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Introduction

The white-spotted sandbass (Paralabrax albomaculatus (Jenyns, 1840)) is a bony fish endemic to the Galápagos Islands. They have a pelagic larval stage that has the potential of wide dispersal throughout the archipelago. The white-spotted sandbass was listed as endangered in the IUCN Red List in 2001 following an estimated 70% decline in its population size, due mostly to overfishing (Robertson et al., 2010). Despite its endemism and importance for the Galápagos artisanal fishing community, very little research has focused on this species.

Fishing pressure is increasing for the white-spotted sandbass due to a rising human population (including an increase in tourist numbers; Galapagos National Park, 2010) and declines of previously favored species such as the sail-fin grouper (Mycteroperca olfax; Ruttenberg, 2001). Despite this decline, management regulations are severely limited, mostly by the lack of information about this endemic species. Species-specific genetic tools would vastly improve our ability to define population structure. In this study, we present 10 polymorphic microsatellites markers that have been developed specifically for this species. These primers can be used to determine genetic diversity, population structure and connectivity among populations in the archipelago.

Materials and Methods

All samples used in this study were collected with permission of the Galapagos National Park (research permit number: PC-28-13). Muscle or liver samples were collected from fishing boats at two distinct geographical locations: “Banco Ruso” (n = 30), south of San Cristobal island and “Bolivar” (n = 40) west of Isabela island. These fishing sites are approximately 220 km apart and are separated by the landmass of Isabela.

DNA was extracted from liver or muscle tissue using the DNEasy Blood and Tissue Kit (Qiagen, Venlo, Netherlands). To obtain microsatellite markers, a next-generation-sequencing approach was used (Castoe et al., 2012a). A paired-end library of genomic DNA was made with the Nextera® DNA Preparation Kit using 50 ng of DNA (following the manufacturers protocol) and sequenced on the Illumina MiSeq. The library construction and sequencing was carried out by the Genetics Core Facility at the University of Manchester. Read lengths were 2 × 250bp and there were 2 × 4,238,835 sequence reads obtained in the raw data. Microsatellites and their primers were then designed from reads filtered by Trimmomatic. Reads were trimmed using the ‘sliding window’ function based on quality scores with a 4bp window size and quality threshold of 20. Leading and trailing were both set to 3 and the minimum length was set to 50bp (see Lohse et al., 2012). There were 2 × 3,930,136 reads remaining after these filtering steps. Microsatellites with sufficient flanking region were screened for using PAL_finder v.0.02 (Castoe et al., 2012b). The primer settings were selected using the recommended criteria in the Qiagen Type-it Microsatellite PCR Kit protocol in order to increase amplification success for the development of primers when using this kit. These settings include: optimum base-pair length (bp) of 20–30pb, 40–60% GC content, optimum melting temperature (Tm) 68 °C, minimum Tm 60 °C and maximum difference in Tm between paired primers 2 °C. PAL_finder was set to search for sequences with a minimum of 8 repeat units ranging from di—to hexa—nucleotide repeats.

A total of 37 loci were selected for screening containing 8 tri-, 15 tetra-, 12 penta- and 2 hexa- nucleotides using 6 individuals to check for successful amplification and variation. Di-nucleotide repeats were not selected as allele scoring is generally more complicated for this repeat motif due to ‘stutter bands’ on either side of an allele peak (ascribed to enzyme slippage during amplification). This simulates allele peaks and therefore may lead to difficult and inaccurate scoring of alleles (Guichoux et al., 2011).

PCRs were carried out using the Type-it_Microsatellite PCR Kit (Qiagen, Venlo, Netherlands), with the recommended cycling conditions (5 min at 95 °C, 28× (30 s at 95 °C, 90 s at 60 °C, 30 s at 72 °C) and a final extension of 30 min at 60 °C). PCR products were initially analyzed using agarose gel electrophoresis, and loci were considered successful if one or two bands were present. Of the 37 initial loci, 10 successfully amplified according to these criteria. These loci were tested using labeled primers with florescent dyes VIC or 6-FAM in duplex PCRs (Table 1). A 3730 DNA Analyzer (Applied Biosystems, Carlsbad, California, USA) was used for the fragment length analysis of the PCR products with the Genescan™ 500 LIZ® size standard. Allele peaks were scored using GeneMapper® Software Version 3.7 (Applied Biosystems, Carlsbad, California, USA) following the procedure recommended by Selkoe & Toonen (2006). Null alleles and scoring errors were checked using Microchecker version 2.2.3 (Van Oosterhout et al., 2004) and information regarding Hardy–Weinberg equilibrium (HWE) was tested using GenoDive (Meirmans & Van Tienderen, 2004). Finally, estimates of allele frequency for the set of microsatellites with null alleles were provided using FreeNA (Chapuis & Estoup, 2007).

Table 1:
Characterization of ten polymorphic microsatellite loci for Paralabrax albomaculatus.
Locus Genbank number Primer sequence (5′–3′) Repeat motif Dye Ta (°C) Size range (bp) Na Ho He
PCR duplex set 1
Paxalb_ 10 KP997010 F: ACAAGTGCATCAAATACATGTCGG ATCT (32) 6-FAM 63.8 404–480 24 0.919 0.944
R: AAGGAATTCAATCTTAGTGGACACG
Paxalb_ 4 KP997008 F: GCCTTATTCTCTCCTTTATCCCC AAGAG (70) VIC 63.4 408–485 24 0.895 0.925
R: CAAAGTTTTGAGACTGAGCAGGG
PCR duplex set 2
Paxalb_ 32 KP997015 F: ATGTCTTGCCTTATCTGTTGTGG AAATT (45) 6-FAM 63.8 295–373 26 0.718 0.927
R: ACTAAACAGCGACGTTATACGAGG
Paxalb_ 22 KP997013 F: TCCCAACCAACACCATTTTATGGC TTTC (56) VIC 66.2 305–454 21 0.914 0.922
R: TCCCTCTCGTTCTCTCCGACTTGC
PCR duplex set 3
Paxalb_ 11 KP997011 F: GAGATGCTGGAGAACTCAGAGGGC TGC (24) 6-FAM 68.2 189–259 19 0.964 0.871
R: AACGACTCCGGCGATTCAGC
Paxalb_ 1 KP997007 F :AACCATGATCACACCTCCATCTTCC ATCT (88) VIC 67.4 305–445 44 0.935 0.966
R: AGCCTTTATGTGGTGAAGGGGTGC
PCR duplex set 4
Paxalb_ 20 KP997012 F: CTGCATTGACAATCTATTGTTCTGG AAAAC (75) 6-FAM 63.3 359–474 49 0.882 0.98
R: GCACGGTGCAATATTTTCTTTCC
Paxalb_ 24 KP997014 F: GTTTTGGTCCAGATGCTTTTAATGG AAT (54) VIC 64 419–477 23 0.575 0.9
R: ACTGTACTGGCTCCAACTGCTGC
PCR duplex set 5
Paxalb_ 8 KP997009 F: GATGTAGCCAGCACAGCAAATGACC AAAG (68) 6-FAM 66.5 316–415 36 0.956 0.963
R: CCTCCATCCTCAACTTTCTCAATTAAATCC
Paxalb_35 KP997016 F: TGTTCCTCGCCTCAAAGTAGGACG AAT (39) VIC 68.2 382–414 11 0.844 0.815
R: CACCGATACAGACCTTTGACAGGC
DOI: 10.7717/peerj.1253/table-1

Notes:

Duplex set

primers that were combined in one PCR

F

forward sequence

R

reverse sequence

Repeat motif

number of times the nucleotide motif is repeated

Dye

fluorescent dye used to label each primer

Ta

optimal annealing temperature

Na

number of alleles

Ho

observed heterozygosity

He

expected heterozygosity

Results

The 10 loci show high levels of polymorphism with 11–49 alleles per locus (Table 1). Microsatellites Pax_alb20, Pax_alb24 and Pax_ alb32 were characterized as containing possible null alleles, and deviated from HWE. Allele frequencies estimates for these 3 microsatellites were of 0.0987, 0.0401, and 0.1634, respectively.

Discussion

As no previous work has been carried out on this endemic species, these loci will be useful for further research to investigate population connectivity, structure and genetic diversity as well as help with the implementation of informed fisheries management.

Although three loci showed evidence for null alleles, estimates of null allele frequencies show that these loci are nonetheless useful for estimating genetic diversity. The high number of alleles per locus could mean that these 10 primers would be very useful to show variation between populations.