Cross species amplification and characterization of microsatellite markers in Leporinus friderici (Characiformes, Anostomidae)

Amplificação cruzada e caracterização de marcadores microssatélites em Leporinus friderici (Characiformes, Anostomidae)

ABSTRACT:

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.

Key words:
neotropical fish; molecular markers; fish genetics; genetic conservation

RESUMO:

Leporinus friderici é um peixe neotropical migratório com elevada importância ecológica e econômica no Brasil. Os marcadores microssatélites são conhecidos por sua importância em estudos genéticos populacionais, programas de manejo e conservação, no entanto, não existem marcadores disponíveis para esta espécie. Neste estudo, sete locos microssatélites, previamente desenvolvidos para Megaleporinus obtusidens foram amplificados com sucesso em L. friderici. Dentre esses loci, cinco apresentaram variabilidade genética moderada a alta, com quatro a sete alelos por loci e heterozigosidades esperadas variando de ≥ 0,574 a 1.000. Esses marcadores representam uma ferramenta valiosa para futuros manejos e estudos ecológicos envolvendo esta espécie e este grupo de peixes neotropicais.

Palavras-chave:
peixes neotropicais; marcadores moleculares; genética de peixes; genética da conservação

With a current estimate of 9100 species, South American ichthyofauna is the most diverse on Earth (REIS et al., 2016). The genus Leporinus is among the most diverse fish groups in this region, with approximately 90 described species (BIRINDELLI et al., 2013; BURNS et al., 2014). Leporinus friderici, popularly known as “piau-três-pintas”, 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).

Microsatellite markers are intensively used in population genetic studies, mainly due to their wide distribution along the eukaryotic genome, high levels of polymorphism, codominance, and high reproducibility (CHISTIAKOV et al., 2006). The development and standardization of molecular markers, such as microsatellites, are cornerstones for the conservation of natural resources (ALLENDORF et al., 2017) and the implementation of adequate and sustainable management in aquaculture. In this study, the cross-species amplification of 15 microsatellite loci, previously developed for Megaleporinus obtusidens (VILLANOVA et al., 2015), were tested for L. friderici with the aim to provide new markers for future genetic studies. After a systematic and phylogenetic review, L. obtusidens was classified as M. obtusidens by RAMIREZ et al., (2017) due to possible synapomorphies, including a unique ZZ/ZW sex chromosome system, in addition to other characters. This new classification; was subsequently, used in the present study.

Twenty-three L. friderici individuals were collected from the Sapucai-Mirim River (Upper Paraná River Basin, São Paulo state, Brazil). Genomic DNA was extracted from fin clips using the “Wizard Genomic DNA Purification Kit” (Promega, Madison, USA). The DNA integrity and its approximate quantification were checked in 1% agarose gel using the “Low DNA Mass Ladder” of 100 to 2.000 base pairs (bp) (Invitrogen, California, USA). The gel was stained with GelRed (Biotium, Fremont, USA) and observed in a transilluminator under UV light (Loccus Biotecnologia).

The 15 loci developed by VILLANOVA et al. (2015) were amplified by polymerase chain reaction (PCR) in a final volume of 15 µL, containing 100 µM of each dNTP, 1.5 mM MgCl₂, 1 × Taq buffer, 0.1 units of Taq polymerase (Invitrogen Carlsbad, California, USA), 10 pmol of both forward and reverse PCR primers labeled with fluorescence (NED, PET, VIC, and FAM), and 10-30 ng/µL of genomic DNA. Thermal cycling was performed on a Verity 96-Well Thermal Cycler (Applied Biosystems) with an initial denaturation at 95 °C for 10 min, 35 cycles of denaturation at 94 °C for 45 s, annealing temperature (between 56 ºC and 60 ºC; Table 1) for 50 s, and extension at 72 °C for 50 s, followed by a final extension step at 72 °C for 10 min. PCR products were visualized on 2.5% agarose gel using a 100 bp DNA Ladder (Invitrogen, California, USA), and were stained with GelRed (Biotium Fremont, USA) and observed in a transilluminator under UV light (Loccus Biotecnologia).

Table 1
Genetic characterization of seven microsatellite loci for Leporinus friderici.

Locus	Motif	Size range (bp)	Primer sequences (5’ - 3’)	Ta (ºC)	PIC	A	H O	H E	F IS
LoLAR5	(ATCC)6	245-480	F:AACCTCCATCTGACACCCATAFAM R:GGGACAAAGCGGAGGATAAT	60	0.28	4	0.235	0.233	-0.073
LoLAR6	(CTGTT)5	205-230	F:GACGGTGACGAATGTGTGTCNED R:TGCAGAAGGCAAAGAACTGA	58	0.75	6	0.650	0.808	0.176**
LoLAR7	(CTCCG)5	200-230	F:CAGAGCTGCTCCATGACTGAPET R:CTCATGGGAAGCGAGTGTTT	60	0.51	5	0.476	0.574	0.174
LoLAR17	(TTAT)6	210-255	F:GCTTGTGTGATTCTCCAGCAVIC R:TGCACGAGCTACATCAAAGG	58	0.08	2	0.095	0.093	-0.023
LoLAR22	(CAA)9	120-150	F:AGCGAGGAGGAAAACAACAANED R:AGCGCCAACTCTTCACTGAG	60	0.56	4	0.650	1.000	0.562**
LoLAR27	(TTA)9	110-155	F:ACACCACCATGCTGCCTTATFAM R:TTCAGCTGCATCATTTCAGG	60	0.59	7	0.644	0.579	0.104
LoLAR37	(TG)14	115-135	F:TTAGAGCCCAAGCTCTCGACPET R:TGGGCCTCGCGTATTTATAG	60	0.48	6	0.532	0.632	-0.193

• Base pairs (bp), annealing temperature (T _a), polymorphic content information (PIC), number of alleles per locus (A), observed (H _O) and expected (H _E) heterozygosities, and fixation index (F_IS). Asterisks indicate significant deviation from the HWE (^** P = 0.000).

Each PCR product was loaded on an automated DNA analyzer (ABI 3730; Applied Biosystems, Foster City, USA) using the 500 LIZ Size Standard (Applied Biosystems, Foster City, USA). Allele sizes were scored using Gene Mapper 3.7 (Applied Biosystems, Foster City, USA). Cervus 3.0 (KALINOWSKI et al., 2007) was used to analyze the polymorphic information content (PIC), total number of alleles per locus (A), allelic range, and expected (H _E) and observed (H _O) heterozygosity. Deviations from the Hardy-Weinberg Equilibrium (HWE) and fixing index values (F_IS ) were tested in Genepop 4.0 (ROUSSET, 2008) following the methods of WEIR and COCKERMAN (1984). Microchecker 2.2.3 was used to verify the possibility of genotyping errors or null alleles (VAN OOSTERHOUT et al., 2004).

Among the 15 loci tested, six were successfully amplified in L. friderici using different annealing temperatures and proved to be polymorphic (Table 1), while the other nine loci were monomorphic and excluded from the analysis. The PIC proved to be very informative (> 0.05) for loci LoLAR6, LoLAR7, LoLAR22, and LoLAR27, moderately informative (0.25-0.50) for locus LoLAR 37, and not significant (< 0.25) for loci LoLAR5 and LoLAR17. The number of alleles per locus (A) ranged from two (LoLAR1) to seven (LoLAR27), while the highest H _E values were observed for LoLAR6 and LoLAR22 (0.808 and 1.000, respectively) (Table 1). These same loci presented significant deviations from the HWE (P<0.000) due to homozygote excess (F _IS = 0.176 and 0.562, respectively) (Table 1). The relatively low number of individuals included in the study might explain these deviations as LoLAR6 and LoLAR22 loci were the most diverse and sampling was probably insufficient to calculate their H _E levels. No evidence for null alleles was suggested by Microchecker, corroborating the results obtained by VILLANOVA et al., (2015).

Cross-species amplification of heterologous microsatellite loci is common when performed for species of the same genus or taxonomically-related genera, as observed in this study and for other Characiformes species (MORELLI et al., 2006). Considering the costs of genomic sequencing and several additional technical steps necessary for the development of new microsatellite markers, data obtained in this study also represent a new possibility of application for other species included in this genus and family.

Overall, the results obtained here make available new useful markers for application in population genetic studies and the conservation of wild populations of L. friderici. In the field of genetic management, genetic tools are essential for determining genetic variability, inbreeding, and parentage, and for establishing selective breeding programs in captivity, which are considered essential for sustainable aquaculture (BEAUMONT et al., 2010, ARANEDA et al., 2017).

ACKNOWLEDGEMENTS

Funding for this study was provided by CTG Brasil (China Three Gorges Corporation), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo).

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• CR-2020-0072.R1

Publication Dates

• Publication in this collection
  11 Dec 2020
• Date of issue
  2021

History

• Received
  27 Jan 2020
• Accepted
  21 July 2020
• Reviewed
  11 Sept 2020