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Mendes NJ, Cruz VP, Ashikaga FY, Camargo SM, Oliveira C, Piercy AN, Burgess
GH, Coelho R, Santos MN, Mendonça FF, Foresti F. 2016. Microsatellite loci in
the tiger shark and cross-species amplification using pyrosequencing
technology. PeerJ 4:e2205 https://doi.org/10.7717/peerj.2205
Microsatellite loci in the tiger shark and cross-species
amplification using pyrosequencing technology
Natália J Mendes, Vanessa P Cruz, Fernando Y Ashikaga, Sâmia M Camargo, Claudio Oliveira, Andrew N Piercy, George H
Burgess, Rui Coelho, Miguel N Santos, Fernando F Mendonça, Fausto Foresti
The tiger shark (Galeocerdo cuvier) has a global distribution in tropical and warm
temperate seas, and is caught in numerous fisheries worldwide, mainly as bycatch. It is
currently assessed as near threatened by the International Union for Conservation of
Nature (IUCN) Red List. In this study we identified 9 microsatellite loci through next
generation sequencing (454 pyrosequencing) using 29 samples from the western Atlantic.
The genetic diversity of these loci was assessed and revealed a total of 48 alleles ranging
from 3 to 7 alleles per locus (average of 5.3 alleles). Cross-species amplification was
assessed in three other species: Carcharhinus longimanus, C. acronotus and Alopias
superciliosus. Given the potential applicability of genetic markers for biological
conservation, these data may contribute to the population assessment of this and other
species of sharks worldwide.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2042v1 | CC-BY 4.0 Open Access | rec: 13 May 2016, publ: 13 May 2016
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Microsatellite loci in the tiger shark and cross-species amplification using pyrosequencing
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technology
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Mendes NJ1, Cruz VP1, Ashikaga FY1, Camargo SM
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Coelho R4, Santos MN4, Mendonça FF5, Foresti F1
1-5,
Oliveira C1, Piercy A2, Burgess G3,
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1Laboratório
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Paulo, Brazil.
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2Lake
de Biologia e Genética de Peixes, Universidade Estadual Paulista, Botucatu, São
Nona Campus, Valencia College, Florida, USA.
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3Florida
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Florida, USA.
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4Portuguese
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5Laboratório
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Federal de São Paulo, Santos, São Paulo, Brazil.
Program for Shark Research, Florida Museum of Natural History, University of Florida,
Institute for the Ocean and Atmosphere, IPMA, Algarve, Portugal.
de Genética Pesqueira e Conservação, Marine Sciences Institute, Universidade
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Corresponding Author: Fernando Fernandes Mendonça
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Universidade Federal de São Paulo – UNIFESP, Instituto do Mar, Laboratório de Genética
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Pesqueira e Conservação, Av. Almirante Saldanha da Gama, 89. Ponta da Praia, Santos, SP –
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Brasil - CEP 11030-400 e-mail: fernando.mendonca@unifesp.br
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Running title: Microsatellite in the tiger shark by pyrosequencing
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Abstract
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The tiger shark (Galeocerdo cuvier) has a global distribution in tropical and warm temperate
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seas, and is caught in numerous fisheries worldwide, mainly as bycatch. It is currently assessed
27
as near threatened by the International Union for Conservation of Nature (IUCN) Red List. In
28
this study we identified 9 microsatellite loci through next generation sequencing (454
29
pyrosequencing) using 29 samples from the western Atlantic. The genetic diversity of these loci
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was assessed and revealed a total of 48 alleles ranging from 3 to 7 alleles per locus (average of
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5.3 alleles). Cross-species amplification was assessed in three other species: Carcharhinus
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longimanus, C. acronotus and Alopias superciliosus. Given the potential applicability of genetic
33
markers for biological conservation, these data may contribute to the population assessment of
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this and other species of sharks worldwide.
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Introduction
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The tiger shark Galeocerdo cuvier (Péron & Lesueur 1822), is a shark from the order
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Carcharhiniformes and family Carcharhinidae. It has a worldwide distribution in tropical and
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temperate seas, and is considered a top predator generally requiring large foraging areas (Heupel
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et al. 2014). Recent data show that this species can move long distances and occupies different
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habitats, including coastal regions and is therefore more susceptible to anthropogenic threats
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(Heupel et al. 2014).
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Captured in many world fisheries as bycatch, the tiger shark is currently classified as
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"Near Threatened" by the International Union for Conservation of Nature (IUCN). However,
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some basic information, such as the characterization of population genetic structure,
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identification of geographical restrictions to gene flow with possible local populations remain
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broadly unknown. In light of this, information on their conservation status is difficult to assess.
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For this reason, molecular markers have been increasingly used in species conservation and
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management programs, including microsatellite molecular markers (Simple Sequence Repeats -
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SSR). A range of SSR markers have been developed using the pyrosequencing technique,
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generating information with millions of base pairs in a single run and in a short period of time.
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Specifically for the tiger shark, nine SSR markers were previously developed on
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specimens from the Hawaiian archipelago (Bernard et al. 2015), but cross-application was not
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tested for other shark species. Thus, the objectives of this study were to identify other
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microsatellites for the tiger shark in specimens from the Atlantic, and design additional
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molecular markers that can be used in this and other shark species for population genetics
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studies.
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Material and Methods
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Sampling
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In fulfillment of data archiving guidelines (Baker 2013), primary data have been
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deposited with Dryad. Samples of tiger shark were collected in landings of the fishing fleet from
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São Paulo coast (n=12) and in scientific cruises in the Fernando de Noronha archipelago (n=6)
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by researchers from the Biosciences Institute of Botucatu, São Paulo State University, and
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Marine Sciences Institute of the São Paulo Federal University, in Brazil. Additionally, 11
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samples were collected from the east coast of Florida, by the Florida Program for Shark
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Research, University of Florida, USA. For evaluating the cross-amplification we used 6 samples
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of Carcharhinus acronotus collected in São Paulo coast, 5 samples of C. longimanus and 5
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samples of Alopias superciliosus, collected in the northeast Atlantic by onboard observers from
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the Portuguese Institute for the Ocean and Atmosphere (IPMA), Portugal. All sampled tissues
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were stored in 95% ethanol to ensure the integrity and quality of tissue for molecular analysis.
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454 GS-FLX Pyrosequencing and Microsatellite Discovery
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The total genomic DNA was extracted from each sample following the protocol
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described by Ivanova et al. (2006). A sample of 100 µg of tiger-shark DNA from São Paulo coast
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was sequenced on a Roche 454 GS FLX sequencer with Titanium platform “Genome sequencer
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20 System” (Instituto Agrobiotecnológico de Rosário – INDEAR, Argentina), following
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procedures described in Margulies et al. (2005).
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To isolate microsatellites and design primers for population genetics all sequences of the
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SSR were compiled using Primer3 (Rozen & Skaletsky 2000) and BatchPrimer3 (You et al.
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2008). Primers were designed based on the following criteria: primer size of 20 bp (min = 18,
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max = 22 bp), ideal annealing temperature of 60°C degrees (min = 55 ° C, max = 63 °C), GC
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optimum of 60% (= 40% min, max = 80%) and the size of the amplified product ranging from
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50-500 bp. The sequences were then grouped and aligned in the Clustal W software (Thompson
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et al. 1994), identifying duplicated sequences for the same locus.
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Novel Microsatellite Markers
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The PCR amplifications to test the synthesized primers were performed in a Thermal
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Cycler VeritiTM (Applied Biosystems™, Life Technologies) under the following conditions:
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initial denaturing for 10 min at 95 °C; 30 cycles of 94 °C for 45s, the primer annealing
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temperature (TA) was tested from of 51 °C to 57 °C for 50 s; 72 °C for 50s, and a final extension
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at 72 °C for 20min. The total reaction volume was 10 µL and composed of 0.20 X PCR Buffer,
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0.25 mM MgCl2, 0.05 mM of each dNTP, 0.5 units of Platinum Taq DNA polymerase
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(Invitrogen™, Life Technologies, EUA), 0.10 µM reverse primer, 0.10 µM forward primer, and
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30 ng of template DNA.
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To verify the effectiveness of the reaction and the amplification of the fragments, 1.5 l
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of the PCR product were subjected to electrophoresis on a 1% agarose gel. The amplified
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products were compared with a 1Kb plus ladder (Invitrogen), subsequently visualized on a
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transilluminator and photographed with a digital camera using the Kodak Digital Science
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software.
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Genotyping was done with the M13-tail PCR method of Schuelke (2000). The best loci,
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that showed high polymorphism and quality of bands, were selected and further analyzed on an
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ABI 3130xl sequencer (Applied Biosystems™, Life Technologies). The allele sizes were
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determined using ROX 500 (Applied Biosystems) as an internal standard with the software
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package GeneMapper 3.7 (Applied Biosystems).
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We used the software GenAlex analysis 6.1 (Peakall & Smouse, 2006) to convert our
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data to run in other analysis programs. Arlequin 3.5 (Guo & Thompson, 1992) was used to
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calculate heterozygosity, number of alleles, Hardy-Weinberg equilibrium and linkage
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disequilibrium. The program Cervus v.3.0.7 (Marshall et al. 1998) was used to test for the the
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presence of null alleles and estimate, the inbreeding coefficient (Fis) and polymorphism
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information content (PIC).
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Results and Discussion
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From the genomic material generated by the pyrosequencing technology, a total of
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71,059 reads with an average size of 367 bp was obtained, consisting of 26,075,405 nucleotides,
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which accounts for approximately 0.75% of the G. cuvier genome, assuming a genome size of
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3.44Gb (estimated from the size of Rhincodon typus, Read et al. 2015). For the identification of
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microsatellite sequences, the online software Batch Primer3 was used, and 615 microsatellite
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loci were identified. A second filtration was subsequently performed with the software Primer
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3.0 which identified 159 microsatellite loci. From these, we selected 30 loci which contained the
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best scores of each primer pair with a size of 15 - 20bp, a GC of 40-50% and little variation in
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the annealing temperature in the PCR reaction. Of these, 20 pairs of primers were synthesized
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and tested, 9 being polymorphic with 1 trinucleotide and 8 dinucleotide (Table 1). The sequences
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with polymorphic microsatellite markers in this study have been deposited in GenBank
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(Accession numbers: KT598263-KT598271).
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The application of the developed markers resulted in 48 alleles, with a minimum of 3
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(TIG_25) to 7 (TIG_1, TIG_7, TIG_12) and average of 5.3 alleles per loci. Transferability tests
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of the markers in other species showed positive amplification in C. longimanus, Alopias
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superciliosus and C. acronotus. For the C. acronotus, two loci were polymorphic (TIG_17,
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TIG_5), and for C. longimanus and A. superciliosus only one polymorphic locus were observed
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in 5 samples of each species, TIG_15 and TIG_7 respectively (Table 2).
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In tiger shark the observed heterozygosity (Ho) and expected heterozygosity (He) ranged
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from 0.16 (TIG_17) to 1.0 (TIG_10) and 0.20 (TIG_25) to 0.72 (TIG_7) respectively. The Ho
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was higher than He, suggesting an excess of heterozygotes relative to the model of Hardy-
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Weinberg equilibrium (HWE). Significant differences from Hardy-Weinberg equilibrium after
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Bonferroni correction (p<0.01) were detected in only 2 loci (TIG_10 and TIG_17). The deviation
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in the Hardy-Weinberg equilibrium for locus TIG_17 (0.715) can be explained by a significant
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value in intrapopulation inbreeding coefficient (Fis) (Kordicheva et al. 2010). This locus was the
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only one with a positive value for the Fis, and may be evidence of a heterozygous deficiency
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(Holsinger & Weir 2009), resulting in a decrease in genetic variability.
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Imbalance values in Hardy-Weinberg equations when considering microsatellite locus
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may be due to the presence of null alleles (Kordicheva et al. 2010). However, the presence of
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null allele was not detected in the present study, indicating that the markers developed are of
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high quality. Further, the polymorphism information content (PIC) was highly informative for all
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the loci (PIC > 0.5), also indicating a high quality marker (Botstein et al. 1980).
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In the present study, the average expected heterozygosity was approximately 0.50 and the
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average observed heterozygosity was 0.55. The levels of genetic variability seen in this study
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may be due to population differences resulting from remote sample locations. This is to be
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expected given that the samples are coming from different oceans and the finding of significant
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differences in the levels of heterozygosity among different groups would not be unforeseen.
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Supplementary Material
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Supplementary material can be found at http://www.ncbi.nlm.nih.gov/genbank/
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Acknowledgements
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The authors thank all those who contributed to this study, including the researchers who
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carried out the sample collection in Fernando de Noronha archipelago and the fishermen for
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facilitating tissue collection during landings in the São Paulo coast. Sampling and data collection
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from the Portuguese fishery were obtained and supported by the Programa Nacional de
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Amostragem Biologica (PNAB) within the scope of the EU Data Collection Framework (DCF).
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The authors are grateful to all fishery observers and longline skippers that helped collect samples
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for this study.
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design. BMC Bioinformatics. 9: 253.
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Table 1. General information about the microsatellite analysis.
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230
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Table 2. Data for microsatellite loci of the cross-amplification in Carcharhinus longimanus,
Carcharhinus acronotus and Alopias superciliosus.
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233
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Table 3. Data for microsatellite loci in the tiger shark, Galeocerdo cuvier.
Table 1(on next page)
General information about the microsatellite analysis.
1
Analyzes
Number of reads
71.059
Selection of microsatellites (using BatchPrimer3)
615
Secondary selection of microsatellite (using Primer 3.0)
159
Amplification and control of PCR product on agarose gel
30
Microsatellite loci to synthesize with fluorescent dye
20
Polymorphism test with capillary sequencer
10
Microsatellite loci in linkage equilibrium
2
N of sequences
9
Table 2(on next page)
Data for microsatellite loci of the cross-amplification in Carcharhinus longimanus,
Carcharhinus acronotus and Alopias superciliosus.
1
2
C. acronotus
3
4
C. longimanus
A. superciliosus
Loci
Na
Range(bp)
Na
Range(bp)
Na
Range(bp)
TIG_1
2
116-118
2
118-134
2
104-118
TIG_5
3
260-264
2
331-335
2
265-273
TIG_7
2
170-180
2
162-170
3
152-170
TIG_10
2
251-253
1
304
1
307
TIG_12
2
296-364
2
246-296
2
372-418
TIG_15
1
336
3
290-310
2
288-312
TIG_17
3
242-270
2
210-224
1
268
TIG_19
0
0
1
316
2
386-394
TIG_25
1
396
1
358
2
388-398
Na: number of alleles
Table 3(on next page)
Data for microsatellite loci in the tiger shark, Galeocerdo cuvier.
1
LOCI
PRIMER SEQUENCE (5’→3’)
MOTIF
T ºC
N
NA
RANGE(bp)
HO
HE
HWE
FIS
PIC
F(NULL)
-0.194
TIG_1
F_ CTCTTGACGGTGCTCGATC
(AC)10
53
29
7
116 - 154
0.758
0.642
0.711
-0.184
0.710
TIG_5
R_AATGGCAACTTTTCCTGTCC
F_GCCAGCATCCATTCATACAG
(CT)8
51
26
4
203-257
0.384
0.337
1.000
-0.141
0.589
(AC)15
57
27
7
169-183
0.925
0.726
0.318
-0.280
0.811
-0.101
(GT)10
59
29
5
256-276
1.000
0.655
0.000
-0.539
0.608
-0.245
(CA)11
53
28
7
364-376
0.535
0.520
0.543
-0.030
0.682
-0.213
(TG)15
55
25
6
231-241
0.520
0.463
0.675
-0.124
0.650
-0.233
(GT)11
57
25
4
268-286
0.160
0.554
0.000
0.715
0.734
-0.138
(TG)10
53
27
5
337-353
0.555
0.443
0.677
-0.260
0.627
-0.214
(CCT)5
55
27
3
331-349
0.222
0.206
1.000
-0.075
0.511
-0.285
-0.239
R_AGAGGGAAGTGGTGTGTGGT
TIG_7
F_CACCAACCTCCCCATCAC
R_CAGACATTCCTCCTCCATCC
TIG_10
F_CTCAGCAGGTCTGGACAACA
R_GGTGGTAGGAACATGGAACG
TIG_12
F_TGCCATGAGTGCTGTTTTTC
R_TGCCGCATTGTTACTGCTAC
TIG_15
F_AACTGCCAAAAGGGACAAGA
R_GTAAGCCCAACAGACCATCC
TIG_17
F_TGAAGCTAACGAGGGGTCTG
R_AGCGCAGAAGATCAAGAGGA
TIG_19
F_TGCTTGTGTCTGAGGTGAGTG
R_TTGGAGGTTCAATCCGAGAC
TIG_25
F_CCGTGCCTATGTGGATTTCT
R_CTTGAAGAGAGTGGGCGAAG
2
3
4
T ºC: primer annealing temperature, N: number of individuals analyzed, NA: number of alleles, He: expected heterozygosity, Ho: observed heterozygosity,
HWE: probability of departure from Hardy–Weinberg equilibrium, Fis: inbreeding coefficient, PIC: polymorphism information content, F(Null): null alleles.