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Development of a microsatellite multiplex genotyping tool for the fish Gilthead seabream (Sparus aurata): Applicability in population genetics and pedigree analysis
Aquaculture Research
Abstract
In spite of the numerous studies performed in the aquacultured fish gilthead seabream, the overall structure of wild and cultivated stocks is rather confusing. In this study, we developed a 10 microsatellite genotyping tool SaGT(6+4), conformed by two polymerase chain reaction-multiplex reactions (SaGT6 and SaGT4), which can be simultaneously combined in automatic sequencers. The utility of this tool was proven through the following applications: (i) characterization and differentiation of wild and cultivated populations; (ii) pedigree reconstruction and estimation of the effective size in a cultivated stock; (iii) ability for pedigree reconstruction under different simulated situations; and (iv) determination of genetic relationships in the absence of data from parents. Based on our results, some recommendations have been provided on the management of the screened stocks. Our results also support the use of this tool in a standardized way, to understand the actual status of gilthead seabream from both wild and cultivated populations.
... Broodstock held in captivity under natural conditions typically start vitellogenesis in September-November, spawning begins during December-January and lasts for 3-5 months with daily spawning, leading to an annual fecundity of 2,000,000 eggs/kg (diameter<1 mm) with a fertilization rate of 80-85% (Barbaro et al., 1997;Arabaci et al., 2010;Mylonas et al., 2011). However, the reproductive behaviour of gilthead seabream has not been reported, despite an increasing need to understand the factors that influence a breeders participation in spawning in order to control the families produced from a broodstock (Gorshkov et al., 1997;Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012). Reproductive behaviour has been described in some species of the family Sparidae, kept in captivity, including silver seabream (Chrysophrys auratus) (Smith, 1986;Mylonas et al., 2011), santer seabream (Cheime-2 to investigate and describe the particularities of reproductive behaviour of the gilthead seabream in rearing conditions. ...
... A number of studies have examined gilthead seabream parental contribution to spawning events as there is a need to genetically improve cultured gilthead seabream to obtain desirable traits such as faster growth that will reduce production costs (Gorshkov et al., 1997;Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012;Duncan et al., 2013). Gilthead seabream spawning success was low when held in pairs (22% success) or groups of 15 females with a single male (44% success) and gilthead seabream were difficult to strip spawn for artificial fertilisation (Gorshkov et al., 1997). ...
... Different authors have concluded that large groups of breeders are required for successful spawning of gilthead seabream (Gorshkov et al., 1997;Duncan et al., 2013) and Sparidae in general (Pankhurst, 1998;Mylonas et al., 2011). Parental assignment of progeny using microsatellites identified that although large broodstocks produce large volume spawns to which many breeders contributed the participation of breeders was variable and a proportion of breeders did not participate in spawning (Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012). Consequently, the effective spawning population size was reduced compared to the actual number of breeders in the broodstock, inbreeding was higher than expected and the families obtained were not predictable. ...
The present study aimed to describe the reproductive behaviour of gilthead seabream (Sparus aurata) in captivity. Twenty-four mature gilthead seabream, divided in two tanks, were utilized for the present study. Reproductive behaviour was recorded using submersibles cameras. A total of 67 spawning events were analysed. The mean duration time that gilthead seabream spent spawning was 54 ± 4 min/day, during which mean number of individual spawning events was 5.6 ± 0.2. The mean volume of eggs produced by both broodstocks was 405 ± 13.4 mL with a fertilization rate of 91.6 ± 0.4%. The reproductive behaviour began with a schooling behaviour and then forming light aggregations. From an aggregation or an encounter while swimming freely a female initiated a spawning rush followed by one or more males to gametes liberation. The spawning rush was brief, 1.6 ± 0.5 sec, over an approximately 1.7 ± 0.2 m distance from the tank bottom to the water surface. Pair spawning, between a single female and male, was the most common (71.6%). Group spawning was less common and involved a single female spawning with two males (22.5%) or three males (4.9%). Spawning rushes involving more than one female were not observed. Gilthead seabream in the present study presented a tendency to pair spawn and eggs collected as a " spawn " were actually the sum of many separate spawning events over a short time period. This is the first description of gilthead seabream spawning and the findings help to understand microsatellite based observations of spawning kinetics.
... Broodstock held in captivity under natural conditions typically start vitellogenesis in September-November, spawning begins during December-January and lasts for 3-5 months with daily spawning, leading to an annual fecundity of 2,000,000 eggs/kg (diameter<1 mm) with a fertilization rate of 80-85% (Barbaro et al., 1997;Arabaci et al., 2010;Mylonas et al., 2011). However, the reproductive behaviour of gilthead seabream has not been reported, despite an increasing need to understand the factors that influence a breeders participation in spawning in order to control the families produced from a broodstock (Gorshkov et al., 1997;Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012). Reproductive behaviour has been described in some species of the family Sparidae, kept in captivity, including silver seabream (Chrysophrys auratus) (Smith, 1986;Mylonas et al., 2011), santer seabream (Cheime-2 to investigate and describe the particularities of reproductive behaviour of the gilthead seabream in rearing conditions. ...
... A number of studies have examined gilthead seabream parental contribution to spawning events as there is a need to genetically improve cultured gilthead seabream to obtain desirable traits such as faster growth that will reduce production costs (Gorshkov et al., 1997;Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012;Duncan et al., 2013). Gilthead seabream spawning success was low when held in pairs (22% success) or groups of 15 females with a single male (44% success) and gilthead seabream were difficult to strip spawn for artificial fertilisation (Gorshkov et al., 1997). ...
... Different authors have concluded that large groups of breeders are required for successful spawning of gilthead seabream (Gorshkov et al., 1997;Duncan et al., 2013) and Sparidae in general (Pankhurst, 1998;Mylonas et al., 2011). Parental assignment of progeny using microsatellites identified that although large broodstocks produce large volume spawns to which many breeders contributed the participation of breeders was variable and a proportion of breeders did not participate in spawning (Brown et al., 2005;Porta et al., 2009;Chavanne et al., 2012). Consequently, the effective spawning population size was reduced compared to the actual number of breeders in the broodstock, inbreeding was higher than expected and the families obtained were not predictable. ...
The present study described the spawning behaviour of gilthead seabream (Sparus aurata), an important quaculture species. Observations were made over a month, of 24 seabream held in 2x18m 3 tanks (7 females and 5 males tank1), utilizing submerged video cameras. The seabream spawned daily usually from 08:0011:00 and eggs were collected from 11:00–12:00. Generally the fish swan in a loose school around the tank.
Spawning started when the fish formed a group and swim in tight circles forming a “ball” that became smaller as the fish swam closer together. Two fish would then exit rapidly from the group, a female followed by a male and during the 12 second spawning rush a cloud of gametes was released approximately 12m from the group. Immediately after gamete release the two fish separated and returned to swim around
the tank. The group of fish left behind by the two spawners gradually broke up as the fish returned to swim around the tank coinciding with the two fish that had spawned. The grouping of fish lasted less than 1 minute. Often various groupings and subsequent pared spawning were observed during the period before egg collection. No evidence was observed of more than two fish spawning together. These observations
indicate the group behaviour was important, but unlike what is often assumed the act of gamete liberation was between an individual female and male.
... ee and five dinucleotide markers) and one (four dinucleotide markers) multiplex reactions respec- tively. Navarro et al. (2008) developed two robust interspecific multiplex reactions, RimA and RimB, of 10 and seven redesigned microsatellite markers respectively, achieving 100% of success in parental assignments despite the presence of null alleles. Porta et al. (2010) proposed two specific multiplex reactions of four (SaGT4) and six (SaGT6) dinucleotide microsatellite markers to successfully assign paternity to 95% of the studied offspring. Borrell et al. (2011) developed a multiplex PCR (OVIDORPLEX) of nine microsatellite markers (four of which were interspecific) that successfully assigned 99.2%. V ...
... Microsatellites also report information about the genetic status of the population. In populations of cultured gilthead seabream, the mean heterozygosity is high (Castro et al. 2007; Navarro et al. 2008; Porta et al. 2010; Borrell et al. 2011), such as in this study, and similar to wild populations ( Segvi c-Bubi c et al. 2011) due to the high rate of gene flow and scarce intensity of selection. The PIC value is also a measure of informativeness (Botstein et al. 1980; Hearne et al. 1992), is commonly used in linkage mapping and is really useful in conducting parentage analyses (Pi~ nera et al. 2006). ...
... loci with >25 alleles or 85% heterozygosity) may present complications such as an increase in the frequency of genotyping errors (dropout and stutter) and high mutation rates. The mean number of alleles and heterozygosity values from the multiplex PCRs in this study were high and similar to those reported by other gilthead seabream multiplex PCRs (Launey et al. 2003; Navarro et al. 2008; Porta et al. 2010; Vogiatzi et al. 2011), although lower than those described by Borrell et al. (2011). In addition, the level of polymorphism of the microsatellite markers also depends on the characteristics of the population (Vandeputte et al. 2011). ...
The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.
... Different results were obtained from two surveys in Greece, which showed a significant reduction of the number of alleles, gene diversity (HE) and allelic richness (Rs) in farmed stocks 43,47,54 . A similar loss of alleles was observed in Spain in farmed F1 progeny based on 10 microsatellite loci 53 . Genetic analysis revealed the origin of broodstock from at least two different populations in Israel 55 and a mixed and highly heterogeneous origin of Italian broodstocks and a massive presence of Atlantic individuals among the breeders 56 . ...
... Offspring analysis from several mass-spawning events demonstrated the consistently low effective population size, a differential average contribution by male and female parents and a high variance in family size 53,59 . To maximize the possible genetic variance within each group and minimize inbreeding and genetic drift, investigations on the selection of individuals to establish broodstocks were performed 59 . ...
The gilthead sea bream, Sparus aurata, is an important demersal commercial species, highly appreciated as food fish for its flesh. It prefers warm coastal euryhaline waters and its life-cycle is determined by protandrous hermaphroditism. It has been traditionally cultured in Mediterranean coastal lagoons for centuries, and it is now reared intensively both in sea cages and in land based farms. Global production has reached 167,827 tonnes I 2012, primarily from aquaculture. It is the main premium marine aquaculture species in the Mediterranean region, together with European sea bass, Dicentrarchus labrax. The population genetic characteristics throughout the whole range have not yet been clearly established. There is a lack of information in some geographic areas and inconsistencies in the degree of the structuring pattern. Some surveys report the absence of or subtle genetic structure, whereas others suggest evident population subdivision, within and among different geographic areas. However no evidence of simple isolation by distance was found, though some effects of oceanographic processes on population connectivity were revealed in some populations. This unclear picture requires further studies to determine the genetic and phenotypic structure of the gilthead sea bream over its whole geographical range in order to develop strategies for the conservation of wild populationsand for the genetic-based management of farmed stocks. As far as aquaculture practices are concerned, there is little information on the origin of broodstock and cultured stocks, on the exchange of breeders, eggs and juveniles, and on the nature and extent of their genetic differentiation. This can be attributed to the lack of commercial traceability tools. Escapees from farms, gametes released by cultured fish at sea and hatchery based restocking in coastal lagoons all contribute to the presence of farmed samples in the natural environment. The risk of genetic impact of the accidental or intentional release of individuals from aquaculture facilities (culture cages, ponds and hatcheries) on wild local populations is still difficult to determine, both for the lack of information on the number of escapees and because the fitness in the wild of escapees and of introgressed offspring is largely unknown. The development of "wild vs. farmed" reliable genetic markers would introduce new tools and indicators useful for environmental monitoring and enforcement. Breeding programmes are in progress to select for growth, morphological traits, product quality and disease resistance. The development of highly informative genetic markers and of genome-wide association studies in gilthead sea bream has paved the way for marker assisted selection.
... In fact, Navarro, Badilla, Zamorano, Pasamontes, Hildebrandt, S anchez and Afonso (2008) reported that the cost of genotyping by a multiplex PCR based on 10 microsatellite markers is no more than one-sixth, which it performed by single reactions loaded in a just one run in the automatic sequencer. Many multiplex PCRs have been developed in gilthead seabream to successfully determine family relationships (Launey, Krieg, Haffray, Bruant, Vanniers & Guyomard 2003; Brown, Tsalavouta, Terzoglou, Magoulas & Mcandrew 2005; Navarro et al. 2008; Porta, Porta, B ajar & Alvarez 2010; Borrell, go, Garc ıa-Fern andez, Mazzeo, P erez, Asturiano, Carleo, V azquez, S anchez & Blanco 2011). Moreover , Vogiatzi, Lagnel, Pakaki, Louro, Canario, Reinhardt, Kotoulas, Magoulas and Tsigenopoulos (2011) proposed five multiplex PCRs with 32 microsatellites found in coding sequences while Lee- Montero, Navarro, Borrell, Garc ıa-Celdran, Mart ın, Negr ın-B aez, S anchez, Armero, Berbel, Zamorano, S anchez, Est evez, Ramis, Manchado and Afonso (2013) developed two multiplex PCR reactions (SMsa1 and SMsa2) and proposed them as an international panel in this species. ...
... For QTL searching, these authors used additional markers by single PCR reactions. Other multiplex PCRs have been developed in gilthead seabream for parentage assignment and/ or population genetic studies (Launey et al. 2003; Brown et al. 2005; Navarro et al. 2008; Porta et al. 2010; Borrell et al. 2011; Vogiatzi et al. 2011; Lee-Montero et al. 2013), but none have included as many microsatellites as this study. Actually, a genetic linkage map with a high level of saturation of molecular markers is needed for an effective QTL detection (Lynch & Walsh 1998). ...
The growth and consolidation of the gilthead seabream (Sparus aurata) industry require improvement based on permanent and cumulative aspects, such as those derived from genetic breeding programmes. Marker Assisted Selection (MAS) by Quantitative Trait Loci (QTL) can be usefully implemented with the appropriate tools. In this study, 138 microsatellite markers from the genetic map of gilthead seabream were redesigned to be amplified under the same PCR conditions. A final set of 13 multiplex PCRs (named ReMsa) with 106 of these markers was developed to cover 100% of the linkage groups. These effective multiplex PCRs enable to optimize QTL searching with a critical reduction in costs and errors. Results showed that the mean value of the number of alleles for 106 markers was 6.9. The mean observed heterozygosity ranged from 0.53 to 0.86, and 74.5% of markers were highly informative according to their polymorphic information content. The correct inheritance and segregation of alleles of each locus was confirmed after genotyping 62 individuals of a full-sib family by these multiplex PCRs. In addition, genetic features of the 20 microsatellite markers that worked correctly but were not included in any multiplex PCR are also reported to provide geneticists with the possibility of including them in more comprehensive screening studies.
... lt of the mixing of distinct populations / broodstocks / generations ( Hartl and Clark , 1997 ) . Indeed , the Bisceglie coast is characterized by a massive presence of floating cages for aquaculture , and frequent escapes have been documented ( CoProMar , pers . comm . ) . As cultured seabream show reduced variability ( Karaiskou et al . , 2009 ; Porta et al . , 2010 ; Loukovitis et al . , 2011 ) that could also have been obtained by allochthonous breeders ( Porta et al . , 2010 ) , their accidental release may have altered allelic frequencies of the local wild genetic assemblage . However , the homogeneity detected among the three temporal replicates from Lesina supports previous evidence , based o ...
... ceglie coast is characterized by a massive presence of floating cages for aquaculture , and frequent escapes have been documented ( CoProMar , pers . comm . ) . As cultured seabream show reduced variability ( Karaiskou et al . , 2009 ; Porta et al . , 2010 ; Loukovitis et al . , 2011 ) that could also have been obtained by allochthonous breeders ( Porta et al . , 2010 ) , their accidental release may have altered allelic frequencies of the local wild genetic assemblage . However , the homogeneity detected among the three temporal replicates from Lesina supports previous evidence , based on a lower number of microsatellite loci ( Rossi et al . , 2009 ) , excluding chaotic ...
Franchini, P., Sola, L., Crosetti, D., Milana, V., and Rossi, A. R. 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.
The gilthead sea bream, Sparus aurata, is a coastal, commercially important fish. Contrasting results concerning the genetic structure of the species at different geographic scales have been reported. Here, an investigation is made into the population genetic structure of S. aurata along the coast of Italy, using samples analysed previously and material from new sampling sites (12) and using different microsatellite loci (10). One sample from the eastern Atlantic and three temporal replicates from one site were also included. The presence of a weak (overall FST = 0.0072), but significant, genetic population subdivision was detected by F-statistics. Temporal replicates indicate genetic data consistency over time. Isolation by distance between the Atlantic and the coast of Italy is suggested by a Mantel test. The distributional pattern of genetic variance obtained by analysis of molecular variation reflects the geographic sampling areas, but is only partially congruent with the results obtained with fewer sites and loci. The dispersal of passive eggs/larvae by the main currents appears to contribute to shaping the gene flow. Given the intensity of sea bream aquaculture activities in Italy, the possibility that aquaculture may have partially contributed to the population genetic pattern detected cannot be excluded.
... For this reason, the implementation of breeding programs in this kind of industrial production systems needs a combination of both physical tagging and genetic identification of the cultured animal (Navarro et al., 2008) to facilitate the manipulation of the broodstock families. In abalone species, it is possible to assign parentage and to study parent-offspring relationships by analyzing enough microsatellite markers Van den Bergb and wilding, 2010;Lafarga de la Cruz et al., 2015) that can be combined in multiplex PCRs to reduce costs, time and handling errors (Neff et al., 2000;Ezaz et al., 2004;Porta et al., 2006Porta et al., , 2009Navarro et al., 2008;Lee-Montero et al., 2013). ...
Haliotis tuberculata coccinea is a promising candidate species for sustainable and integrated aquaculture development of the Canary Islands, a lot of studies focused on the technical improvement of culture systems of this species have been conducted in the PCTM of ULPGC. However, genetic studies investigating the genetic resources of its broodstock population to allow improved manipulation of the seed production procedures, to keep an acceptable genetic variability level and to prevent a possible genetic drift for such a broadcast spawning species, were still to be developed. Therefore, the present study was designed to have insights into the genetic structure of the existing broodstock. A group of microsatellite markers, belonging to other abalone species, were selected and grouped in to two multiplex PCRs which were used to develop one single multiplex PCR (MULTHAB) optimized for amplification of only 8 microsatellite primers after the exclusion of monomorphic and non-amplified primer pairs. The aim of this study is to assess the usefulness of using this multiplex to study the genetic diversity of H. tuberculata coccinea broodstock. 316 DNA samples were used and included: 16 samples from F1 animals that are part of the breeder group composed of different hatchery generations and 300 DNA samples that belonged to the next F2 generation obtained from the F1 breeder group. A total number of 80 alleles were detected for the 8 selected microsatellite markers in MULTHAB multiplex in all analyzed samples. They showed good allelic polymorphism with moderate allelic number per locus (NA) and expected heterozygosity(He) values, (mean= 10 and 0.6299, respectively). High PIC number values (mean =0.5753) for all used loci in this study were observed, suggesting that the selected markers showed a high capability to detect polymorphisms in a given population of H. tuberculata coccinea. The 8 microsatellite markers were successfully segregated in two identified families produced within this study. The optimized MULTHAB succeed to assign 32% of the F2 analyzed individuals to their possible parent pair or single parent. The mean FST value (0.0103) obtained in this study shows a little genetic differentiation between the parent group (F1) and the offspring group (F2). The high unbalanced samples size of F1 population (16 individual) and F2 population (300 individuals) resulted in unexpected observation for the allelic number per locus and the expected heterozygosity which were higher in F2 animals than those of F1 animals.There was high deviation from the linkage equilibrium and the H-W equilibrium for the studied microsatellite loci in all the broodstock population. Additionally, the mean FIS was positive (0.0899), this results indicates that the studied population was affected by a somehow loss of genetic diversity due to heterozygosity deficiency, inbreeding and founder problems, which was expected due to the crossing between parents and offspring of different generations for seed production. This study concludes the successful applicability of the developed MULTHAB multiplex for genetic studies for H. tuberculata coccinea brood stock, there is little genetic differences between the parent animals (F1) and their offspring (F2) with marks of heterozygote deficiency probably due to inbreeding and small number of founder animals, leading to reduction of genetic variability on the long run. This results indicate that adequate genetic maintenance should be implemented for the H. tuberculata coccinea broodstock population.
... In conclusion, data concerning interbreeding between wild and cultured fish in the Mediterranean, are variable and sometimes contradictory and this fact has been recently stated (Porta et al., 2010). This is mainly due to the different methodological approaches in the various studies, but also maybe because of the delayed beginning of the monitoring when compared to the long existence of interbreeding and the pre-existed genetic changes in wild populations. ...
... Concurrently, the development of multiplex PCRs allows geneticists to reduce the economic cost per reaction. Several multiplex PCRs have been described for gilthead sea bream; Batargias et al. (1999), Launey et al. (2003), Brown et al. (2005); and more recently Navarro et al. (2008), Porta et al. (2010) and Borrell et al. (2011). In our study, we used the first reproducible, uniform and standardized gilthead sea bream panel (SMsa1; Super Multiplex Sparus aurata) consisting of eleven highly polymorphic and specific markers (Lee-Montero et al. 2013). ...
The gilthead sea bream (Sparus aurata L.) is one of the most important Sparid farmed in Europe, especially in the Mediterranean area. However, efficient breeding programs for this species are scarce and very little, and it is known concerning their population structure. The present study was mainly designed to genetically characterize, by microsatellite markers, three gilthead sea bream populations sampled along the Spanish coast (Cantabrian Sea, the Atlantic Ocean and Mediterranean Sea) and their progeny with the aim of studying its genetic variability and its genetic structure. Moreover, we evaluated different strategies of broodstocks management (breeders’ number, origin and sex combination) on parental contributions and on effective breeding numbers. That number of breeders in the stock was of great importance to the maximization of contribution since the larger broodstock resulted in larger proportion of parents contributing. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire was translated into reduced effective breeding numbers and consequently into expected increased inbreeding rates. Our results highlight the high genetic variability of the studied sea bream populations, as well as the existence of three genetically differentiated populations along the Spanish coast. These findings should be relevant for the establishment of successful breeding programs in aquaculture of the gilthead sea bream.
... Структурно мікросателіти можуть являти собою як однорідні, так і різнотипні послідовності. Також відомий варіант, коли мікросателіти містять вставні послідовності (так звані спейсери) між типовими повторами [22,23]. ...
Purpose. The application of molecular genetic markers has been widely used in modern experimental fish-farming in recent years. This methodology is currently presented by a differentiated approach with individual mechanisms and clearly defined possibilities. Numerous publications in the scientific literature that are dedicated to molecular genetic markers for the most part offer purely practical data. Thus, the synthesis and analysis of existing information on the general principles of action and the limits of the main methods of using molecular genetic markers is an actual problem. In particular, such a description will make it possible to plan more effectively the experiment and to obtain the desired results with high reliability.
Findings. The main types of variable parts of DNA that can be used as molecular genetic markers in determining the level of stock hybridization, conducting genetic inventory of population and solving other problems in modern fish-farming are described in this paper. Also, the article provides an overview of principal modern methods that can be used to identify molecular genetic markers.
Originality. This work is a generalization of modern ideas about the mechanisms of experiments with molecular genetic markers in fish-farming. Information is provided in the form of consistent presentation of the principles and purpose of each method, as well as significant advances during their practical application.
Practical value. The proposed review of classic and modern literature data on molecular genetic markers can be used for planning, modernization and correction of research activity in modern fish-farming.
... In all cases, the DNA was kept at 4 @BULLET C until utilisation. All 376 offspring and 131 breeders were genetically characterised using microsatellite markers (multiplex) and familial assignments, determined according to Porta et al. [22]. ...
Growth is a priority trait from the point of view of genetic improvement. Molecular markers linked to quantitative trait loci (QTL) have been regarded as useful for marker-assisted selection in complex traits as growth. Polymorphisms have been studied in five candidate genes influencing growth in gilthead seabream (Sparus aurata): the growth hormone (GH), insulin-like growth factor-1 (IGF-1), myostatin (MSTN-1), prolactin (PRL), and somatolactin (SL) genes. Specimens evaluated were from a commercial broodstock comprising 131 breeders (from which 36 males and 44 females contributed to the progeny). In all samples eleven gene fragments, covering more than 13,000 bp, generated by PCR-RFLP, were analyzed; tests were made for significant associations between these markers and growth traits. ANOVA results showed a significant association between MSTN-1 gene polymorphism and growth traits. Pairwise tests revealed several RFLPs in the MSTN-1 gene with significant heterogeneity of genotypes among size groups. PRL and MSTN-1 genes presented linkage disequilibrium. The MSTN-1 gene was mapped in the centromeric region of a medium-size acrocentric chromosome pair.
... In conclusion, data concerning interbreeding between wild and cultured fish in the Mediterranean, are variable and sometimes contradictory and this fact has been recently stated (Porta et al., 2010). This is mainly due to the different methodological approaches in the various studies, but also maybe because of the delayed beginning of the monitoring when compared to the long existence of interbreeding and the pre-existed genetic changes in wild populations. ...
Aquatic farming has been considered, during the last decades, as the fastest growing food production industry powered by governmental and technological impulsion. Compensation for fisheries decline, creation of new jobs and source of financial windfall are the most important benefits. However, similar to most of the human food-production activities, aquaculture raised several issues related to the environmental welfare and consumer safety. An effort to record the aquaculture-environment and -human safety interactions with regard to the Mediterranean mariculture, is attempted herein. We focused on this geographical area due to its individualities in both the hydrological and physicochemical characteristics and the forms of aquaculture activities. The cage farming of euryhaline marine fish species and more recently of bluefin tuna and mollusk farming are the dominating aquaculture activities. The impacts of these activities to the environment, through wastes offloads, introduction of alien species, genetic interactions, disease transfer, release of chemicals, use of wild recourses, alterations of coastal habitats and disturbance of wildlife, are analytically considered. Also the consumer safety issues related to the farming are assessed, including generation of antibiotic-resistant microorganisms, contaminants transferred to humans though food chain and other hazards from consumption of aquacultured items. Within these, the major literature findings are critically examined and suggestions for scientific areas that need further development are made. The major tasks for future aquaculture development in this region are: (i) to ensure sustainability and (ii) to balance the risks to public or environmental health with the substantial economical benefits. In regard with monitoring, tools must be created or adapted to predict the environmental costs and estimate consumer impact. At a canonistic and legal basis, the establishment of appropriate legal guidelines and common policies from all countries involved should be mandatory.
Aquaculture finfish production based on floating cage technology has raised increasing concerns on the genetic integrity of natural populations. Accidental mass escapes can induce the loss of genetic diversity in wild populations by increasing genetic drift and inbreeding. Farm escapees likely represent an important issue in the gilthead sea bream, which accounted for 76.4% of total escapes recorded in Europe during a three‐year survey. Here, we investigated patterns of genetic variation in farmed and wild populations of gilthead sea bream from the Western Mediterranean, a region that faced a long‐history of gilthead sea bream farming. We focused our attention on the role that genetic drift may play in shaping these patterns. Results based on microsatellite markers matched those observed in previous studies. Farmed populations showed lower levels of genetic diversity than wild populations and were genetically divergent from their wild counterparts. Overall, farmed populations showed the smallest population effective size and increased levels of relatedness compared to wild populations. The small broodstock size coupled with breeding practices that may favor the variance in individual reproductive success likely boosted genetic drift. This factor appeared to be a major driver of the genetic patterns observed in the gilthead sea bream populations analysed in the present study. These results further stress the importance of recommendations aimed at maintaining broodstock sizes as large as possible and equal sex‐ratios among breeders, as well as avoiding unequal contributions among parents.
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The implementation of any sustainable breeding program requires a maximization of effective size (Ne) to maintain inbreeding rate at minimum levels. However, the control of Ne remains a major challenge for those species whose reproduction in captivity is based on a spawning strategy where there is no control over the established matings. The present study aims to assess the spawning dynamic of gilthead sea bream (Sparus aurata) at different dates from two mini-broodstocks (4 ♀ and 4 ♂). Here we report the use of a simple protocol based on the genotyping of fertilized eggs from a reduced set of microsatellite loci which provides a quick evaluation of daily spawning in terms of family structuring (number of active breeders and number and family size). According to our results, the daily spawning dynamics is characterized by a fluctuation in the number of active breeders, reaching maximum values (until 100%) during peak production phase. However, the proportion of possible mating (families) in a single daily spawning never exceeded more than 81 %. Unbalanced parental contributions were highly frequent, with extreme cases involving the assignment of 50 - 90% of the offspring to a single family. Therefore, the use of mini-broodstocks and the grouping of spawning harvested at different dates, are proposed as strategies to minimize the inbreeding risk by providing a more optimal pattern of family structuring (increase in the number of families obtained and a more balanced parental contributions) that entails a more conservative Ne/N ratio.
The gilthead sea bream (Sparus aurata L.) is one of the most important marine fish species produced in southern European countries. Over the last two decades, microsatellites have become a powerful tool for DNA studies of fish populations, making it possible to establish existing genealogical relationships between individuals and to perform reliable estimates of genetic parameters. In the present study, a total of 7959 fish (494 breeders and 7465 fry) were genotyped using two rounds of multiplex reactions with four microsatellite markers each. Offspring were assigned based on the Mendelian exclusion principles, using FAP program. Genealogical information was successfully obtained for more than 83 % of the progeny. Heritability estimates were close to 0.40 in all cases and for all considered traits. The lowest value was for the length at first age (350 ± 6 dph) and the highest for the length at the second age (500 ± 7 dph). The heritabilities for the first and the second weight were similar although somewhat higher for the second age, but not significant. The highest genetic correlations were found between weight and length at the same age and the lowest between weight and length at different ages. Based on the results of this study, it seems reasonable to expect that the implementation of a breeding program for improved growth in this population of gilthead sea bream will offer comparable performances to those achieved in other populations and species of great success in aquaculture around the world.
Purpose. The goal of this work was the selection of microsatellite markers to study the genetic structure of populations of silver (Hypophthalmichthys molitrix) and bighead (Hypophthalmichthys nobilis) carps of fish farm JSC "Donrybkombinat" of Donetsk region.
Methodology. Products of amplification were separated in 2 % agarose gel in 1×TBE buffer. Visualization was performed with help of transilluminator in UV-region of the spectrum and photographing of electrophoregrams by digital camera. Processing and analysis of gels were performed using program TotalLab v2.01. The frequency of each amplicon by particular locus was determined as a percentage from the total number of amplicons at this locus. Statistical analysis of results was carried out using programs Excel, «Biosys-1».
Findings. At the results of investigations of populations silver carp and bighead carp were analyzed genotypes of individuals by using three microsatellite DNA loci: MFW 15, MFW 23, MFW 06.
Originality. It has been first shown that to investigate genetic structure of Ukrainian silver and bighead carps of JSC "Donrybkombinat" the most informative microsatellite loci were MFW 15 and MFW 23.
Practical value. Microsatellite loci MFW 15 and MFW 23 used in research were suitable for interspecies differentiation of Ukrainian silver and bighead carps and can be applied in population-genetic investigations of fishes.
Genetic diversity at nine isozyme loci was surveyed in an endangered tree species, the argan tree, endemic to south-western Morocco. The species is highly diverse (3.6 alleles/locus) with populations strongly differentiated from each other (F ST=0.25). This example is used to illustrate a method for standardizing measures of allelic richness in samples of unequal sample sizes, which was developed for the estimation of the number of species and relies on the technique of rarefaction. In addition, it is shown that the measure of subdivision, θ ST, obtained when allelic richness is used in place ofh (Nei's index of diversity), is much larger than the F ST [e.g. θ ST(40)=0.52, where (40) indicates the specified sample used to estimate the allelic richness]. This suggests that rare alleles (which strongly influence measures of allelic richness) have a more scattered distribution than more frequent ones, a result which raises special conservation issues for the argan tree.
Determination of seed origin is essential for a sustainable use of aquatic resources, in order to protect local biodiversity in restocking programmes or culture-based fisheries and as a precautionary approach to mitigate the genetic impact of accidental escapees from fish farms. Tracking the geographical origin of individual breeders is also useful to fish farmers, whose information on their own broodstocks is sometimes fragmentary. Gilthead sea bream (Sparus auratus) broodstocks from two Italian commercial hatcheries were genetically characterized using four microsatellite loci. The most likely geographical origin among five natural populations was inferred for each single breeder through Bayesian statistic-based methods. Results show a mixed and highly heterogeneous origin of the broodstocks analysed, and revealed a high percentage of Atlantic individuals among breeders.
SUMMARY - Use of molecular markers is specially interesting for the genetic study of the Iberian pig, whose morphologic and physiologic variability prevents the knowledge of his populational structure. Crossbreeding, mainly with Duroc, is a common practice to increase the carcass performance which leads to the attainment of worse quality cured products. Molecular analysis techniques allow the estimation of genetic variability and divergence between species and populations, and thus they can be used for phylogenetic studies, conservation programs and control of the genetic origin of products. This work presents the results obtained with the application of three different techniques of DNA polymorphisms detection to samples from different populations of Iberian pigs. The RAPD technique was used for the identification of diagnostic markers which allowed the detection of Duroc genes in Iberian pig samples. AFLP technique was used for the attainment of specific markers of one Iberian pig population, with a high inbreeding level, in order to study its possible utilization in a conservation program. Finally, seven populations have been analysed with 19 microsatellite markers. The analysis of these polymorphic loci genotypes enabled the estimation of different parameters of genetic diversity (heterozygosity, genetic distances).
This study represents the first large-scale population genetic analysis of the marine fish gilthead sea bream (Sparus aurata), one of the most significant species in the South European aquaculture. Six wild and five cultivated sample sets covering the South Atlantic and Mediterranean European area have been screened for allozyme, microsatellite and mitochondrial DNA (mtDNA) variation. Microsatellites showed higher levels of polymorphism than allozymes. The low variability of mtDNA offered no basis for population differentiation. The results reveal levels of variability for S. aurata above those from other sparids. Cultivated populations show a slight decrease of variability related to the wild ones, but not sufficient to document inbreeding depression effects, thus suggesting a fairly proper management. Wild populations reveal a slight degree of differentiation more pronounced with microsatellites than with allozymes, but not apparently associated with geographic or oceanographic factors. The cultivated populations seem to be highly divergent as a result of genetic drift caused by different factors pertaining to their respective histories. With both markers, the two cultivated Spanish sample sets are the most divergent. The high differentiation between cultivated and wild populations from the same area might indicate no evidence for significant genetic flow between them.
The present study checks on the validity of the hypothesis that heterozygosity and the fluctuating asymmetry (FA), common measure of the developmental stability, are linked in populations of wild and cultured stocks of Sparus
aurata from five countries. Muscle and liver samples were analysed for variation in 26 allozymes and three microsatellite loci. Pectoral fin rays and upper and lower gill rakers of the first branchial arch were counted on the left and on the right sides of each fish. Fluctuating asymmetry existed in the majority of the samples although their values were consistently low, (0·305±0·147), but higher in the cultured samples. The allozyme heterozygosity values were always high, but lower in the cultured samples. The microsatellite DNA analysis produced similar results. Heterozygosity was higher in cultured individuals (except for the Greek samples). These findings seem to be early evidence that the reared samples are losing some genetic variation, especially due to the loss of the rarest alleles (which were present in the wild populations). Genetic drift, probably caused by propagation practices, is most likely responsible for the decrease of the genetic variation. No distinct pattern of geographic separation was identified.
This study was carried out to investigate the genetic variability and the population genetics of Sparus auratus. In fact, despite its importance in Mediterranean fisheries and aquaculture, very little it is known concerning its population structure. Samples of wild gilthead sea bream were collected in seven different localities along the Mediterranean and Atlantic coasts and were genetically characterized by means of microsatellite markers. Genotyping at four microsatellite loci revealed high polymorphism (7–38 alleles/locus) and expected heterozygosities, which ranged from 0.80 to 0.85. A slight but significant population structure was found (FST = 0.010). In fact, at least three populations of gilthead sea bream within the Western Mediterranean Sea were identified (Sardinian Sea, Sardinian Channel and Central Tyrrhenian Sea), which are also genetically differentiated from those of the Atlantic Ocean and the Adriatic Sea.
From a total of 524 microsatellite loci considered in nearly 40 000 individuals of 78 species, freshwater fish displayed levels of population genetic variation (mean heterozygosity, h=0·46, and mean numbers of alleles per locus, a=7·5) roughly similar to those of non-piscine animals (h=0·58 and a=7·1). In contrast, local population samples of marine fish displayed on average significantly higher heterozygosities (h=0·79) and nearly three times the number of alleles per locus (a=20·6). Anadromous fish were intermediate to marine and freshwater fish (h=0·68 and a=11·3). Results parallel earlier comparative summaries of allozyme variation in marine, anadromous, and freshwater fishes and probably are attributable in part to differences in evolutionarily effective population sizes typifying species inhabiting these realms.
Gilthead seabream (Sparus aurata) is one of the most important species among intensively reared fish in the Mediterranean region. Although there is a considerable interest in the genetic improvement of this species, many of the genetic parameters of commercial stocks have yet to be investigated. The effective population size (Ne) of a commercial gilthead seabream broodstock was determined using microsatellite analysis. Eight microsatellite loci were used to ensure a high confidence to the parental assignment of offspring from several mass-spawning events. The Ne was consistently low, ranging from 14 to 18, between photoperiod-controlled broodstock groups. This equated to an inbreeding rate of 2.7–3.5% per generation. The primary constraint on Ne was the high variance in family size and fewer males than females contributing to each spawning. The contribution of male parents to spawning was also more variable than females. Log-linear modelling of offspring counts revealed a significant quadratic relationship, with intermediate parental weights being optimum. The relationship between parental weight and contribution may be evidence of an optimum age structure to the broodstock, or stabilising selection. Low Ne in commercial stocks of gilthead seabream could lead to an increased risk of inbreeding through the practice of broodstock replacement from within same-farm populations.
Six new microsatellite loci were isolated and characterized in 32 individuals from a farm population of gilthead seabream (Sparus aurata). Expected heterozygosity at all loci was high, ranging from 0.835 to 0.958 with between 10 and 27 alleles per locus. A multiplex polymerase chain reaction protocol was developed using four of the loci. Cross-species amplification of the loci was tested in six species of the Sparidae family and four loci were successfully amplified in two or more related species.
The gilthead seabream, Sparus aurata, represents an important economic resource for Mediterranean aquaculture. In spite of its wide geographic distribution and
economic importance, only recently studies have been carried out on the genetic composition of natural populations, which
have revealed a picture of a heterogeneous degree of genetic differentiation among S. aurata populations. In this study an allozyme analysis of samples from six different collecting sites along the Italian and Croatian
coasts was carried out, covering an area in the Central Mediterranean sea that has yet to be investigated through gene-enzyme
systems. Data on 26 gene loci, 10 of which are polymorphic, indicate a slight but significant genetic structure (FST = 0.0167) of the species. A hierarchical analysis of population subdivision made it possible to identify three different
assemblages found in the Adriatic Sea, Tyrrhenian Sea and Sardinian Channel, though an isolation by distance model can be
rejected. The results are discussed in the light of previous literature and taking conservation into consideration.
Formulae are given for estimators for the parameters F, θ, f (FIT, FST, FIS) of population structure. As with all such estimators, ratios are used so that their properties are not known exactly, but they have been found to perform satisfactorily in simulations. Unlike the estimators in general use, the formulae do not make assumptions concerning numbers of populations, sample sizes, or heterozygote frequencies. As such, they are suited to small data sets and will aid the comparisons of results of different investigators. A simple weighting procedure is suggested for combining information over alleles and loci, and sample variances may be estimated by a jackknife procedure.
Gilthead seabream (Sparus aurata) is one of the most important species of intensively reared fish in the Mediterranean region. Its short history of domestication, along with the need to develop markets, new products and efficiency in the production process, has resulted in an increased interest in the potential genetic improvement of this species. Little work has so far been directed at establishing the procedures for selective breeding in gilthead seabream at a commercial level, although genetic parameters calculated in other studies have indicated that there is a large potential for improvement in certain traits. Selective breeding of commercial gilthead seabream populations is constrained by aspects of the biology that complicate the production of genetic groups and the maintenance of same-age offspring populations. The aim of this thesis was to develop a protocol for the selective breeding of gilthead seabream, specifically to serve a commercial hatchery and on-growing unit in Cyprus, where the fieldwork was carried out. The hatchery broodstock was monitored over a three-year period to identify the rate of sex reversal in introduced fish and to quantify the sex ratio of the stock over time. The analysis of the egg production records was used to evaluate the success of photoperiod manipulation in each group. Size variation in the larval and juvenile stages is a common problem in the rearing of gilthead seabream, leading to cannibalism and labour-intensive sorting operations. Studies on larval populations, from first feeding through to metamorphosis, indicated the origin of size variation was the differences in early feeding ability. The size advantages could be maintained throughout the larval period. During the juvenile stage of the farm production system, a method to standardise the size sorting of populations by grading was developed in order to counter environmental effects of separating groups of fish. Using this method, grading would be suitable to form the first stage of a selection programme for growth rate. The potential gain of selection for growth rate during the on-growing stage was very high, using a simulated criterion and previous estimates of heritability. Other possible quality traits for selection were also examined and quantified in the hatchery populations. Existing and specifically developed microsatellite markers were used for the assignment of offspring to parents from mass spawning of the hatchery broodstock. The effective population size of single spawning events were found to be low and determined by a high variation in contribution to mass spawning. Contribution was found to be significantly linked to body size, which led to the formation of a replacement policy for the broodstock to maximise spawning performance. Survival of individual families through the larval period was also examined. Based on the results of the experimental work, a two-stage selection programme was designed, along with the presentation of specific procedures for each stage of the production system. This project makes recommendations on various strategies that can increase the effective population size within a selection programme and these are discussed as part of the genetic management of hatchery populations. Significant progress has also been made in the use of genetic markers in monitoring the rate of inbreeding and contribution of individual broodfish, which are considered essential in this species.
Note that an updated reference for Genepop is Rousset (2008) genepop’007: a complete re-implementation of the genepop software for Windows and Linux (DOI: 10.1111/j.1471-8286.2007.01931.x)
This study investigates the use of microsatellite loci for estimating relatedness between individuals in wild, outbred, vertebrate populations. We measured allele frequencies at 20 unlinked, dinucleotide-repeat microsatellite loci in a population of wild mice (Mus musculus), and used these observed frequencies to generate the expected distributions of pairwise relatedness among full sib, half sib, and unrelated pairs of individuals, as would be estimated from the microsatellite data. In this population one should be able to discriminate between unrelated and full-sib dyads with at least 97% accuracy, and to discriminate half-sib pairs from unrelated pairs or from full-sib pairs with better than 80% accuracy. If one uses the criterion that parent-offspring pairs must share at least one allele per locus, then only 15% of full-sib pairs, 2% of half-sib pairs, and 0% of unrelated pairs in this population would qualify as potential parent-offspring pairs. We verified that the simulation results (which assume a random mating population in Hardy-Weinberg and linkage equilibrium) accurately predict results one would obtain from this population in real life by scoring laboratory-bred full- and half-sib families whose parents were wild-caught mice from the study population. We also investigated the effects of using different numbers of loci, or loci of different average heterozygosities (He), on misclassification frequencies. Both variables have strong effects on misclassification rate. For example, it requires almost twice as many loci of He = 0.62 to achieve the same accuracy as a given number of loci He = 0.75. Finally, we tested the ability of UPGMA clustering to identify family groups in our population. Clustering of allele matching scores among the offspring of four sets of independent maternal half sibships (four females, each mated to two different males) perfectly recovered the true family relationships.
An enrichment cloning method was evaluated for the isolation of microsatellite loci from eastern white pine and the resulting markers were examined for polymorphisms. A 200-fold enrichment was achieved for highly abundant (AC)n repeats, but for much less abundant (ACAG)n repeats an enrichment of only 20-fold was obtained. Using a single set of PCR conditions, 19 microsatellite loci were identified from 77 primer pairs evaluated. Genotyping of 16 (AC)n loci in 16 unrelated white pines from the north-central United States revealed an average of 5.4 alleles per locus and an average observed heterozygosity of 0.515. Five loci were scored among megagametophytes from a single pine to obtain a haploid genotype of the segregating female meiotic products. All loci segregated according to Mendelian expectations and linkage was established for two of the loci. It was concluded that (AC)n loci are highly variable in this species and that SSR (simple sequence repeat) markers can be efficiently developed for genome mapping and population genetics studies.
Applications of quantitative genetics and conservation genetics often require measures of pairwise relationships between individuals, which, in the absence of known pedigree structure, can be estimated only by use of molecular markers. Here we introduce methods for the joint estimation of the two-gene and four-gene coefficients of relationship from data on codominant molecular markers in randomly mating populations. In a comparison with other published estimators of pairwise relatedness, we find these new "regression" estimators to be computationally simpler and to yield similar or lower sampling variances, particularly when many loci are used or when loci are hypervariable. Two examples are given in which the new estimators are applied to natural populations, one that reveals isolation-by-distance in an annual plant and the other that suggests a genetic basis for a coat color polymorphism in bears.
GENECLASS2 is a software that computes various genetic assignment criteria to assign or exclude reference populations as the origin of diploid or haploid individuals, as well as of groups of individuals, on the basis of multilocus genotype data. In addition to traditional assignment aims, the program allows the specific task of first-generation migrant detection. It includes several Monte Carlo resampling algorithms that compute for each individual its probability of belonging to each reference population or to be a resident (i.e., not a first-generation migrant) in the population where it was sampled. A user-friendly interface facilitates the treatment of large datasets.
The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60-90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.
A new method is described for estimating genetic relatedness from genetic markers such as protein polymorphisms. It is based on Grafen's (1985) relatedness coefficient and is most easily interpreted in terms of identity by descent rather than as a genetic regression. It has several advantages over methods currently in use: it eliminates a downward bias for small sample sizes; it improves estimation of relatedness for subsets of population samples; and it allows estimation of relatedness for a single group or for a single pair of individuals. Individual estimates of relatedness tend to be highly variable but, in aggregate, can still be very useful as data for nonparametric tests. Such tests allow testing for differences in relatedness between two samples or for correlating individual relatedness values with another variable.
From a total of 524 microsatellite loci considered in nearly 40 000 individuals of 78 species, freshwater fish displayed levels of population genetic variation (mean heterozygosity, h=0.46, and mean numbers of alleles per locus, a=7.5) roughly similar to those of non-piscine animals (h=0.58 and a=7.1). In contrast, local population samples of marine fish displayed on average significantly higher heterozygosities (h=0.79) and nearly three times the number of alleles per locus (a=20.6). Anadromous fish were intermediate to marine and freshwater fish (h=0.68 and a=11.3). Results parallel earlier comparative summaries of allozyme variation in marine, anadromous, and freshwater fishes and probably are attributable in part to differences in evolutionarily effective population sizes typifying species inhabiting these realms. (C) 2000 The Fisheries Society of the British Isles.
A new method is described for estimating genetic relatedness from genetic markers such as protein polymorphisms. It is based on Grafen's (1985) relatedness coefficient and is most easily interpreted in terms of identity by descent rather than as a genetic regression. It has several advantages over methods currently in use: it eliminates a downward bias for small sample sizes; it improves estimation of relatedness for subsets of population samples; and it allows estimation of relatedness for a single group or for a single pair of individuals. Individual estimates of relatedness tend to be highly variable but, in aggregate, can still be very useful as data for nonparametric tests. Such tests allow testing for differences in relatedness between two samples or for correlating individual relatedness values with another variable.
We have developed an octaplex and a hexaplex polymerase chain reaction (PCR) genotyping system for rainbow trout (Oncorhynchus mykiss). During the development of the two multiplex systems, we encountered numerous allele scoring difficulties. The variable `plus A' modification (non-templated adenylation of the 3′ end of the amplified sequence by Taq polymerase) of PCR products across various DNA templates, in addition to the preferential amplification of smaller alleles over larger alleles (differential amplification) for certain loci, resulted in misidentified genotypes. The time spent correcting misidentified alleles greatly inhibited the efficiency of the semi-automated genotyping systems. We have determined that minimizing primer concentrations (ranging from 0.016 to 0.325 μM) and increasing Taq polymerase concentration (up to 3 units in a 15-μl sample volume) resulted in consistent `plus A' modification of alleles for all loci. Furthermore, increasing the KCl concentration from 50 to 100 mM during PCR amplification, alleviated the problem of differential amplification. These modifications virtually eliminated the need for manual editing of misidentified alleles for both multiplex PCR systems despite variation in template DNA quality and concentration. The information presented in this study will prove useful for those wishing to optimize a multiplex PCR system in the same or other species.
SUMMARY - The use of molecular genetic markers to address questions related to aquaculture management has steadily grown over the last two decades. These markers can provide valuable information for various aspects of aquaculture practice, such as: (i) genetic identification and discrimination of aquaculture stocks; (i¡) monitoring of inbreeding or other changes in the genetic composition of the stocks that may result from such phenomena as breeding programmes, founder events and genetic drift; (iii) comparisons between hatchery and wild stocks; (¡v) assessment of the impact on natural populations of escaped or released cultured fish; (v) assignment of progeny to parents through genetic tags, so that animals from different families can be reared together in breeding programmes; (vi) identification of marker genetic loci associated with quantitative trait loci (QTL) and use of these markers in selection programmes (marker assisted selection); and (vii) assessment of successful implementation of genetic manipulations such as induction of polyploidy and gynogenesis. Allozymes were the first molecular genetic tools used in aquaculture management. Mitochondrial DNA (mtDNA) provided another valuable marker, because of increased resolution and discrimination power in studies of population structure. Nuclear DNA markers were more recently introduced in aquaculture studies and their main attractiveness lay in their abundance in the genome, their Mendelian inheritance, and their potential to detect high levels of polymorphism. Especially microsatellite DNA analysis revolutionized the use of molecular genetic markers in aquaculture and seems to be the marker destined to dominate this type of studies in the coming years. The application of genetic markers to aquaculture management is demonstrated here by the analysis of genetic variability in cultivated and wild populations of gilthead sea bream (Sparus aurata).
The Spanish aquaculture sector has undergone a rapid and continuous growth becoming one of the biggest European producers. A wide range of species are farmed and, among them, the sea bass, sea bream and turbot make a significant contribution to the total of the Spanish fish farming output. In this paper, we analyse and predict the medium-term trend of the production and price level for the three leading species previously mentioned. The results obtained show a significant increase in the production of sea bream, sea bass and turbot. The results show also a slight decrease in the sea bream average price, a stable trends for the sea bass and increasing prices for the turbot.
Twelve new microsatellite markers were isolated from the gilted seabream Sparus aurata L., a seawater fish distributed throughout the Mediterranean Sea and on the East Atlantic Coast and exploited in both fisheries and aquaculture. Characterization in 16 individuals from a hatchery population from France reveals three to 17 alleles and a mean expected heterozygosity of 0.752. Inheritance and linkage were assessed in a bi-parental cross, and using polymorphism and linkage information, two PCR-multiplex were developed for routine analysis of Sparus aurata populations.
The gilthead sea bream (Sparus aurata) is one of the main European aquaculture products and a prospective model species for the Sparidae, which includes several other commercially important species. Future selective breeding of aquaculture stocks will be heavily underpinned by molecular genetic techniques, especially by marker-assisted selection (MAS). Gene marker resources in marine fish species, however, lag behind those of other agricultural animals, and only scanty information exists about the genetic source of phenotypic variation and the identity of quantitative trait loci (QTL). In order to develop molecular resources in gilthead sea bream, complementary DNA libraries were constructed from liver and mixed embryo and larval stages by unidirectional cloning. A long-read expressed sequence tag (EST) database was generated, containing 1394 cDNA clones representing 852 unique cDNA sequence-reads. Tissue-specific patterns of gene expression were determined when grouped using the proposal for characterizing cellular component put forward by the Gene Ontology consortium. Transcripts encoding cytoskeletal proteins were most abundant in the embryonic/larval library, while the most abundant transcripts in the liver library encoded secreted and extracellular proteins. Of both libraries, 505 clones were sequenced in both orientations (5′ and 3′ end sequencing), where 226 clones were determined as full-length sequence reads. Cluster analysis of 3′end-sequenced clones from both libraries revealed that alternative polyadenylation signals were utilized, although no evidence of alternative splicing was found. We report for the first time for gilthead sea bream or any sparid a transcriptional analysis of two tissues and briefly consider the utility of ESTs for characterizing tissue-specific expression profiles.
"Wright's views about population genetics and evolution are so fundamental and so comprehensive that every serious student must examine these books firsthand. . . . Publication of this treatise is a major event in evolutionary biology."-Daniel L. Hartl, BioScience
Tractable forms of predicting rates of inbreeding (DeltaF) in selected populations with general indices, nonrandom mating, and overlapping generations were developed, with the principal results assuming a period of equilibrium in the selection process. An existing theorem concerning the relationship between squared long-term genetic contributions and rates of inbreeding was extended to nonrandom mating and to overlapping generations. DeltaF was shown to be approximately (1)/(4)(1 - omega) times the expected sum of squared lifetime contributions, where omega is the deviation from Hardy-Weinberg proportions. This relationship cannot be used for prediction since it is based upon observed quantities. Therefore, the relationship was further developed to express DeltaF in terms of expected long-term contributions that are conditional on a set of selective advantages that relate the selection processes in two consecutive generations and are predictable quantities. With random mating, if selected family sizes are assumed to be independent Poisson variables then the expected long-term contribution could be substituted for the observed, providing (1)/(4) (since omega = 0) was increased to (1)/(2). Established theory was used to provide a correction term to account for deviations from the Poisson assumptions. The equations were successfully applied, using simple linear models, to the problem of predicting DeltaF with sib indices in discrete generations since previously published solutions had proved complex.
Four major problems can affect the efficiency of methods developed to estimate relatedness between individuals from information of molecular markers: (i) some of them are dependent on the knowledge of the true allelic frequencies in the base population; (ii) they assume that all loci are unlinked and in Hardy-Weinberg and linkage equilibrium; (iii) pairwise methods can lead to incongruous assignations because they take into account only two individuals at a time; (iv) most are usually constructed for particular structured populations (only consider a few relationship classes, e.g. full-sibs vs. unrelated). We have developed a new approach to estimate relatedness that is free from the above limitations. The method uses a 'blind search algorithm' (actually simulated annealing) to find the genealogy that yield a co-ancestry matrix with the highest correlation with the molecular co-ancestry matrix calculated using the markers. Thus (i and ii) it makes no direct assumptions about allelic frequencies or Hardy-Weinberg and linkage equilibrium; (iii) it always provide congruent relationships, as it considers all individuals at a time; (iv) degrees of relatedness can be as complex as desired just increasing the 'depth' (i.e. number of generations) of the proposed genealogies. Computer simulations have shown that the accuracy and robustness against genotyping errors of this new approach is comparable to that of other proposed methods in those particular situations they were developed for, but it is more flexible and can cope with more complex situations.
1^9 Development of a microsatellite multiplex genotyping tool in Sparus aurata J Porta et al
Aquaculture Research, 2009, 1^9 Development of a microsatellite multiplex genotyping tool in Sparus aurata J Porta et al.
- R Franch
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Franch R., Louro B., Tsalavouta M., Chatziplis D., Tsigenopoulos C.S., Sarropoulou E., Antonello J., Magoulas A.,
Mylonas C.C., Babbucci M., Patarnello T., Power D.M., Kotoulas G. & Bargelloni L. (2006) A genetic linkage map of
the hermaphrodite teleost ¢sh Sparus aurata L. Genetics
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Genetic effects of domestication, culture and breeding of fish and shellfish, and their impacts on wild populations. Gilthead seabream âSparus aurata
- L Sola
- A Moretti
- D Crosetti
- N Karaiskou
- A Magoulas
- A R Rossi
- M Rye
- A Triantafyllidis
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