Genetic Characterization of Mediterranean Atlantic Populations of Spartus aurata

and

Cristbal Gallardo, Diego Lpez* and Daniel Lpez Departamento de Biologa Celular, Fisiologa y Gentica, Facultad de Ciencias, Universidad de Mlaga, Campus de Teatinos, E-29071, Mlaga, Spain.

Spartus aurata is a very popular fish in the Mediterranean cuisine, having acquired a great importance in aquaculture. However in recent years, production is decreasing, which has never happened before in the history of seabream aquaculture in Spain, having returned the production level at 2006 values. There are various causes that may be behind this negative trend such as allelic diversity decreased as a result of high rates of inbreeding. Through the use of screening techniques and biotechnology it is possible to improve the quality of cultivated species, which could allow the industry regain competitiveness as a result of increased production and improved product quality. In this work we have analyzed the genetic structure of the Mediterranean and Atlantic populations, in order to determine their genetic variability, with the aim of designing future strategies to increase the aquaculture farms productivity.
Keywords Spartus aurata aquaculture microsatellite seabream

Introduction
The seabream (Sparus aurata L.), seabass (Dicentrarchus labrax) and turbot (Psetta maximum) are the most important species of fish marine breeding produced in the southern European countries. Total aquaculture production of sea in Europe and the rest of the world in 2010 was 139,925 tons, according to FEAP statistics. There are aquaculture productions of seabream in 19 countries. The main producers are Greece with approximately 72.000 t. (representing 51,5% of total), Turkey with 21.000 tons (15,0%) and Spain with 20.360 (14,6%). Although seabream fishing

continues in the Mediterranean and the Atlantic, its medium-term volume remains relatively constant, whereas seabream breeding keep growing and account for 95% of the total [3] . The aquaculture production of seabream in Spain in 2010 was 20.360 tons, 14,1% lower than 2009, when 23.690 tons. This decline in production has never happened before in the history of seabream aquaculture in Spain, having returned the production level at 2006 values. Compared with the remarkable growth of aquaculture in third countries, the evolution of this activity in the members states of the European Union in the past decade shows a pessimistic future, which is reflected in a stagnation of production [3]. For this reason, the industry needs to breed better quality individuals to maintain a good price and performance. Through the use of screening techniques and biotechnology it is possible to improve the quality of cultivated species, which could allow the industry regain competitiveness as a result of increased production and improved product quality. The levels of genetic differentiation or similarity inferred by neutral molecular markers, such as microsatellites, represent a basic source of information for reconstructing the evolutionary history of a species and for depicting the actual situation in terms of genetic structure and gene flow [2]. In this study we have characterized the genetic structure of the Mediterranean and Atlantic populations of S. aurata through an analysis of microsatellites polymorphism, and compared them with a reference population to determine their variability, with the aim to determine which population is more suited to introduce new alleles in fish farms populations to increase their genetic diversity.

* To whom correspondence should be addressed. Email: Dieguinpillin@hotmail.com

Material and methods

Sampling and microsatellite genotyping In all, 300 adult seabream, ~ 1622 cm total length, were collected from different localizations in the Mediterranean sea, the Atlantic Ocean and the reference population farm. Genomic DNA was obtained from n clips using the salting-out extraction method described by Aljanabi and Martinez (1997), then used as a template in a polymerase chain reaction (PCR) for ten microsatellite loci: Sau140A, Sau140b, Sai10a, Sai10b, Sai19a, Sai19b, Sau47a, Sau47b, SauANa, SauANb, Sai14a, Sai14b, Sai15a, Sai15b, Sau97a, Sau97b, Sai12a, Sai12b, Sau82a and Sau82b. The forward primers for each locus were labeled with 5-uorescent dye (6-FAM, HEX, or TAMRA), and the amplied products were processed for polymorphism detection on an ABI 3730 automated sequencer.

Genetic variability and differentiation Expected heterozygosities and allele frequencies were calculated using the software CERVUS version 2.0, independently for each population and for populations as a whole. The software GENETIX version 4.03 was used to conduct a correspondence factor analysis in two dimensions. Finally, to calculate the genotypic frequencies, the number of alleles, the allelic richness, the gene diversity, the reduction in heterozygosity due to genetic drift in subpopulations (Fst) and the reduction of heterozygosity due inbreeding in the total population (Fis) , the software FSTAT version 2.9.3.2. was used.

Results
The analysis of the genetic variability showed that the allelic diversity of the reference population is higher that the Mediterranean and Atlantic populations, showing k values of 10 for all analyzed loci (Table 1-3).

Likewise, statistical analysis showed that the differences between the allele number of the Mediterranean and Atlantic populations were significant (Table 4). On the other hand, the polymorphic information content (PIC) values showed that both in the reference population and in the Mediterranean the genetic marker that provides more information is Sai19 ( PIC values of 0,877 and 0,860 respectively), while that in the Atlantic population the marker which more information provides is Sau140 (PIC = 0,809). The Kruskal-Wallis test values indicated (Table 5) that the differences in the fixation index median values among the populations are not great enough to exclude the possibility that the difference is due to random sampling variability, that is, there is not a statistically significant difference (P = 0,823). However, in the case of the reference population highlights the fixation index value obtained for the marker Sai12 (F = -1139.251), which reflects the great excess of heterozygotes for this locus. In addition, two other locus should be highlighted, Sau97, which has excess of heterozygotes in the reference population but defect in the Atlantic, and Sau82, which shows defect of heterozygotes in the reference population and excess in the Mediterranean. Regarding to Fis values, the results revealed that none of the three populations showed a considerable degree of inbreeding, since in any case the RHV values were less of 0,05 (Table 6). However, in the different populations did appear markers with heterozygosis excess, according with the RLV values (Table 6). Concretely, in the reference population were Sai10, Sai12 and Sau97, in the Atlantic population Sai19, SauAN and Sai12, while in the Mediterranean population were SauAN and Sai12. Fst values calculated between the three populations showed that in all cases the results were significant (P-value = 0,0166). These results imply that there are genetic divergences among populations (Table 7). Finally, if we analyze the factor analysis plot , it is easy to verify that the different populations are not homogeneously distributed, so that each occupies different regions of space (Figure 1).

Table 1. Genetic parameters that characterize the reference population

Locus Sau140 Sai10 Sai19 Sau47 SauAN Sai14 Sai15 Sau97 Sai12 Sau82
k: number of alelles

k 10 10 10 10 10 10 10 10 10 10
N: number of individuals

N 100 100 100 100 100 100 100 100 100 100

Ho 0.800 0.940 0.920 0.820 0.920 0.850 0.940 0.940 1.000 0.880

He 0.888 0.885 0.892 0.853 0.869 0.867 0.890 0.889 0.877 0.884
He: expected heterozygosity

P-valor 0.6650 0.0550 0.2133 0.8833 0.0567 0.7533 0.0817 0.0550 0.0017* 0.6483

PIC 0.827 0.869 0.877 0.833 0.850 0.848 0.874 0.873 0.859 0.867

F 0.099 -0.062 -0.031 0.039 -0.059 0.020 -0.056 -0.057 -1139.251 0.005
F: fixation index

Ho: observed heterozygosity

PIC: polymorphic information content

Table 2. Genetic parameters that characterize the Atlantic population

Locus Sau140 Sai10 Sai19 Sau47 SauAN Sai14 Sai15 Sau97 Sai12 Sau82
k: number of alelles

k 8 7 6 7 7 6 7 7 7 7
N: number of individuals

N 100 100 100 100 100 100 100 100 100 100

Ho 0.820 0.860 0.880 0.730 0.900 0.720 0.880 0.810 0.940 0.800

He 0.835 0.815 0.784 0.788 0.829 0.786 0.819 0.817 0.790 0.802
He: expected heterozygosity

P-valor 0.7033 0.1383 0.0067* 0.9633 0.0250 0.9417 0.0650 0.6383 0.0017* 0.5733

PIC 0.809 0.785 0.747 0.756 0.801 0.749 0.790 0.787 0.755 0.773

F 0.018 -0.055 -0.122 0.074 -0.086 0.084 -0.074 0.009 -0.190 0.002
F: fixation index

Ho: observed heterozygosity

PIC: polymorphic information content

Table 3. Genetic parameters that characterize the Mediterranean population.

Locus Sau140 Sai10 Sai19 Sau47 SauAN Sai14 Sai15 Sau97 Sai12 Sau82
k: number of alelles

k 7 8 10 10 9 8 8 8 8 8
N: number of individuals

N 100 100 100 100 100 100 100 100 100 100

Ho 0.800 0.860 0.880 0.800 0.920 0.780 0.890 0.790 0.960 0.860

He 0.841 0.853 0.878 0.828 0.851 0.802 0.846 0.810 0.813 0.836
He: expected heterozygosity

P-valor 0.9067 0.4750 0.5450 0.8400 0.0267* 0.7900 0.1717 0.7150 0.0017* 0.2733

PIC 0.816 0.832 0.860 0.804 0.829 0.766 0.823 0.780 0.785 0.812

F 0.049 -0.008 -0.002 0.034 -0.081 0.027 -0.052 0.025 -0.181 -0.029
F: fixation index

Ho: observed heterozygosity

PIC: polymorphic information content

Table 4. ANOVA performed on the allelic diversity of the Mediterranean and Atlantic populations

Source of Variation Between Groups Residual Total

DF: degree of freedom SS: sum of squares

DF 1 18 19
MS: mean square

SS 11,25 11,3 22,55

F: F static

MS 11,25 0,628

F 17,92

P <0,001

Table 5 Kruskal-Wallis one way analysis of variance on ranks performed on fixation index values

Group Reference Atlantic Mediterranean

N 10 10 10

Missing 0 0 0

Median -0,0435 -0,0265 -0,005

25% -0,059 -0,086 -0,052

75% 0,02 0,018 0,027

H = 0,390 with 2 degrees of freedom. (P = 0,823)

Table 6. Fis values obtained through FST software analysis, and its associated P-values.

Marker Sau140 Sai10 Sai19 Sau47 SauAN Sai14 Sai15 Sau97 Sai12 Sau82 All

Reference population Fis RLV RHV 0.009 0.6950 0.4317 -0.062* 0.0433 0.9850 -0.032 0.2517 0.8400 0.039 0.9083 0.1800 -0.059 0.0867 0.9500 0.020 0.7400 0.3467 -0.057 0.0717 0.9583 -0.058* 0.0500 0.9717 -0.141* 0.0017 1.000 0.004 0.6167 0.5067 -0,034* 0.0017 1.000

Atlantic population Fis RLV RHV 0,018 0.7183 0.3583 -0.055 0.1683 0.9050 -0.123* 0.0067 0.9967 0.074 0.9667 0.0667 -0.086* 0.0367 0.9850 0.083 0.9483 0.0767 -0.075 0.0667 0.9617 0.009 0.6333 0.4850 -0.190* 0.0017 1.000 0.003 0.5733 0.5350 -0,034* 0.0150 0.9900

Mediterranean population Fis RLV RHV 0.049 0.9100 0.1617 -0.008 0.5133 0.6067 -0.003 0.5167 0.5867 0.034 0.8167 0.2850 -0.081* 0.0350 0.9917 0.027 0.7667 0.3267 -0.051 0.1583 0.8967 0.025 0.7750 0.3133 -0.181* 0.0017 1.000 -0.029 0.3233 0.7867 -0.022 0.0567 0.9550

Significant values are marked with an asterisk RLV: Proportion of randomisations that gave a lower Fis than the observed

RHV: Proportion of randomisations that gave a higer Fis than the observed

Table 7. Fst values obtained in pairs among the three populations. Significant values are marked with an asterisk.

Population A Population B Population C

Population A Fst P-value 0.0000 0.0528* 0.01667 0.0457* 0.01667

Population B Fst P-value 0.0528* 0.01667 0.0000 0.0718* 0.01667

Population C Fst P-value 0.0457* 0.01667 0.0718* 0.01667 0.0000 -

Discussion
In this study we have employed ten microsatellite loci to analyze the differences in genetic structure existing in Mediterranean and Atlantic populations of Spartus aurata, with respect to a reference population, with the aim to determine if there is any genetic factor responsible for the decline in production in recent years. Despite expectations, results indicated that inbreeding levels in the reference population were not significant, which allows a priori rule out that this factor is one of the causes of the decline in production. It should be noted that the allelic diversity of the reference population was even greater than those of the Mediterranean and Atlantic populations, showing k values of 10 for all locus (Table 2). When we analyzed the fixation indexes, it was very striking the obtained value of F = -1139.251 for the locus Sai12, which seems to reflect the strong selection against homozygous individuals. This phenomenon should be analyzed in depth in further researches to determine their possible effects on the yield of farms. Likewise, it should be also considered the possible effect on the production of an increase of heterozygotes for the genes associated with the

Figure 1. Plot of the factorial analysis performed on the reference, Mediterranean and Atlantic popu lations. The black points correspond to Atlantic population, white points to reference population and gray points to Mediterranean population.

marker Sau82, which showed defect of heterozygotes in the reference population and excess in the Mediterranean. Finally, the obtained Fst valued indicated that there are genetic divergences among populations, as shown by factorial analysis plot, so that should be examined the potential positive impact of the addition to farms of individuals from the Atlantic Ocean and Mediterranean Sea. As concluding remarks, the data generated in this study provide useful information on the population structures, and allow us to reject that the loss of genetic variability in fish farms is a key factor in the decline in production, although additional experiments are needed to determinate if the frequency shift in certain genes have had any effect.

[2] Franchini P, Sola L, Crosetti D, Milana V, Rossi AR. (2012). Low levels of population genetic structure in the gilthead sea bream, Sparus aurata,along the coast of Italy. ICES Journal of Marine Science 69:41-50. [3] Gobierno de Espaa. La acuicultura marina de peces en Espaa. Informe APROMAR. 2011. [4] Porta J, Porta JM, Martnez-Rodrguez G, lvarez MC. 2006. Genetic structure and genetic relatedness of a hatchery stockof Senegal sole (Solea senegalensis) inferred by microsatellites. Aquaculture 251:46-55.

References
[1] Aljanabi SM and Martinez I. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR based techniques. Nucleic Acids Res 25: 46924693