Genetic Characterization at Mediterranean and Atlantic Populations of Spartus aurata

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Spartus aurata is a very popular fish in theMediterranean cuisine, having acquired a greatimportance in aquaculture. However in recentyears, production is decreasing, which has neverhappened before in the history of seabreamaquaculture in Spain, having returned theproduction level at 2006 values. There arevarious causes that may be behind this negative

trend such as allelic diversity decreased as aresult of high rates of inbreeding. Through theuse of screening techniques and biotechnology itis possible to improve the quality of cultivatedspecies, which could allow the industry regaincompetitiveness as a result of increasedproduction and improved product quality. Inthis work we have analyzed the genetic structureof 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 inEurope 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 theproduction 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.

Genetic Characterization of Mediterranean and

Atlantic Populations of Spartus aurata

Cristóbal Gallardo, Diego López* and Daniel López Departamento de Biología Celular, Fisiología y Genética, Facultad de Ciencias, Universidad de Málaga,

Campus de Teatinos, E-29071, Málaga, Spain.

* To whom correspondence should be addressed. Email: [email protected]



Material and methods

Sampling and microsatellite genotyping

In all, 300 adult seabream, ~ 16 – 22 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 fin 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′-fluorescent dye (6-FAM, HEX, or TAMRA), andthe amplified 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 referencepopulation 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 RHVvalues 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 implythat 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).



Locus k N Ho He P-valor PIC F

Sau140 10 100 0.800 0.888 0.6650 0.827 0.099Sai10 10 100 0.940 0.885 0.0550 0.869 -0.062Sai19 10 100 0.920 0.892 0.2133 0.877 -0.031Sau47 10 100 0.820 0.853 0.8833 0.833 0.039

SauAN 10 100 0.920 0.869 0.0567 0.850 -0.059Sai14 10 100 0.850 0.867 0.7533 0.848 0.020Sai15 10 100 0.940 0.890 0.0817 0.874 -0.056Sau97 10 100 0.940 0.889 0.0550 0.873 -0.057

Sai12 10 100 1.000 0.877 0.0017* 0.859 -1139.251Sau82 10 100 0.880 0.884 0.6483 0.867 0.005


Sau140 8 100 0.820 0.835 0.7033 0.809 0.018Sai10 7 100 0.860 0.815 0.1383 0.785 -0.055Sai19 6 100 0.880 0.784 0.0067* 0.747 -0.122Sau47 7 100 0.730 0.788 0.9633 0.756 0.074SauAN 7 100 0.900 0.829 0.0250 0.801 -0.086Sai14 6 100 0.720 0.786 0.9417 0.749 0.084Sai15 7 100 0.880 0.819 0.0650 0.790 -0.074Sau97 7 100 0.810 0.817 0.6383 0.787 0.009

Sai12 7 100 0.940 0.790 0.0017* 0.755 -0.190Sau82 7 100 0.800 0.802 0.5733 0.773 0.002


Sau140 7 100 0.800 0.841 0.9067 0.816 0.049Sai10 8 100 0.860 0.853 0.4750 0.832 -0.008Sai19 10 100 0.880 0.878 0.5450 0.860 -0.002Sau47 10 100 0.800 0.828 0.8400 0.804 0.034

SauAN 9 100 0.920 0.851 0.0267* 0.829 -0.081Sai14 8 100 0.780 0.802 0.7900 0.766 0.027Sai15 8 100 0.890 0.846 0.1717 0.823 -0.052Sau97 8 100 0.790 0.810 0.7150 0.780 0.025

Sai12 8 100 0.960 0.813 0.0017* 0.785 -0.181Sau82 8 100 0.860 0.836 0.2733 0.812 -0.029

Source of Variation DF SS MS F P

Between Groups 1 11,25 11,25 17,92 <0,001Residual 18 11,3 0,628Total 19 22,55

Table 1. Genetic parameters that characterize the reference population

Table 2. Genetic parameters that characterize the Atlantic population

Table 3. Genetic parameters that characterize the Mediterranean population.

k: number of alelles N: number of individuals Ho: observed heterozygosity He: expected heterozygosity PIC: polymorph ic information content F: fixation index

k: number of alelles N: number of individuals Ho: observed heterozygosity He: expected heterozygosity PIC: polymorphic information content F: fixation index

k: number of alelles N: number of individuals Ho: observed heterozygosity He: expected heterozygosity PIC: polymorphic information content F: fixation index

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

DF: degree of freedom SS: sum of squares MS: mean square F: F static



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

Group N Missing Median 25% 75%

Reference 10 0 -0,0435 -0,059 0,02

Atlantic 10 0 -0,0265 -0,086 0,018

Mediterranean 10 0 -0,005 -0,052 0,027

Reference population Atlantic population Mediterranean population

Marker Fis RLV RHV Fis RLV RHV Fis RLV RHV

Sau140 0.009 0.6950 0.4317 0,018 0.7183 0.3583 0.049 0.9100 0.1617Sai10 -0.062* 0.0433 0.9850 -0.055 0.1683 0.9050 -0.008 0.5133 0.6067Sai19 -0.032 0.2517 0.8400 -0.123* 0.0067 0.9967 -0.003 0.5167 0.5867

Sau47 0.039 0.9083 0.1800 0.074 0.9667 0.0667 0.034 0.8167 0.2850SauAN -0.059 0.0867 0.9500 -0.086* 0.0367 0.9850 -0.081* 0.0350 0.9917Sai14 0.020 0.7400 0.3467 0.083 0.9483 0.0767 0.027 0.7667 0.3267Sai15 -0.057 0.0717 0.9583 -0.075 0.0667 0.9617 -0.051 0.1583 0.8967

Sau97 -0.058* 0.0500 0.9717 0.009 0.6333 0.4850 0.025 0.7750 0.3133Sai12 -0.141* 0.0017 1.000 -0.190* 0.0017 1.000 -0.181* 0.0017 1.000Sau82 0.004 0.6167 0.5067 0.003 0.5733 0.5350 -0.029 0.3233 0.7867All -0,034* 0.0017 1.000 -0,034* 0.0150 0.9900 -0.022 0.0567 0.9550

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

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

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

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

Fst P-value Fst P-value Fst P-value

Population A 0.0000 - 0.0528* 0.01667 0.0457* 0.01667

Population B 0.0528* 0.01667 0.0000 - 0.0718* 0.01667Population C 0.0457* 0.01667 0.0718* 0.01667 0.0000 -



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Genetic Characterization at Mediterranean and Atlantic Populations of Spartus aurata