Genetic diversity of Prochilodus lineatus stocks using in the stocking program of Tietê River , Brazil Diversidad genética de lotes de Prochilodus lineatus utilizados en el programa de repoblamiento del río Tietê , Brasil

Objective. Assess the genetic diversity in four brood stocks and one juvenile stock of curimba Prochilodus lineatus in a Hydropower plant in São Paulo Brazil, using the Tietê River stocking program. Materials and methods. Five RAPD primers were used to amplify the extracted DNA from 150 fin-clip samples. Results. Fifty-nine fragments were polymorphic, 52 had frequencies with significant differences (p<0.05), 45 had low frequencies, 54 were excluded, and two were fixed fragments. High values for polymorphic fragments (71.19% to 91.53%) and Shannon index (0.327 to 0.428) were observed. The genetic divergence values within each stock were greater than 50%. Most of the genetic variation was found within the groups through the AMOVA analysis, which was confirmed by the results of the identity and genetic distance. High ancestry levels (FST) among the groups value indicated high and moderate genetic differentiation. The estimates of number of migrants by generation (Nm) indicated low levels of gene flow. High and moderate genetic divergence between groups (0.58 to 0.83) was observed. Conclusions. The results indicate high variability within the stocks, and genetic differentiation among them. The fish stocks analyzed represent a large genetic base that will allow the fish technicians to release juveniles without genetic risks to wild populations present in the river. These genetic procedures may be used as models for other migratory species, including those threatened by extinction.


INTRODUCTION
The Tietê is an important river in Brazil, located in the State of São Paulo.This River runs through the metropolitan area of São Paulo City and continues through 1.136 km up to the Paraná River, on the border with Mato Grosso do Sul.Along the way, several dams have been installed.These dams favor local and regional economic development, but they also bring serious and irreversible alterations of the natural hydrologic regime of rivers; affecting habitat quality, the dynamics of the biota and aquatic fauna, including fish (1).Recently, there has been a decline in the population of various wild fish like the Prochilodus lineatus (curimba) in this River.P. lineatus (Prochilodontidae, Characiformes) is a migratory species of great economic importance for both fisheries and aquaculture, found throughout the Jacuí, Paraíba do Sul, Paraná, Paraguay and Uruguay River basins in South America (2).According to the Brazilian Ministry of Fisheries and Aquaculture (MPA), in 2009, 4.469 ton of Prochilodus were produced (3).The main factors responsible for the production decline in the Tietê River, have been the intense deforestation of the river banks, excessive fishing of juveniles, water pollution, introduction of exotic fish and hydropower dams (4)(5)(6).
The Tietê River has more than 10 hydropower plants.These dams have broken migratory paths, interfered with the life cycle of numerous aquatic organisms, and produced major changes in ecosystems (7).
In dams, the passage ladders have been built to allow an alternative route of the natural flow of the migratory species.However, these ladders have been criticized for their unidirectional behavior (8), for their selectivity of the fish and their inefficiency in allowing downstream return of adults, eggs, and larvae (9).Another decision that has promoted the preservation of fish fauna is the regional development of stocking programs (10).
Although stocking programs have been used as a conservation method, risks associated with reducing the population size, increasing ecological competition between wild and captivebred individuals, impact the ecosystem and reduce the genetic diversity of wild stocks; which are questionable (11,12).
In this context, the genetic variation of broodstocks used in these programs must be monitored to increase the preservation of wild fishing resources in stocking programs.With this purpose, the RAPD molecular marker (Random Amplified Polymorphic DNA) has been applied to estimate genetic diversity in broodstocks (13)(14)(15).
The objective of the present study was to evaluate the genetic diversity of Prochilodus lineatus stocks used in the stocking program of Tietê River, Brazil, by using RAPD markers.Molecular methods.The DNA was isolated from the 0.5 cm 2 fin-clips, and extraction was based on the previously described methods (16).The samples were treated with 550 ml lise buffer (50 mM Tris-HCl, 50 mM EDTA, 100 mM NaCl, and 1%(w/v) SDS), 7 ml proteinase K (200 μg/ ml) per sample, and incubated overnight at 50°C.Then, 600 μl 5 M NaCl was added to each sample before being centrifuged for 10 min at 12000 rpm.The aqueous layer was removed carefully into new micro tubes where the DNA was precipitated with 700 μl of freezing ethanol and incubated later at -20°C for 2 h.The DNA samples were centrifuged again, washed with 700 μl 70% (v/v) ethanol, re-suspended in TE buffer (10 mM Tris and 1 mM EDTA), and treated with 6 ml RNAse (30 μg/ml) at 38°C for 40 min.The DNA was quantified in the Shimadzu spectrophotometer (UV-1601, USA) with absorbance at 260 nm.The samples were diluted to the concentration of 10 ng/μl.The DNA quality was checked using agarose gel electrophoresis (1% w/v) buffered with TBE 1X (500 mM Tris-HC1, 60 mM boric acid and 83 mM EDTA) at 70 volts for 1 h.Thereafter, the gels were photographed using the L-PIX Image Transiluminator software 1.3 -Loccus Biotechnology -Molecular Image, USA (Figure 1).

Collection
The genomic DNA was amplified in the reaction volume of 15 ml using buffer Tris-KCl 1X (Tris-HCl 20 mM pH 8.4 and KCl 50 mM), 2.5 mM MgCl 2 , 0.46 mM primer (oligonucleotides), 0.2 mM from every dNTPs, one unit of Platinum Taq DNA Polymerase and 10 ng of DNA.The RAPD reactions were amplified in the "Eppendorf Mastercycler ® Gradient" thermocycler, programmed to 40 cycles, an initial step of denaturation at 94°C for 4 min, and a final step of extension at 72°C for 5 min.Every cycle consisted of 1 min at 94 o C, 90 s at 40°C and 2 min at 72°C.
We evaluated 60 primers from the Operon Kit (Operon Technologies Inc. in Alameda, California, USA).Different stocks were evaluated for five selected primers.The product from the amplification was separated in agarose gel at 1.4% (w/v).We used 15 ml of the amplified product and 2 ml of the sampling buffer (40% (w/v) sucrose and 0.25% (w/v) bromophenol blue) in horizontal electrophoresis.The electrophoresis was carried out at 70 V for 4 h (3 V/cm) using the TBE 1X buffer.Every reaction had a negative control (N) where all the previous components but DNA were added to the solution.A bath with ethidium bromide at 0.5 mg/ml for 30 min was used to reveal the gel.Thereafter, the gels were photographed using the L-PIX Image Transiluminator software 1.3.

RAPD statistical analysis.
The size of the fragments was determined by comparison with a 100 pb DNA Ladder (Invitrogen ® , USA).The presence or absence of identical molecular-sized fragments was used to construct a similarity matrix based on the calculation of the Jaccard similarity coefficient, codifying 1 if a fragment was present and 0 if absent.
Based on this matrix, the genetic divergence was estimated by the Mantel test, using the Monte Carlo method, by Mantel-Struct program (17).The percentage of polymorphic fragments and the Shannon index were obtained from the POPGENE software version 1.31 (18).The software TFPGA 1.3 (19) was used to estimate the identity and genetic distance among the groups.The frequency of fragments was estimated by the exact test.The ARLEQUIN 3.0 software (20) was used to determine the genetic differentiation using the estimates of the ancestry coefficient (F ST ), the number of migrants per generation (Nm) and the molecular analysis of variance -AMOVA.The molecular variance between the groups was evaluated after combining all of them in the following groups: BAxBB, BAxBC, BAxBD, BAxJS, BBxBC, BBxBD, BBxJS, BCxBD, BCxJS and BDxJS.The significance of these tests was determined by the random permutation method using from 1.000 to 10.000 permutations.The significance of the F ST was tested by the X 2 [c²=2n F st (k-1); GL = (k-1) (s-1)] after (21) in which n is the number of individuals in the two groups, k is the number of alleles and s is the number of groups.The magnitude of the genetic differentiation between these groups was based on the Wright definition ( 22) as having little differentiation when F ST is ranging between 0 and 0.05, moderate when F ST is ranging between 0.051 and 0.15, high when the F ST is ranging between 0.151 and 0.25 and very high when the F ST is above 0.25.
The F ST showed high ancestry between groups.These results suggest a high and moderate genetic differentiation.Corroborating this hypothesis, the Nm was low in all the groups suggesting a low gene flow.High and moderate genetic divergence between groups was also observed (Table 4).

DISCUSSION
The RAPD analysis was appropriate to evaluate the genetic diversity within and between P. lineatus stocks.
The main problems towards the dominant performance of this technique (23) were overcome through the amplification tests and standardization of samples carried out in conjunction with the negative controls in the different amplifications.
Forty-five fragments with low frequency and fifty-five excluded fragments were found in the five stocks (Table 2).According to Jacometo et al (24), the presence of low frequencies and absence of these fragments within each stock may be explained by the bottleneck effect or founder effect.The bottleneck effect is the reduction of an existing population to a small number of individuals.These decrease causes the loss of genetic and allele variability (25).The founder effect is the formation of stocks with few individuals that normally do not represent the genetics of natural populations.This effect may result from the presence of low frequency or absent alleles (24).Two fragments in BA and BD were fixed.This result showed that, despite the presence of fragments with low frequency or absent, there is was a high genetic variability within both stocks.
The results for genetic variability and divergence, calculated by FP, SI and GDIV exhibited high genetic variability in four broodstocks.These results showed that broodstocks were formed from a large number of individuals with high genetic variation, showing also a high variability.
According to the analysis of molecular variance (AMOVA), most of the genetic variations occurs within every group of P. lineatus and not between them.These results were confirmed by the estimates of identity and genetic distance (Table 3).The F ST showed high ancestry between groups.These results suggest a high and moderate genetic differentiation.Corroborating this hypothesis, the Nm was low in all the groups suggesting a low gene flow.High and moderate genetic divergence between groups was also observed (Table 4).Lopera-Barrero -Genetic diversity of Prochilodus lineatus using restocking program which allowed the maintenance of variability.The gene pool of fish broodstocks in aquaculture is sometimes characterized by reduced diversity (26).inadequate reproductive management as well as a small number of effective contributing parents (N e ) (27), inappropriate selection of parents, founder effect, misuse of the reproduction period ( 15) and the breeding system used in mating (28) are also some constraints capable of inducing significant loss in the genetic variability.
Juvenile stock also exhibited high genetic variability.
Reproductive management is highly important to preserve the genetic diversity of juveniles, which will participate in the stocking programs.An inadequate reproductive management may cause less survival of juveniles and consequently, the wild populations will be permanently affected.Based on these results, it is suggested that the reproductive and genetic management of four P. lineatus broodstocks analyzed in this study, should be considered as genetically different stocks.This suggestion should be applied especially when stocks are used in repopulating or breeding programs.This way, the first step in the establishing hatcheries and stocking programs, is to investigate the genetic variability of broodstocks (30).The correct selection of the individuals to be used in the formation of broodstock is a crucial step (27), and their genetic evaluation can offer important bases for formulating reproductive management strategies.These strategies will allow safe exchange of broodstocks among fish farms to break cycles of endogamy which are common in controlled environments (25).
Conversely, the decline in the genetic variability may induce adaptability and survivorship problems for these offspring, and negatively influences on the ecosystem (28).In a worst-case scenario, species can go extinct (4).Thus, all the fish released during stocking programs should represent wild populations (5), and both must undergo genetic analysis.
Genetic monitoring using molecular markers such as the RAPD on the wild populations, broodstocks and progenies that have participation in stocking programs is fundamental to avoid declines in the genetic variability, avoiding effects on wild populations, and allowing for insights into preservation, management and reproduction of the P. lineatus.
In summary, the current results indicated high variability within the stocks and genetic differentiation among them.The fish stocks analyzed represent a large genetic base that will allow fish technicians to release juveniles without genetic risks to wild populations.Few studies have been conducted in this species, genetically evaluating broodstocks and offspring (29,30).Therefore, the results are important, especially when are used in stocking, breeding or conservation programs.
of biological samples.The experiment took place between May 2010 and February 2011.We collected 120 fin-clip samples from four captive stocks of Prochilodus lineatus reared at the AES Hydropower plant (21°29'98''N and 49°78'40"W), São Paulo, Brazil.These stocks were kept in captivity for six years, and are important because they have been enhancing the fish stocking program in the Rivers of São Paulo.Were also collected 30 samples of juveniles (90 days old) to assess the genetic variability of individuals being released in the stocking process.The origin of stocks: Broodstock A (BA): second generation (F1) bred from wild populations collected from the Tietê River, São Paulo.Broodstock B (BB): first generation (F0) from wild populations collected in the Pardo River, São Paulo.Broodstock C (BC) and D (BD): are of unknown origin.However, it is known that the BC has the first generation of individuals in captivity (F0), and BD has second-generation (F1) individuals in captivity.Juvenile stock (JS): unknown origin.

Table 1 .
Nucleotide sequence in the primers, percentage of G+C bases, number of fragments (NF), number of polymorphic fragments (NPF), and size of the amplified fragments from the P. lineatus stocks.

Table 2 .
Characterization of size and fragments frequency with significant values, using the exact test (p<0.05) in P. lineatus stocks.

Table 3 .
Molecular analyses of variance (AMOVA), genetic distance (D) and genetic identity (I) for the different groups of P. lineatus.

Table 4 .
(22) , X 2 test for the F ST , genetic differentiation according to Wright(22), number of migrants (Nm) and genetic divergence in the different groups of P. lineatus.
Due to the absence of management information, it is not possible to state that they have a common origin or individual exchange between the broodstocks.Moreover, BC showed larger genetic variability than BA and BB (%FP: 91.53, IS: 0.428).