Article

Short Communication Heterologous amplification and characterization of microsatellite markers in the Neotropical fish Leporinus friderici

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Abstract

Leporinus friderici, native to the Amazon Basin and popularly known as "piau-três-pintas", has great ecological and economic importance; it is widely fished and consumed throughout much of tropical South America. Knowledge of the genetic diversity of this native species is important to support management and conservation programs. We evaluated microsatellite loci amplification, using heterologous primers, in 31 individuals of L. friderici. These samples were collected from natural populations of the Araguaia River basin, in central Brazil, and the DNA was extracted from samples of muscle tissue. Eight loci were successfully analyzed. Six of them were polymorphic, and the number of alleles ranged from three to 10. Values of expected heterozygosities for these polymorphic loci ranged from 0.488 to 0.795. Exclusion probability (0.983), the identity probability (0.000073), and the mean genetic diversity values were high, showing that these microsatellite markers are suitable for assessing the genetic variability of L. friderici populations. There is a growing interest in studies that evaluate the genetic variability of natural populations for various purposes, such as conservation. Here, we showed that a viable alternative to the costly development of specific primers for fish populations is simply testing for heterologous amplification of microsatellite markers available from research on other species.

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... Leporinus friderici, popularly known as "piau-trêspintas", naturally occurs in Suriname, the Amazon, Paraná and Paraguay River Basins (GODOY, 1987). As a herbivorous fish, this species plays an important ecological role in its ecosystem, and has social and economic importance for local fisheries (OLIVATTI et al., 2011). However, their natural populations are declining in response to several anthropogenic disturbances, such as habitat modifications, water pollution, construction of dams, and the absence of adequate conservation practices (ABELL et al., 2008;BARLETTA et al., 2010). ...
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Leporinus friderici is a migratory neotropical fish with elevated ecological and economic importance in Brazil. Microsatellite markers are highly important in population genetic studies, management, and conservation programs; however, no markers are available for this species. In this study, seven microsatellite loci, previously developed for Megaleporinus obtusidens, were successfully cross-amplified in L. friderici. Among these loci, five presented moderate to high genetic variability levels, with four to seven alleles per loci and expected heterozygosities varying from ≥ 0.574 to 1.000. These markers represent a valuable tool for the future management and ecological studies involving this species and group of neotropical fishes.
... Some empirical studies with micosatellite loci have allowed to reconstruct the fish population genealogy and their family that are mixed when they hatch; this resulted in 99% right assignations of male and female parents and their offsprings (Norris et al., 2000;. Morelli, 2007;Zhan et al., 2009;Olivatti et al., 2011). used these DNA markers to distinguish the group of cod fish family (Gadus morhua). ...
Thesis
Rainbowfishes (Melanotaeniidae) are widely distributed throughout New Guinea and Australia. They are very famous for ornamental trade because of their vivid coloration. They display amazing species richness and some of them are on the red list of endangered species. The species Melanotaenia boesemani, one of the most popular within this family, is.facing great threats. Rearing of this species in aquaculture setups thus appears as a promising solution to limit capture of wild specimens. Yet, the number of farms that raise M. boesmani is very low. This is probably due to the problems reported by the farmers, i.e. higher proportion of females per spawning, loss of coloration, lower growth rate and fecundity, frequent morphological abnormalities. In this context, this study aimed at gathering new genetic information that would be useful for the aquaculture and conservation of the Melanotaeniidae family. Specifically, the objectives of the research were: 1) to develop new microsatellite DNA markers from the endangered M. boesemani, 2) to evaluate the genetic diversity of wild populations of Melanotaenia and refine their taxonomy, 3) to describe the geographic origins of M. boesemani reared by ornamental fish farmers in Indonesia, and evaluate the inbreeding pressure resulting from this domestication. Using next generation sequencing, 12 microsatellite DNA markers were developed and validated from M. boesemani. All microsatellite loci revealed polymorphic and cross-breeding experiments showed that they followed a Mendelian inheritance pattern. These new markers were subsequently implemented to evaluate the genetic variability of 44 wild populations (corresponding to 1152 fish specimens). Multilocus Fis values revealed that 5 species significantly departed from Hardy-Weinberg expectations and suggested the possible occurrence of genetically differentiated subpopulations. Combined with a phylogenetic analysis performed on the cytochrome oxydase I (COI) gene and with the observation of several diagnostic morphological characters, the 12 microsatellite markers also enabled to characterize 8 new species previously undescribed. Finally, these microsatellite markers were applied to analyze and compare the genetic variability of M. boesemani samples obtained from 6 aquaculture farms around Jakarta with that of the two native populations of this species , i.e. from Ayamaru and Uter Lakes (West Papua). Results indicated that all reared strains originated from Ayamaru Lake. No deficit in heterozygotes was evidenced, suggesting that there was no major inbreeding in these reared populations. Genotype analysis also suggested that M. boesemani species consists of a metapopulation composed of genetically differentiated populations. Altogether, these results indicated that the problems experienced by the farmers are obviously not due to inbreeding depression and are probably caused by other factors such as unsuitable management and/or poor water quality. In conclusion, these new microsatellite markers proved useful to evaluate the genetic structure and diversity of a large number of rainbowfish species, among which many are endangered. The results presented here on one of the most threatened species (M. boesemani) show that it is still possible to prevent its extinction. This, however, implies to increase its aquaculture production in order to quickly alleviate the overfishing pressure. This, in turn, involves a better management of rearing practices.
... However, differentiation of genetically related skate population by distance has been observed in northeast Atlantic continental shelf [32]. To reduce the time and cost of MS marker development, pre-existing markers from related species have been applied to the study of many animal, fungus, plant and fish species [33,34]. Moreover, cross-species markers can be used for the identification of invading species and the identification of parental origin in hybrid fish [35]. ...
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The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.
... friderici), reflecting high levels of differentiation (Chiari and Sodré, 1999). A second study of L. friderici based on microsatellite markers also revealed high levels of heterozygosity (0.54) and genetic diversity (0.750) (Olivatti et al., 2011), providing additional evidence for the differentiation of fishes of the genus Leporinus. ...
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DNA barcoding is a useful complementary tool for use in traditional taxonomic studies due to its ability to detect cryptic species, and may be particularly efficient in the identification of fish species. The fish fauna of the Itapecuru River represents an important fishery resource in the Brazilian State of Maranhão, although it is currently suffering increasing degradation as a result of anthropogenic impacts. Therefore, DNA barcoding was used in the present study to identify fish species and establish a database of the rich freshwater fish fauna of Maranhão. A total of 440 specimens were analyzed, corresponding to 64 species belonging to 59 genera, 31 families, and 10 orders. Overall, 92.19% of these species could be identified by DNA barcoding, and were characterized by low levels (average 0.80%) of intra-specific divergence. However, five species (Anableps anableps, Gymnotus carapo, Sciades couma, Pseudauchenipterus nodosus, and Leporinus piau) presented values of mean genetic divergence above 3%, indicating the existence of cryptic diversity in these fishes. The DNA barcoding approach permitted the analysis of a large number of specimens and facilitated the discrimination and identification of closely related fish species in the Itapecuru Basin.
... Several studies have empirically used microsatellite loci to successfully reconstruct pedigrees in fish populations with families mixed from hatching (Norris et al. 2000;Morelli 2007;Zhan et al., 2009;Olivatti et al.,2011). ...
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Microsatellites or Simple Sequence Repeats (SSRs) represent an abundant source of genetic markers that are highly abundant and dispersed evenly throughout eukaryotic genomes. They have become the markers of choice for a wide range of applications in population genetic, conservation, and evolutionary biology. Microsatellites represent ideal molecular markers because they have multiple alleles that are highly polymorphic among individuals. Polymorphism is achieved by having variable numbers of tandem repeat motifs resulting in size variation which can then be visualized by PCR with pairs of locus-specific flanking primers, followed by electrophoresis of the amplification product. Microsatellite motifs occur once every 10kb in fishes. They are inherited in a co-dominant fashion and are fast and easy to assay. They are co-dominant in nature with high levels of polymorphism and can reproduce very well. Hence, giving better information than the dominant marker. This makes them a choice-maker for estimating population structure and genetic diversity.
... Finally, 11 microsatellite loci agreed with Hardy-Weinberg equilibrium that could be well used for studies on population genetic diversity and genetic structure. The reason of five loci which deviated from Hardy-Weinberg equlibrium, could be due to the presence of null alleles, allelic dropout, size homoplasy, or the limited sample size [45]. ...
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The blue swimming crab (Portunus pelagicus) is a valuable marine fishery resource in Indo-West Pacific Ocean. So far, rare genetic resource of this species is available. In this report, the restriction-site associated DNA (RAD) approach was employed to mine the genomic information and identify molecular markers in P. pelagicus. A total of 0.82 Gbp clean data were generated from the genome of individual “X2A”. De novo assembly produced 85,796 contigs with an average length of 339 bp. A total of 45,464 putative SNPs and 17,983 microsatellite loci were identified from the genomes of ten individuals. Furthermore, 31 pairs of primers were successfully designed, with 16 of them exhibiting polymorphism in a wild population. For these polymorphic loci, the expected and observed alleles per locus ranged from 1.064 to 7.314 and from 2 to 11, respectively. The expected and observed heterozygosity per locus ranged from 0.0615 to 0.819 and from 0.0626 to 1.000, respectively. Nine loci showed high informative with polymorphism information content (PIC) > 0.5. Five loci significantly deviated from Hardy–Weinberg equilibrium in the samples analyzed. No linkage disequilibrium was found among the 16 polymorphic microsatellite loci. This study provided massive genetic resource and polymorphic molecular markers that should be helpful for studies on conservation genetics, population dynamics and genetic diversity of P. pelagicus and related crab species.
... Deviation from the Hardy-Weinberg equilibrium (HWE) (p < 0.0024) was evident at four loci (CR-11, CR-20, CR-21 and CR-43) with having null alleles. Generally, heterozygote deficiency could be due to allelic dropout, the limited sample size, size homoplasy, or the presence of null alleles (Olivatti et al., 2011). ...
Article
The neritid species Clithon retropictum (Gastropoda), an oviparous snail, is distributed in freshwater and brackish water in southern Korea, south-east Japan, southern China and Taiwan. This species is listed as a class II endangered species in Korea because of its decreasing population size. It has led to an increase in the need for genetic information about this species. For this aim, in this study, we developed microsatellite markers for C. retropictum by using nextgeneration sequencing. A total of 185,972 sequences containing motifs with a minimum of five repeats motifs were identified from 3,392,119 reads. Of the 46 loci screened among 49 individuals, 36 were successfully amplified and 21 were polymorphic among 49 individuals, with 14 tri-nucleotide repeats and 7 tetra-nucleotide repeats. All loci exhibited relatively high genetic variability except one locus (CR08), with an average of 10.33 alleles per locus, and the mean observed and expected heterozygosities were 0.661 and 0.758, respectively. Our results demonstrated the utility of next-generation sequencing as a method for the rapid and cost-effective identification of microsatellites. These 21 newly developed microsatellite markers will be informative tools for investigating the genetic structure and diversity among populations of this endangered species and will help facilitate effective strategies for its conservation.
... The "piau" Leporinus friderici (Characiformes, Anostomidae) is one of the most widely distributed species in the Neotropical region. In addition, it is considered to be one of the most abundant species in the various water systems of the Amazon region with relevant economic importance Olivatti et al. [18]. The species of the genus Leporinus present great potential for fish farming because they have good commercial acceptance Baldisserotto & Gomes et al. [19]. ...
... Deviation from the Hardy-Weinberg equilibrium (HWE) (p < 0.01) was evident at one locus (Mic44) (Table IV). Generally, it could be due to the limited sample size, size homoplasy, or the presence of null alleles (Olivatti et al., 2011). No significant evidence for linkage disequilibrium existed in any comparisons by location or by locus after Bonferroni correction (p < 0.01). ...
... In fish, however, cross-species amplification varies, showing a tendency to amplify homologous loci in species such as flatfish 34) and non-orthologous loci in catfi sh 31) . Heterologous microsatellites have been suggested for use in genetic polymorphism tests and genetic variability evaluations 35,36) . The present analysis also disclosed that the repeat motifs are conserved in Pangasiid catfishes but not in Clariid catfi shes. ...
Article
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Thirty microsatellite markers developed for River catfish (Hemibagrus nemurus) were screened for cross amplification in six species of catfish. Out of 30 primers, only 3 (10) to 6 (20) produced successful amplifications in six catfish species. Six different loci could be amplified in Pangasius pangasius and Clarias batrachus, 5 loci in Clarias gariepinus, 4 loci in Pseudomystus siamensis and 3 loci in Clarias macrocephalus and Pangasius nasutus. The low rate of amplification in these 6 closely related species proved the primers are locus-specific. Sequence analysis of locus MnRm7-1 showed differences in the flanking region and repeat motif in certain species, suggesting the existence of indel mutations in catfish species in that particular region. The examined DNA sequence revealed that the repeat motif is conserved within Pangasiid catfishes and not conserved within Clariid catfishes. Catfish are an important fisher y resource both as a source of income and an alternative protein for most Southeast Asian nations 13). Indeed, in the most recent accounting, catfi sh species made up for 67 of total freshwater aquaculture production in Malaysia 4). Catfi sh have also been used as an animal model in reproductive, physiological and genetic studies. Species from three major familiesnamely, Bagriidae, Clariidae and Pangasiidaeare of particular commercial importance and widely cultured in Southeast Asia. The study of genetics has been of major importance for the aid aquaculture sector and assist conservation programme. Microsatellite DNA markers are short tandem repeat motifs with high levels of polymorphism 5,6) , and are distributed in both coding and non-coding regions 7). These markers have been a subject of great interest due to their co-dominant characteristics, which make them more informative than dominant markers such as RAPD and AFLP. In aquaculture, microsatellites have become one of the most useful molecular markers in fish population studies and have supplemented conventional markers such as allozymes and mitochondrial DNA that show totally low overall levels of variation 8). Microsatellite markers also show high allelic variation that makes them effective as markers in many genetic studies. Microsatellite markers are widely applied in studies on population genetic structures and genetic diversity of species. However, the development of microsatellite markers is very time-consuming and labor-intensive. As a potential alternative , some microsatellite markers have been shown to be conser ved within related taxa 9,10). Through cross-species amplification, microsatellite markers
... A number of studies have verified the use microsatellite loci to reconstruct pedigrees in fish populations with families mixed from hatching [46][47][48][49]. SSR has gained it fame due to its high variability amidst individuals of the same strain. ...
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Fisheries and aquaculture has evolved and metamorphosed into so many stages, however, compared with the constant rise in population. hence the quest to meet the ever growing demand of fish in the world gave rise to the advent of the application of biotechnology in fish; molecular markers are identifiable DNA sequence and they have found application in different parts which includes breeding, population genetics, and environmental management. They produce more accurate information, microsatellite markers has become indispensable among other markers because of its unique characteristics such as co-dominance, high polymorphism, short stretches and it is widely distributed in the genome of organisms. Microsatellite has wide variety of application which includes parental and pedigree analysis, population genetics, conservation of stocks, assessment of wild and cultured population, marker assisted selection and breeding, however high cost of developing species specific markers has been one of the major challenges of microsatellite but multiplexing is effective in reducing its cost production. The unique attributes of microsatellite marker has made it an indispensable marker over all other dominant markers.
... The effort with the development of this tool allows, from now on, genetic and molecular approaches in the study of P. macropilis species. In addition, the success of cross amplification observed from species of origin to correlated species represents time and economic savings in the development of new markers, regardless of the taxa studied (Bech et al. 2010;Canales-Aguirre et al. 2010;Olivatti et al. 2011;Telles et al. 2011). This study provided an important first step in the development of molecular markers suitable for multispecies studies of phytoseiid mites, from a set of SSR loci of P. macropilis. ...
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Phytoseiid mites are efficient predators of mites and small pest insects. Understanding the dispersion and distribution pattern of phytoseiid mites is essential to promote the conservation of these natural enemies and support their use in biological control. Population genetic studies using molecular markers such as microsatellites have proved to be extremely informative to address questions about population structure and dispersion patterns of predatory mites. The objective of this work was to develop specific microsatellite markers for the predatory mite Phytoseiulus macropilis, aiming at improving field dispersion studies. For this purpose, the genomic DNA was extracted from the whole body of a pool of 260 adult females and used to build the genomic microsatellites-enriched library, using biotinylated probes (CT) 8 and (GT) 8. In total 26 pairs of primers were synthesized and screened across 30 adult females of P. macropilis for characterization. Seven loci were polymorphic, revealing from two to six alleles per locus. Cross amplifications were successfully obtained in the species Phytoseiulus persimilis, Amblyseius swirskii and Proprio-seiopsis sp. The molecular markers obtained are the first developed for P. macropilis-they are effective for the detection and quantification of genetic variation, and show high trans-ferability, thus can be used in genetic and molecular studies of this and other species of the same genus and also of close genera.
... The transference of microsatellites from the source species to closely related species is a subject of interest due to the difficulties and economic investment needed irrespective of the taxons studied (Bech et al. 2010;Canales-Aguirre et al. 2010;Olivatti et al. 2011;Telles et al. 2011). In Tephritid flies, a species group of economic interest as the mites used in this study, microsatellites are transferable between species with variable degree of relatedness, ranging from 49 to 24% when species belong to a different genus from the one of the source species (Augustinos et al. 2008). ...
Article
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Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.
Chapter
The genetic information contained in structural genes, gene regulatory regions, and noncoding DNA is what makes living beings similar and also what differentiates all the species on planet Earth. The rich diversity of life is still in the process of discovery; it has been the focus of increasing attention by sectors of society addressing developmental policies that often are detrimental to the environment. Conservation of genetic resources does not imply only conservation of species diversity, but also the genetic differences between populations of the same species or between animal breeds, strains and varieties. Therefore, knowledge of population differentiation and the temporal and spatial extent of gene flow are essential to management decision-making. As the methodologies for genetic diversity assessment have been developed in the last decades, genetic diversity estimation within and among populations has assumed central importance in conservation programs for genetic resources. The aim is to reveal putative genetic units so that genetic erosion of genetically unique populations can be prevented (Bijlsma and Volker Loeschcke 2012).
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Jaú (Zungaro jahu): um peixe mais ameaçado que os outros? Bol
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Alves CBM (2006). Jaú (Zungaro jahu): um peixe mais ameaçado que os outros? Bol. Soc. Bras. Ictiologia 83: 4-5
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