Lee for help in collecting samples and
Among 48 pairs of primers designed, 21 primer pairs were found
Griffith University Animal Ethics Committee provided approval for
Kathryn Real and Jemma Somerville for assistance in the laboratory. polymorphic and selected for characterization in 32 Australian
this study in accordance with Australian Animal Ethics Approval No.
smelt individuals. Sequences of the selected loci were deposited
ENV/23/14/AEC.
GenBank accession No.
KY124334
KY124335
KY124336
KY124337
KY124338
KY124339
KY124340
KY124341
KY124342
KY124343
KY124344
KY124345
KY124346
KY124347
KY124348
KY124349
KY124350
KY124351
Locus
BS1
BS2
BS3
BS4
BS5
BS6
BS8
BS9
BS11
BS14
BS15
BS16
BS18
BS20
BS21
BS22
BS23
BS24
F: CAGCCCGCACTATGAAGAAT R: GCTTCACCTTGAGCCTGTCT
F: TCTAATCGAACTTGGCGACC R: GCGGTTGGCTAATCTCTGTG
F: ACACATGTAAGCATTGAGAACTTTG R: GGAAGAGCCTGACAGGACAA
F: CCTTCCCTAATGAGACATAGTCA R: TATCATATGAAACCCAGCAAGC
F: CTACGAAAGGGTCGTCCTGA R: CCTGGTGACTCCAGAACCTT
F: TCGAAAGACAGACAGAAGAACC R: GATGGATGGGTGGAATTCAG
F: GTGGAAGTGCTGAAGAGGCT R: GGGTCAATTTGTCTATCCCA
F: TTCTGTGTAACTTAAGGCTATGAGAAT R: GCTACGAGTCCCTCTACGCA
F: CGGAGATAAGGAAGCTGTGG R: AGGTTTGCTGCCTGTGAAAC
F: AATTTCCCAGATTTGGATGAA R: TCAGTTGATTGACTGTGACTTTGA
F: TGCTAATCGCTCAGGCTAAA R: ATCCTCCACACCCAGAGTCA
F: ATGTGGTTCTATGGGCGTTC R: AACCGTTCAGTCCAACCAAC
F: AACTCTGACGTGGGCTCTGT R: TGTCAGAACAGCTTGGATGG
F: TGGGTAGAAGGATGAGACATCA R: GAGCATCAGGGTAGGTTGGA
F: CCAGTAAAGTTAAATACCAAAGACG R: TGAGCCTTAATTTCATTGTCTGAG
F: TCTTTAAAGCAGCTGAAGGAGTT R: AAGGATGAATGAAATGATCTGACA
F: CTGTCTCCTGCTTGGGTGAT R: CAATCCACAGTTTGGTGGTG
F: TTCCAGCTTTGGGAAATGTT R: TCTGGTGAGAACAAGGGTCC
Primer sequence (5′-3′) Forward (F) and Reverse (R)
(AC)18
(AC)15
(AC)21
(AC)19
(AC)17
(AC)15
(AC)18
(AC)15
(AC)16
(AGAT)14
(AATC)11
(AGCC)10
(AGCC)11
(ACTG)16
(ACAG)14
(ACAG)13
(ACGC)13
(AATG)15
Repeat motif 4 19 14 12 14 8 8 4 12 20 5 12 3 11 8 11 7 8
57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57
NA
57
Tm (oC)
153–167
190–206
142–175
157–175
122–160
151–159
148–176
128–148
125–186
126–179
159–174
220–248
126–157
109–168
122–173
140–204
118–207
118–150
Alleles range (bp)
0.793
0.625
0.778
0.704
0.867
0.655
0.969
0.938
0.969
0.875
0.625
0.355
0.906
0.931
0.906
0.935
0.688
0.111
HO
0.738
0.735
0.869
0.778
0.859
0.671
0.863
0.685
0.945
0.872
0.476
0.421
0.845
0.901
0.861
0.901
0.917
0.358
HE
0.698
0.674
0.837
0.73
0.827
0.585
0.833
0.617
0.926
0.843
0.412
0.401
0.812
0.876
0.831
0.876
0.895
0.334
PIC
(Continues)
.662
.783
.111
.075
.663
.822
.393
.000*
.689
.079
.250
.155
.445
.910
.985
.163
.003
.000*
PHWE
T A B L E 1 Characterization of 21 microsatellite loci in Australian smelt (Retropinna semoni), including GenBank accession number, primer sequence, repeat motif, annealing temperature (Tm), number of alleles observed (NA), allele size range, observed (HO) and expected heterozygosity (HE), polymorphic information content (PIC) and probability of Hardy–Weinberg equilibrium (PHWE)
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.175 0.548 0.632 0.813 *Loci deviating from Hardy–Weinberg equilibrium (HWE) at p
