Development, genetic diversity analysis, and transferability of microsatellite markers for Brycon amazonicusAbstract
Brycon amazonicus is a species native to Brazil, with significant socioeconomic importance and immense potential for fish production. It is the second most cultivated species in the Amazon. The lack of specific molecular markers limits genetic research. This study aimed to identify species-specific microsatellite markers for B. amazonicus and analyze the genetic diversity of four fish farms: Nova Mutum, Instituto Nacional de Pesquisa da Amazônia, Jatuarana, and Nova Airão. In addition, the transferability of these markers to species of the same genus (B. orbignyanus, B. falcatus, and B. gouldingi) was evaluated. Seventeen primer pairs were developed using the enriched library method. Eight of these were used for the genetic analysis of B. amazonicus stocks. In total, 47 alleles were identified. The mean endogamy coefficient (FIS) was negative and significant for Nova Mutum stocks. However, the populations of B. amazonicus in Instituto Nacional de Pesquisa da Amazônia (0.179), Jatuarana (0.099), and Nova Airão were positive and nonsignificant. Analysis of molecular variance showed that most of the variation was observed within the populations evaluated (57%), and genetic differentiation (FST = 0.423) among the stocks was high. Bayesian analysis indicated that the best number of genetic clusters was K = 3. Transferability testing showed successful amplification (90%) of the primers by estimating the allele size between 144 and 294 base pairs (bp) and a total of 24 alleles for the related Brycon species. This indicates the high potential of microsatellites for the analysis of diversity and population genetic structure in both the genus Brycon and family Characidae.
Keywords:
molecular genetics; molecular marker; matrinxã; fish
Resumo
Brycon amazonicus é uma espécie nativa do Brasil, de grande importância socioeconômica e com grande potencial para a produção de peixes. É a segunda espécie mais cultivada na Amazônia. A falta de marcadores moleculares específicos tem limitado a pesquisa genética. O objetivo deste estudo foi desenvolver marcadores microssatélites específicos para B. amazonicus e analisar a diversidade genética em quatro fazendas de peixes: Nova Mutum; Instituto Nacional de Pesquisa da Amazônia; Jatuarana; e Nova Airão. Além disso, foi avaliada a transferibilidade desses marcadores para espécies do mesmo gênero (Brycon orbignyanus, Brycon falcatus e Brycon gouldingi). Dezessete pares de iniciadores foram desenvolvidos usando o método de biblioteca enriquecida. Oito desses foram usados na análise genética de estoques de B. amazonicus. Um total de 47 alelos foi identificado. O coeficiente de endogamia médio (FIS) foi negativo e significativo para os estoques de Nova Mutum. No entanto, as populações de B. amazonicus no Instituto Nacional de Pesquisa da Amazônia (0,179), Jatuarana (0,099) e Nova Airão foram positivas e não significativas. A análise de variância molecular mostrou que a diferenciação genética mais significativa foi encontrada dentro das populações (57%), e a diferenciação genética (FST= 0,423) entre os estoques foi alta. A análise bayesiana indicou como número de grupos genéticos como K = 3. Os testes de transferibilidade mostraram uma amplificação bem-sucedida (90%) dos primers, estimando o tamanho dos alelos entre 144 e 294 pares de bases (pb) e um número total de alelos 24 para as espécies Brycon relacionadas. Isso indica o alto potencial dos microssatélites para a análise da diversidade e estrutura genética de populações tanto no gênero Brycon quanto na família Characidae.
Palavras-chave:
genética molecular; marcadores moleculares; matrinxã; peixe
1. Introduction
Fish farming in Brazil increased by 3.1% in 2023, resulting in the production of 887.029 thousand tons of fish. Native fish production, together with aquaculture production, represents an important part of Brazilian pisciculture (Peixe BR, 2024). In 2023, the proportion of native fish produced in Brazil was 29.7% (263.479 thousand tons) (Peixe BR, 2024). In the Central Amazon, fish are an important resource for the local economy and supply food to riverine populations (Cajado et al., 2018).
Brycon amazonicus Spix & Martius, 1829, commonly known as “matrinxã,” belongs to the genus Brycon, is a rheophilic fish species native to the Amazon basin. It has great potential for use in aquaculture in both semi-intensive and intensive systems (Nakauth et al., 2016). With its omnivorous eating habits and good feed acceptance, it is one of the most cultivated fish species in the Amazon and ranks second in terms of production (Ziemniczak et al., 2023). In addition, it possesses valuable zootechnical traits, including efficient feed conversion, high carcass yield, and strong market acceptance by consumers (Lima et al., 2021).
Despite its importance, only a few studies have evaluated the genetic diversity of B. amazonicus. Maintaining genetic diversity is important because it permits the progeny to adapt and respond to environmental changes. This is a crucial factor in captive stocks as it reduces the potential for inbreeding (Urrea-Rojas et al., 2021). Molecular markers can aid in obtaining relevant information regarding the genetic population structure, demographic history, kinship, and mating systems (Penha et al., 2020).
Microsatellites are one of the most commonly used molecular markers for the population genetic analysis of fish (Diyie et al., 2021) and have been demonstrated to successfully describe genetic diversity because of their codominance and polymorphism (Vu et al., 2024). Simple sequence repeats or short tandem repeats are polymorphic markers that are prevalent in both the coding and non-coding regions of the genome and have co-dominant characteristics that permit differentiation between homozygotes and heterozygotes (Schlötterer, 2004).
These markers have been used in several studies analyzing populations of the genus Brycon (Souza et al., 2018a, 2018b; Penha et al., 2020). However, B. amazonicus does not have species-specific primers, and the use of heterologous primers makes it challenging to obtain satisfactory amplification. Thus, the objectives of this study were to develop species-specific primers for B. amazonicus, analyze the genetic diversity of fish stocks, and conduct cross-amplification tests for other species of the same genus.
2. Material and Methods
The research was approved by the Ethics Committee on Animal Use of the State University of Londrina (CEUA / UEL No. 2166.2020.33).
2.1. DNA extraction
Caudal fins (approximately 0.5 cm2) were collected from nine B. amazonicus specimens. Genomic DNA was extracted using the NaCl protocol described by Lopera-Barrero et al. (2008). The integrity of the extracted DNA was confirmed by loading 5 µg of DNA onto a 1% agarose gel (w/v) in 1-Tris-borate-ethylenediaminetetraacetic (TBE) buffer. The gel was subjected to electrophoresis for 60 min at 100 V and 400 mA. The gel containing the DNA fragments was visualized using a transilluminator device with ultraviolet light, and the image was photographed using Kodak EDAS program (1D Image Analysis 3.5, Kodak, USA).
2.2. Development of microsatellite markers
The hybridization capture method described by Billotte et al. (1999) was used to produce an enriched microsatellite library with the probes (AGA) 5, (CT) 8, and (GT) 8 in the enrichment phase. Extracted genomic DNA (5 µg) from B. amazonicus was digested with 50 U of the restriction enzyme, RsaI type II (Promega, Madison, WI, USA) to obtain several fragments with a known termination sequence. These fragments were ligated to the single-stranded adapters (10 mM) Rsa21 (5′-CTTCTTGCTTACGCGTGGACTA-3′) and Rsa25 (5′-TAGTCCACGCGTAAGCAAGAGCACA-3′) using T4 DNA ligase (Promega, Madison, WI, USA). The cells were then incubated at 20 °C for 2 h to ensure ligation.
Hybridization with biotin-linked oligonucleotides complementary to the microsatellite sequences was used to select the relevant fragments. These fragments were captured using streptavidin-coated magnetic beads with high biotin-conjugation capacity (Thermo Fisher Scientific, Waltham, MA, USA). The microsatellite-enriched fragments were amplified via polymerase chain reaction (PCR) using primers for Rsa21, cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), and transformed into competent Escherichia coli JM109 cells (Promega, Madison, WI, USA). Plasmids isolated from single colonies were sequenced using the Big Dye Terminator sequencing kit v. 3.1 (Applied Biosystems, Foster City, CA, USA) on an automated genetic analyzer (ABI 3500xL, Applied Biosystems, Foster City, CA, USA) with the M13 tail primer (5′-TGTAAAACGACGGCCAGT-3′).
The sequences were analyzed using MEGA v. 6.0 software (Tamura et al., 2013). Sequences exhibiting poor quality or low specificity were excluded from analysis. Microsatellite markers were constructed using Primer3 v. 2.0.4.0 software (Rozen and Skaletsky, 1999). All primers were tested for possible primer-primer interactions and overlapping structures (hairpins) using AutoDimer 1.0 software (Vallone and Butler, 2004).
The annealing temperatures of the developed primers were optimized for B. amazonicus. Amplicons were resolved by electrophoresis on a 3% (w/v) agarose gel in 1x TBE buffer for 120 min at 100 V and 200 mA. The gel containing the DNA fragments was visualized using a transilluminator device with ultraviolet light, and the image was photographed using Kodak EDAS program (1D Image Analysis 3.5, Kodak, USA). Primers lacking specificity were discarded, and 17 primers were selected for validation, transferability, and genetic diversity analysis based on the bands in the gel with the best resolution.
2.3. Genetic diversity analysis and validation of microsatellite markers
Ninety-six samples of B. amazonicus were collected from four municipalities: 24 samples each from a fish farm in Nova Mutum, Mato Grosso-BR (13,822° S, 56,083° O); National Institute for Amazon Research (INPA), Amazonas-BR (3,095° S, 59,988° O); Jatuarana, Manaus-BR (3,0553° S, 59,6586° W); and Nova Airão, Amazonas-BR (2,3715° S 60,5638° W), respectively.
Genomic DNA was extracted from the caudal fins of B. amazonicus using the NaCl protocol described by Lopera-Barrero et al. (2008) and quantified using a spectrophotometer SLIPQ 026 - L-Quant Quantifier (Loccus Biotecnologia, Ribeirão Preto, Brazil). DNA was diluted to 30 ng/µL to standardize sample concentrations. The integrity of the extracted DNA was determined by resolving the DNA by electrophoresis on a 1% agarose gel (w/v) in 1x TBE buffer for 1 h at 100 V and 400 mA. The gel containing the DNA fragments was visualized using a transilluminator device with ultraviolet light, and the image was photographed using Kodak EDAS program (1D Image Analysis 3.5, Kodak, USA).
PCR amplification was performed in a final reaction volume of 15 µL containing recombinant "hot start" enzyme Taq DNA polymerase (1.0 U/µL) (LGC Biotecnologia, Cotia - SP); 1x buffer without MgCl2; 2.0 mM MgCl2; 0.2 mM dNTPs; forward primer (0.12 µL); reverse primer (0.48 µL); 0.48 µL of M13 tail primer (5′-TGTAAAACGACGGCCAGT-3′) labeled with FAM, HEX, NED or PET probes (Applied Biosystems); 1 μL of genomic DNA (30 ng/µL), and MilliQ water for a total volume of 15 μL. The PCR cycling conditions were: 4 min of initial denaturation at 94 °C, followed by 35 cycles of 45 s of denaturation at 94 °C, 30 s of annealing at 64 °C, and 1 min of extension at 72 °C, followed by 10 min of final extension at 72 °C.
Capillary electrophoresis was performed on the amplicons using a genetic analyzer (ABI 3500xL) with GS-600 LIZ (Thermo Fisher Scientific) as an internal molecular weight marker. Alleles were visualized using GeneMarker v. 2.6.3 software (SoftGenetics LLC).
2.4. Cross-amplification tests
Cross-amplification tests using the developed microsatellites were performed on three species of the genus Brycon: eight samples from B. gouldingi, eight samples from B. falcatus, and eight samples from B. orbignyanus. The same PCR and capillary electrophoresis conditions as described for B. amazonicus were used. For the amplified primers, the number of alleles produced (Na) and amplification success rate (As%) (percentage of bands with satisfactory amplification) were determined.
2.5. Statistical analysis
Allele number, effective alleles, observed (Ho) and expected (He) heterozygosity, Hardy-Weinberg equilibrium, low-frequency and exclusive alleles, analysis of molecular variance (AMOVA), fixation index (FST) (0.05 significance level), and paired genetic differentiation index (FST+) were calculated using GenAlEx v. 6.5 software (Peakall and Smouse, 2012). Wright's (1978) definition was used for differentiating FST values, where values between 0.00 and 0.05, 0.051 and 0.15, 0.151 and 0.25, and >0.25 indicate small, moderate, high, and very high genetic differentiation, respectively. The inbreeding coefficient (FIS) was calculated using FSTAT v. 2.9.3.2 software (Goudet, 2005).
The presence of null alleles was confirmed using Micro-Checker software (Van Oosterhout et al., 2004). Polymorphic information content (PIC) was calculated using Cervus v. 3.0.7 software (Kalinowski et al., 2007) and ranked according to Botstein et al. (1980). PIC values >0.5, between 0.5-0.25, and <0.25 indicated if the primers were highly, reasonably, and poorly informative, respectively.
Bayesian analysis of the genetic structure was performed using Parallel Structure software (Besnier and Glover, 2013; Pritchard et al., 2000) to verify the existence of possible clusters (K) among populations. A mixed cluster model with a burn-in period of 250.000 and 1.000.000 Markov chain Monte Carlo repetitions was performed, with 20 runs for each hypothetical K value ranging from one to eight (K = 1-8). The number of clusters was determined using the method proposed by Evanno et al. (2005) and implemented on Structure Harvester website (Earl and von Holdt, 2012). A principal coordinate analysis (PCoA) was performed using the cmdscale function of R v. 4.0.1 (R Core Team, 2020).
3. Results
Of the 17 primers developed and tested, eight microsatellite markers showed polymorphic amplification (Table 1). The PIC values of the primers ranged from highly informative (Bram18, Bram21, Bram24, and Bram27) to less informative (Bram6, Bram11, Bram22, and Bram26).
Characterization of the primers, annealing temperature (TA, °C), base pairs, and PIC of the new microsatellite markers used in the analysis of genetic diversity in fish farm stocks of Brycon amazonicus.
The INPA and Jatuarana populations showed the highest Number of low-frequency alleles (p <0.100) with a total of fourteen and nine alleles, followed by the Nova Airão populations with six alleles, and the Nova Mutum populations with five alleles. Exclusive alleles were also identified in the stocks: fifteen in INPA; five in Nova Mutum, and three in both the Jatuarana and Nova Airão populations (Table 2).
Genetic analysis of 96 individuals from the four stocks of B. amazonicus, resulted in the identification of 47 alleles (Table 3). The fragment sizes ranged from 113 base pairs (bp) (Bram27) to 326 bp (Bram21 and Bram26). The Bram22 locus in the Nova Mutum stock showed a monomorphic pattern. However, the primers revealed a polymorphic pattern for the other stocks. The Number of alleles per locus varied from one to eight.
The average Ho ranged from 0.279 in Jatuarana to 0.511 in Nova Mutum populations. The average He was the lowest in Nova Airão (0.300) and the highest in Nova Mutum (0.341). The mean FIS endogamy coefficient was negative and significant for the Nova Mutum stock (-0.149). However, in the INPA (0.179), Jatuarana (0.099), and Nova Airão (0.066) populations, the values were positive and not significant. Deviation from Hardy-Weinberg equilibrium (p<0.05) was found for five loci (Bram11, Bram18, Brm22, Bram24, Bram26) in the INPA samples: four loci (Bram6, Bram11, Bram24, and Bram27) in the Jatuarana stock, three loci (Bram21, Bram24, Bram27) in the Nova Mutum stock, and two loci (Brm24 and Bram27) in Nova Airão samples (Table 3).
AMOVA showed that most of the variation was observed within the populations evaluated (57%). There was a high and significant genetic differentiation (FST: 0.423) according to Wright's (1978) classification (Table 4). The paired genetic differentiation index (FST) among the stocks was very high in five of the combinations, (INPA × Nova Mutum), (Jatuarana × Nova Mutum), (Jatuarana × INPA), (Nova Airão × Nova Mutum), and (Nova Airão × INPA), and moderate in the combination (Nova Airão × Jatuarana). This showed that each stock in captivity had formed its own characteristics and genetic diversity over time, which were established from the initial stock (founder effect) (Table 4 and 5)
Molecular analysis of variance (AMOVA), genetic differentiation (FST), and Wright classification in B. amazonicus stocks.
The analysis of principal coordinates (PCoA) in the stocks of B. amazonicus showed a greater genetic distance between samples from INPA (green) and Nova Mutum (purple) in relation to the four stocks evaluated. However, the samples from Nova Airão (blue) and Jatuarana (orange) had smaller genetic distances between them. By evaluating Figures 1 and 2 together, the Jatuarana and Nova Airão stocks probably had a common origin, thereby resulting in a loss of genetic differentiation. Bayesian analysis of the population structure indicated the presence of three genetic clusters (K =3): i) samples from Nova Mutum; ii) samples from the INPA stock; and iii) samples from the Jatuarana and Nova Airão farms (Figure 2).
Cross-amplification tests showed 100% successful amplification at the Bram2, Bram4, Bram6, Bram15, Bram18, Bram26, and Bram27 loci for all three species tested. The species that presented the highest Number of alleles was B. gouldingi with 17 alleles, followed by B. falcatus with 16 alleles, and B. orbignyanus with 14 alleles. The sizes of the alleles ranged from 126 bp (Bram27) to 307 bp (Bram26) (Table 6).
4. Discussion
The Polymorphic information content values demonstrated that the information provided by the primers was useful for analyzing population genetic diversity in B. amazonicus. Similar PIC values were reported in other species of the genus Brycon. Castro et al. (2017) evaluated genetic diversity by implementing heterologous primers in B. orbignyanus and found PIC values between 0.215 (Bh5) and 0.609 (Bc48-10).
Similar results for the Na were previously reported by Urrea-Rojas et al. (2021) in a study evaluating the genetic diversity of B. amazonicus using heterologous microsatellites, where the highest values were obtained with the primers BoM13 (nine alleles) and Bh5 (eight alleles). Similarly, Oliveira et al. (2018) compared natural and captive populations and reported variations in the Na from seven to nine. Thus, the Na can vary because of several factors, such as sample origin, sample size, and primers used. Null alleles were not detected.
The average values for Ho were expected because the evaluated stocks were kept in captivity for more than 10 years. A deficit in heterozygotes was evident, especially in the INPA and Jatuarana stocks. However, B. amazonicus has a higher average Ho than in the other Brycon species (Oliveira et al., 2018). Therefore, removing B. amazonicus from the wild to implement them as a broodstock in fish farming may have influenced these results, as there were no studies prior to their capture.
The stocks evaluated in this study were taken from populations older than 10 years, for which data on their formation, mating type, and reproductive management were unknown. Thus, the founder effect may be an important factor in the levels of genetic diversity observed, as the initial stocks of the fish farms evaluated may have had a limited size, contributing to a reduction in genetic diversity (Pandolfi et al., 2021).
The deviations presented in the previous loci may be related to a lack of heterozygotes, as reflected by positive FIS values. These results indicate that the evaluated stocks were influenced by allele frequency losses, suggesting inbreeding. Pandolfi et al. (2021) described that stocks are subjected to inbreeding processes owing to the limited number of sires and the artificial selection processes to which they are exposed.
Based on the results of this study, the introduction of new populations with high genetic variability is necessary, particularly for the Nova Mutum stock. For all fish farm stocks reared in captivity, we suggest the following: i) a previous evaluation to determine the genetic diversity of the individuals to be added to the farm, ii) mating should ideally be between individuals with high genetic diversity, and iii) parents should have a high number of effective alleles to guarantee diversity in their progeny.
Other measures to be considered are equalizing the sizes of families and delimiting the duration of generations (Urrea-Rojas et al., 2021). Reproducers should be replaced from supplies near the fish farm. The herd should never be "reinforced" with individuals from other drainage systems or with fish from other farms of unknown origin, and accidental or intentional escape of individuals kept in captivity should be avoided (Oliveira et al., 2018).
The results of cross-amplification tests are like those observed in the stocks of B. amazonicus analyzed in the present study, confirming that the region flanked by the primers had similar sizes despite variations in the ringing site, thereby allowing for heterologous or cross-amplifications (Urrea-Rojas et al., 2021).
One of the greatest challenges faced by the aquaculture industry is the sustainable and non-exhaustive production of natural (wild) species. In fish farming in the Amazon, production and reproduction stocks are obtained by removing fish from nature, which leads to a decline in species variation, especially B. amazonicus, the second most produced species in the Amazon (Oliveira et al., 2018). Therefore, it is necessary to develop methodologies to evaluate genetic diversity. In this study, new microsatellite markers that are useful for targeting and evaluating the genetic structure of both the target and other species of the genus Brycon and family Characidae were highlighted.
Seventeen new microsatellite markers were developed, eight of which were implemented in the genetic diversity analysis of four matrinxã populations. They exhibited medium-to-low genetic diversity. The low genetic diversity may be the result of low genetic variability of the broodstock or the limited number of broodstocks in the starting stock. The importance of developing microsatellite markers in the matrinxã is crucial. It aids in understanding and evaluating the genetic diversity and population structure in breeding programs, genetic improvement, and conservation (restocking) of wild individuals or stocks kept in captivity, both in the genera Brycon and family Characidae.
Acknowledgements
The authors would like to thank the “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),” “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),” “Fundação Araucária,” and “Programa de Pós-Graduação em Ciência Animal (Universidade Estadual de Londrina)” for scholarships and financial support.
References
-
BESNIER, F. and GLOVER, K.A., 2013. Parallel Structure: a R package to distribute parallel runs of the population genetics program STRUCTURE on multi-core computers. PLoS One, vol. 8, no. 7, pp. e70651. http://doi.org/10.1371/journal.pone.0070651 PMid:23923012.
» http://doi.org/10.1371/journal.pone.0070651 - BILLOTTE, N., LAGODA, P.J.R., RISTERUCCI, A.M. and BAURENS, F.C., 1999. Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits, vol. 54, pp. 277-288.
- BOTSTEIN, D., WHITE, R.L., SKOLNICK, M. and DAVIS, R.W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics, vol. 32, no. 3, pp. 314-331. PMid:6247908.
-
CAJADO, R.A., OLIVEIRA, L.S., OLIVEIRA, C.C., PONTE, S.C.S., BITTENCOURT, S.C.S., QUEIROZ, H.L. and ZACARDI, D.M., 2018. Larval distribution of Brycon amazonicus (PISCES: BRYCONIDAE) around the Mamirauá Sustainable Development Reserve: an ecological basis for management. Revista Ibero-Americana de Ciências Ambientais, vol. 9, pp. 78-87. http://doi.org/10.6008/CBPC2179-6858.2018.006.0010
» http://doi.org/10.6008/CBPC2179-6858.2018.006.0010 -
CASTRO, P.L.D., RIBEIRO, R.P., SANTOS, S.C.A.D., GOES, E.S.D.R., SOUZA, F.P., POVEDA-PARRA, A.R., VARGAS, L., URREA-ROJAS, A.M. and LOPERA-BARRERO, N.M., 2017. Cross-amplification of heterologous microsatellite markers in Piracanjuba. Ciência Rural, vol. 47, no. 12, pp. 1-6. http://doi.org/10.1590/0103-8478cr20170374
» http://doi.org/10.1590/0103-8478cr20170374 -
DIYIE, R.L., AGYARKWA, S.K., ARMAH, E., AMONOO, N.A., OWUSU-FRIMPONG, I. and OSEI-ATWENEBOANA, M.Y., 2021. Genetic variations among different generations and cultured populations of Nile Tilapia (Oreochromis niloticus) in Ghana: application of microsatellite markers. Aquaculture (Amsterdam, Netherlands), vol. 544, pp. 1-8. http://doi.org/10.1016/j.aquaculture.2021.737070
» http://doi.org/10.1016/j.aquaculture.2021.737070 -
EARL, D.A. and VON HOLDT, B.M., 2012. Structure Harvester: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources, vol. 4, no. 2, pp. 359-361. http://doi.org/10.1007/s12686-011-9548-7
» http://doi.org/10.1007/s12686-011-9548-7 -
EVANNO, G., REGNAUT, S. and GOUDET, J., 2005. Detecting the Number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, vol. 14, no. 8, pp. 2611-2620. http://doi.org/10.1111/j.1365-294X.2005.02553.x PMid:15969739.
» http://doi.org/10.1111/j.1365-294X.2005.02553.x -
GOUDET, J. 2005 [viewed 08 October 2024]. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.4) [online]. Available from: https://www2.unil.ch/popgen/softwares/fstat.htm
» https://www2.unil.ch/popgen/softwares/fstat.htm -
KALINOWSKI, S.T., TAPER, M.L. and MARSHALL, T.C., 2007. Revising how the computer program Cervus accommodates genotyping error increases success in paternity assignment. Molecular Ecology, vol. 16, no. 5, pp. 1099-1106. http://doi.org/10.1111/j.1365-294X.2007.03089.x PMid:17305863.
» http://doi.org/10.1111/j.1365-294X.2007.03089.x -
LIMA, D.L.G., CAJADO, R.A., SILVA, L.V.F., MAIA, J.L.S. and ZACARDI, D.M., 2021. Morphological description of the initial development of Brycon amazonicus (Characiformes, Bryconidae) from the Lower Amazon, Pará. Biota Amazônia, vol. 11, pp. 60-67. http://doi.org/10.18561/2179-5746
» http://doi.org/10.18561/2179-5746 -
LOPERA-BARRERO, N.M., POVH, J.A., RIBEIRO, R.P., GOMES, P.C., JACOMETO, C.B. and LOPES, T.S., 2008. Comparison of DNA extraction protocols of fish fin and larvae samples: modified salt (NaCl) extraction. Ciencia e Investigación Agraria, vol. 35, pp. 65-74. http://doi.org/10.4067/S0718-16202008000100008
» http://doi.org/10.4067/S0718-16202008000100008 -
NAKAUTH, A.C.S.S., VILLACORTA-CORREA, M.A., FIGUEIREDO, M.R., BERNARDINO, G. and FRANÇA, J.M., 2016. Embryonic and larval development of Brycon amazonicus (SPIX & AGASSIZ, 1829). Brazilian Journal of Biology =. Revista Brasileira de Biologia, vol. 76, no. 1, pp. 109-116. http://doi.org/10.1590/1519-6984.13914 PMid:26909629.
» http://doi.org/10.1590/1519-6984.13914 -
OLIVEIRA, R.C., SANTOS, M.C.F., BERNARDINO, G. and HRBEK, T., 2018. From river to farm: an evaluation of genetic diversity in wild and aquaculture stocks of Brycon amazonicus (Spix & Agassiz, 1829), Characidae, Bryconinae. Hydrobiologia, vol. 805, no. 1, pp. 75-88. http://doi.org/10.1007/s10750-017-3278-0
» http://doi.org/10.1007/s10750-017-3278-0 -
PANDOLFI, V.C.F., YAMACHITA, A.L., SOUZA, F.P., GODOY, S.M., LIMA, E.C.S., FELICIANO, D.C., PEREIRA, U.P., POVH, J.A., AYRES, D.R., BIGNARDI, A.B. and PENAFORT, J.M., 2021. Development of microsatellite markers and evaluation of genetic diversity of the Amazonian ornamental fish Pterophyllum scalare. Aquaculture International, vol. 29, no. 6, pp. 2435-2449. http://doi.org/10.1007/s10499-021-00757-8
» http://doi.org/10.1007/s10499-021-00757-8 -
PEAKALL, R. and SMOUSE, P.E., 2012. ALEx 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics (Oxford, England), vol. 28, no. 19, pp. 2537-2539. http://doi.org/10.1093/bioinformatics/bts460 PMid:22820204.
» http://doi.org/10.1093/bioinformatics/bts460 -
PEIXE BR, 2024 [viewed 08 October 2024]. Anuário Peixe BR da Piscicultura de 2024 [online]. Associação Brasileira de Piscicultura. Available from: https://www.peixebr.com.br/anuario2022/#:~:text=A%20PEIXE%20BR%20come%C3%A7a%20as,de%20peixes%20cultivados%20no%20Brasil
» https://www.peixebr.com.br/anuario2022/#:~:text=A%20PEIXE%20BR%20come%C3%A7a%20as,de%20peixes%20cultivados%20no%20Brasil -
PENHA, D.S., SOUZA, F.P., LIMA, E.C.S., URREA-ROJAS, A.M., PANDOLFI, V.C.F., YAMACHITA, A.L., POVH, J.A., LEITE, N.G., PEREIRA, U.P. and LOPERA-BARRERO, N.M., 2020. Transferability of heterologous primers in Brycon falcatus. Acta Amazonica, vol. 50, no. 3, pp. 232-238. http://doi.org/10.1590/1809-4392201904191
» http://doi.org/10.1590/1809-4392201904191 -
PRITCHARD, J.K., STEPHENS, M. and PETER, D., 2000. Inference of population structure using multilocus genotype data. Genetics, vol. 155, no. 2, pp. 945-959. http://doi.org/10.1093/genetics/155.2.945 PMid:10835412.
» http://doi.org/10.1093/genetics/155.2.945 -
R CORE TEAM 2020 [viewed 08 October 2024]. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing [online]. Available from: https://www.R-project.org/
» https://www.R-project.org/ -
ROZEN, S. and SKALETSKY, H.J., 1999. PRIMER 3 on the WWW for general users and for biologist programmers. In: S. MISENER and KRAWETZ, S.A. eds. Bioinformatics methods and protocols: methods in molecular biology Berlin, Germany: Springer, pp.365-386. http://doi.org/10.1385/1-59259-192-2:365
» http://doi.org/10.1385/1-59259-192-2:365 -
SCHLÖTTERER, C., 2004. The evolution of molecular markers: just a matter of fashion? Nature Reviews. Genetics, vol. 5, no. 1, pp. 63-69. http://doi.org/10.1038/nrg1249 PMid:14666112.
» http://doi.org/10.1038/nrg1249 -
SOUZA, F.P., LIMA, E.C.S., LEITE, N.G., URREA-ROJAS, A.M., YAMACHITA, A.L., PANDOLFI, V.C.F. and LOPERA-BARRERO, N.M., 2018b. Transferability of heterologous microsatellite primers in Brycon gouldingi. Ciência Rural, vol. 48, no. 11, pp. 1-6. http://doi.org/10.1590/0103-8478cr20180412
» http://doi.org/10.1590/0103-8478cr20180412 -
SOUZA, F.P., URREA-ROJAS, A.M., RUAS, C.D.F., POVH, J.A., RIBEIRO, R.P., RUAS, E.A., GIACOMIN, R.M., GOES, B.D., CASTRO, P.L.D. and LOPERA-BARRERO, N.M., 2018a. Novel microsatellite markers for the endangered neotropical fish Brycon orbignyanus and cross-amplification in related species. Italian Journal of Animal Science, vol. 17, no. 4, pp. 916-920. http://doi.org/10.1080/1828051X.2018.1436008
» http://doi.org/10.1080/1828051X.2018.1436008 -
TAMURA, K., STECHER, G., PETERSON, D., FILIPSKI, A. and KUMAR, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, vol. 30, no. 12, pp. 2725-2729. http://doi.org/10.1093/molbev/mst197 PMid:24132122.
» http://doi.org/10.1093/molbev/mst197 -
URREA-ROJAS, A.M., SOUZA, F.P., LIMA, E.C.S., GONÇALVES, L.U., POVH, J.A., AYRES, D.R., BIGNARDI, A.B., PEREIRA, U.P. and LOPERA-BARRERO, N.M., 2021. Genetic diversity of Matrinxã breeding stocks: implications for management and conservation. Semina: Ciências Agrárias, vol. 42, no. 2, pp. 757-768. http://doi.org/10.5433/1679-0359.2021v42n2p757
» http://doi.org/10.5433/1679-0359.2021v42n2p757 -
VALLONE, P.M. and BUTLER, J.M., 2004. AutoDimer: a screening tool for primer-dimer and hairpin structures. BioTechniques, vol. 37, no. 2, pp. 226-231. http://doi.org/10.2144/04372ST03 PMid:15335214.
» http://doi.org/10.2144/04372ST03 -
VAN OOSTERHOUT, C., HUTCHINSON, W.F., WILLS, D.P.M. and SHIPLEY, P., 2004. Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes, vol. 4, no. 3, pp. 535-538. http://doi.org/10.1111/j.1471-8286.2004.00684.x
» http://doi.org/10.1111/j.1471-8286.2004.00684.x -
VU, D.D., NGUYEN, M.T., NGUYEN, M.D., NGUYEN, P.L.H., BUI, T.T.X., PHAN, K.L., VU, D.G., PHAM, Q.T. and NGUYEN, T.P.T., 2024. Genetic population structure of the Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) detected by microsatellite analysis. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, pp. e264369. http://doi.org/10.110.1590/1519-6984.264369 PMid:36287528.
» http://doi.org/10.110.1590/1519-6984.264369 - WRIGHT, S., 1978. Evolution and genetics of populations: a treatise in four volumes 4th ed. Chicago: University of Chicago, vol. 4: Variability within and among natural populations, 590 p.
-
ZIEMNICZAK, H.M., HONORATO, C.A., VALENTIM, J.K., FERREIRA, E., FERRAZ, H.T., RAMOS, D.G.S., VIEIRA, N.T. and SATURNINO, K.C., 2023. Anatomical and histological traits of Brycon amazonicus liver cultivated in a semi-intensive system. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 83, pp. e244784. http://doi.org/10.1590/1519-6984.244784 PMid:34190759.
» http://doi.org/10.1590/1519-6984.244784
Publication Dates
-
Publication in this collection
28 Oct 2024 -
Date of issue
2024
History
-
Received
24 Apr 2024 -
Accepted
09 Sept 2024
