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Latin american journal of aquatic research

versión On-line ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.45 no.1 Valparaíso mar. 2017

http://dx.doi.org/10.3856/vol45-issue1-fulltext-18 

Short communication

 

Utility of five SSR markers for genetic diversity and paternity exclusion analysis in the Patagonian toothfish

 

Cristian Araneda1, Natalia Lam1,2, Patricia Iturra2, Felipe Jilberto1, Valentina Cordova2 & Pablo Gallardo3

1 Departamento de Producción Animal, Facultad de Ciencias Agronómicas, Universidad de Chile, Santiago, Chile
2
Programa Genética Humana, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
3
Escuela de Ciencias y Tecnologías Agrícolas y Acuícolas, Facultad de Ciencias, Universidad de Magallanes, Punta Arenas, Chile


Corresponding author: Pablo Gallardo (pablo.gallardo@umag.cl)
Corresponding editor: Guido Plaza


ABSTRACT. The Patagonian toothfish or Chilean sea bass (Dissostichus eleginoides), found in the Southern Ocean surrounding Antarctica, is an important fishery species for Chile. This high-value species is regarded as overfished, making it an attractive target for aquaculture. When developing a reproduction program for any aquaculture species, it is important to implement genetic tools to evaluate diversity, inbreeding, and parentage. We calculated genetic diversity and paternity/maternity exclusion probabilities based on five commonly-used microsatellite loci in a natural population of Patagonian toothfish from southern Chile (n = 34) in order to evaluate the potential utility of these five markers in stock management. The observed number of alleles per locus (Na) and observed heterozygosities (HO) are within range as described by studies performed in other sub-Antarctic regions. All five loci were strongly polymorphic, with HO > 0.6 and Na > 7, and paternity exclusion probabilities were high (PE > 0.99), indicating the potential utility of these loci in paternity/maternity exclusion analyses of Patagonian toothfish.

Keywords: Dissostichus eleginoides, Chilean sea bass, heterozygosity, microsatellites, genetic diversity.


 

The Patagonian toothfish (Dissostichus eleginoides), also marketed as "Chilean sea bass," is an important commercial fishery species found in the Southern Ocean surrounding Antarctica. It has been regarded as overfished since the 1990s (Marko et al., 2011; Reyes et al., 2012). The species is a benthopelagic fish with a long lifespan and is widely distributed along the southern Chilean coast, Patagonian shelf, and sub-Antarctic islands and seamounts at depths of 1000-2000 m (Appleyard et al., 2002). Commercial fishing of the Patagonian toothfish is on the rise due to its market value (which is higher than that of salmon, for instance), and international regulations govern sustainable fishery of the species (Marko et al., 2011). Therefore, the Patagonian toothfish is an attractive target for Chilean aquaculture (Reyes et al., 2012). Chilean government agencies currently provide strong incentives and funding for aquaculture programs with new marine species. In Chilean Patagonia, aquaculture research projects are establishing Dissostichus eleginoides broodstock as a basis for developing culture technology. This research is focused on obtaining high-quality juveniles, addressing issues such as nutrition and genetics, and maximizing maturation and reproductive performance. In this context, genetic tools are needed to support reproduction programs in assessing genetic diversity, inbreeding, and parentage. Short-sequence repeats (SSR), or microsatellites, with high levels of polymorphism have been the most useful molecular markers for evaluating marine fish populations. We tested five microsatellite markers (cmrDe2, cmrDe4, cmrDe9, cmrDe13, and cmrDe30) (Reilly & Ward, 1999) in an offshore Chilean population. These markers had been commonly used in genetic diversity studies to evaluate the distribution and population structure of the Patagonian toothfish in the Southern Ocean, Antarctic Islands, Patagonian shelf (Falkland/Malvinas Islands), and Atlantic, West and Western Indian Ocean sectors (Smith & McVeagh, 2000; Appleyard et al., 2002, 2004; Shaw et al., 2004; Rogers et al., 2006). Our main objectives were to evaluate genetic diversity and estimate paternity/ maternity exclusion probabilities, in order to evaluate the potential utility of these five markers in broodstock management.

We studied 34 fishes, collected in September-October of 2011 from two sampling points in Chilean Patagonia in front of Trinidad Gulf (Magallanes and Chilean Antarctic region): n = 19 from point one (49°29'41"S, 76°26'17"S) and n = 15 from point two (50°15'05"S, 76°20'15'W). Genomic DNA was extracted using the phenol-chloroform method from fin-clip samples (Taggart et al., 1992). Microsatellite analysis was performed using five markers developed for this species (cmrDe2, cmrDe4, cmrDe9, cmrDe13, and cmrDe30) (Reilly & Ward, 1999) and the M13-tailed primer method (Schuelke, 2000) (Table 1). PCR amplifications were carried out as reactions containing 10x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, Triton X-100 0.1%), 2.5 mM MgCl2, 0.2 mM dNTPs, 8 pmol reverse primer, 8 pmol fluorescent-labeled universal M13 primer, 2 pmol forward primer, 1 U Taq polymerase (Biotools®), and 2-μL DNA samples. The thermal profile was 94°C for 2 min, followed by 30 cycles at 94°C (30 s), specific marker Tm (Table 1) (45 s), 72°C (45 s), 8 additional cycles at 94°C (10 s), 53°C (45 s) to bind M13-labeled primer, and 72°C (45 s) with a final 10-min extension step at 72°C.

 

Table 1. SSR markers and characteristics of primers used in genetic analysis of Patagonian
toothfish. Underlined sequences in forward primer indicate tail to bind M13-labeled primer.

 

Different amplicons labeled with different florescent dyes were pooled in one reaction tube and run with a GeneScan™ 500 LIZ™ dye-size standard (Applied Biosystems®) in an automatic sequencer at external laboratories. Genotypes were identified using GeneMarker® software (SoftGenetics®). Genetic diversity was assessed according to observed (HO) and expected (HE) heterozygosities, number of observed alleles by locus (Na), polymorphism information content (PIC), and paternity/maternity exclusion probabilities (PE), with Cervus 3.0 software (Araneda et al., 2004; Kalinowski et al., 2007). The presence of null alleles was assessed with six different estimators (Dempster et al., 1977; Chakraborty et al., 1992; Brookfield, 1996; Van Oosterhout et al., 2004; Kalinowski et al., 2006), using Micro-Checker 2.2.3 (Van Oosterhout et al., 2004), Cervus 3.0 (Kalinowski et al., 2007) and FreeNA (Chapuis & Estoup, 2007). Genepop 4.2 (Rousset, 2008) was used to evaluate deviation from Hardy-Weinberg expectations along with pairwise FST to evaluate population differentiation between the two collection sites.

Genetic differentiation did not vary significantly between the two sampling points (FST = -0.002; P > 0.05), possibly due to their geographical proximity. There-fore, in subsequent analyses, all 34 samples were used together and considered to belong to the same biologic population.

Na ranged from 8 (cmrDe13) to 25 alleles (cmrDe9) (Table 2). These two loci have also shown the lowest (cmrDe13) and highest (cmrDe9) allele numbers in other studies performed with the five markers used here (Reilly & Ward, 1999; Smith & McVeagh, 2000; Appleyard et al., 2002, 2004; Shaw et al., 2004; Rogers et al., 2006).

 

Table 2. Genetic diversity in Patagonian toothfish collected from Southern
Chile. Na: number of observed alleles by locus, HO: observed heterozygosity,
He:
expected heterozygosity, PIC: Polymorphism information content, PE1:
paternity/maternity exclusion probability for a single parent, PE2: paternity/
maternity exclusion probability for a single parent when one parent is known,
PEi: identity exclusion probability for a single individual.

 

Two loci (cmrDe2 and cmrDe9) showed significant deviations from H-W expectations, both with deficits of heterozygotes, possibly due to the presence of null alleles or the small sample size (Table 3).

 

Table 3. Null allele frequencies for five SSR loci in Patagonian toothfish using six different
estimators

 

Locus cmrDe9 has also shown significant deviation from H-W expectations in previous studies (Appleyard et al., 2002, 2004; Shaw et al., 2004; Rogers et al., 2006), suggesting that this finding may also reflect difficulties in scoring this locus (Rogers et al., 2006). In general, all null allele frequency estimators gave similar results. Only one locus showed a null allele frequency above 10% (cmrDe2) according to all estimators except the Brookfield-2 method, which indicated higher null allele frequencies for three loci (cmrDe2, cmrDe9, and cmrDe30) (Table 3). This method assumes that all individuals that fail in amplifications are null homozygotes (Brookfield, 1996). It is widely accepted that if the null allele frequency is below 10%, relatedness/parentage estimators can be used directly, without adjustment; on the other hand, loci with null allele frequencies above 50% must be excluded, due to very low power (Huang et al. , 2016). To avoid this distortion, and to evaluate the power for paternity/maternity exclusion analysis, two datasets were used, one with and one without genotype adjustment, with the Brookfield-2 method using Micro-Checker 2.2.3.

All genetic diversity estimates were within the ranges published in previous works on Patagonian toothfish from geographic areas other than the Chilean coast, where observed heterozygosities ranged from 0.5261 for locus cmrDe13 (Rogers et al., 2006) to 0.9480 for locus cmrDe9 (Smith & McVeagh, 2000) (Table 2). Our findings were consistent with these previous results showing high levels of genetic diversity. In our work, all loci were strongly polymorphic with observed heterozygosities >0.6 and PIC >0.7, indicating their potential utility in paternity exclusion analysis (Table 2).

The global paternity exclusion probability for one putative parent using only the molecular information from progeny was high enough to perform a paternity/maternity exclusion analysis in a breeding population with five polymorphic SSR loci (PE1 = 0.98999 and PE1 = 0.99245 with adjusted genotypes) (Araneda et al., 2004). However, in a reproduction program, even if no physical tagging is performed, it is always possible to genotype all breeders. Adding genotyping information from the other parent and progeny increases the combined paternity/maternity exclusion probability. When both datasets are available, the paternity/maternity exclusion probability for a single parent can be increased to 0.99897 and 0.99927 (adjusted genotypes) when the other parent is known (PE2). Finally, the combined identity exclusion probability was PEI = 0.99999998 and PEI = 0.9999 9999 (adjusted genotypes), indicating that these five SSR markers show excellent potential for identifying an individual in a sample using its known genotype. While the presence of null alleles decreases the exclusion probability, a comparison of the statistics with and without adjustments reveals this reduction to be minor. In the context of a breeding population, the effect of a null allele is to mistakenly reject a true father, due to a lack of observed shared alleles with the offspring; that is, the number of real parents is underestimated. This scenario (type I error) is preferable to including a nonrelated individual as a parent (type II error) for breeding applications.

The potential application of these markers in paternity/maternity exclusion analysis must be evaluated in real families after Chilean reproduction aquaculture projects begin to produce families with viable offspring.

ACKNOWLEDGEMENTS

This work was supported by FONDEF-CONICYT grant DA09i1002. We wish to thank our technician Raquel Becerra Vera for DNA extraction and general assistance.

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Received: 26 August 2015;
Accepted: 4 April 2016

 

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