Development and characterization of 21 novel

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Nov 22, 2016 - smelt are formally recognised as being two described taxa R. se- moni (Weber) ... analyses have recognized a complex of five or more cryptic species throughout .... ful for assessing the population structure at various spatial scales .... GENETIX 4.05, logiciel sous Windows TM pour la génétique des pop-.
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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.

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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)

ISLAM et al.       3

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.175 0.548 0.632 0.813 *Loci deviating from Hardy–Weinberg equilibrium (HWE) at p