Microsatellite loci development for three catfish species from northwestern South America

The Neotropical catfish species Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus are important fishery resources in Colombia that show historical declines in their capture. This study used next-generation sequencing with 454 FLX technology (Roche Applied Science) and bioinformatics analysis to develop between 18 and 24 microsatellite loci for these species. The novel microsatellite loci showed high values of polymorphic information content -PIC (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876), and the average number of alleles/locus ranged from 7-15 for A. pardalis, 9-30 for P. grosskopfii and 5-14 for S. cuspicaudus. The average observed and expected heterozygosities were respectively, 0.757 ± 0.035 and 0.834 ± 0.015 for A. pardalis; 0.596 ± 0.040 and 0.881 ± 0.009 for P. grosskopfii; and 0.747 ± 0.031 and 0.757 ± 0.025 for S. cuspicaudus. For future studies, these loci can be useful to estimate the genetic diversity and population structure in these three Neotropical catfishes.

Keywords:
Freshwater fish; Molecular markers; Next-generation sequencing; Siluriformes

Authorship

Natalia Restrepo-Escobar

Facultad de Ciencias, Escuela de Biociencias, Laboratorio de Biología Molecular y Celular, Universidad Nacional de Colombia - Sede Medellín. Carrera 65 Nro. 59A - 110 Bloque 19 A Laboratorio 310, 050034 Medellín, Colombia. (NRE) nrestree@unal.edu.co, (EJM) ejmarque@unal.edu.coUniversidad Nacional de ColombiaColombiaMedellín, ColombiaFacultad de Ciencias, Escuela de Biociencias, Laboratorio de Biología Molecular y Celular, Universidad Nacional de Colombia - Sede Medellín. Carrera 65 Nro. 59A - 110 Bloque 19 A Laboratorio 310, 050034 Medellín, Colombia. (NRE) nrestree@unal.edu.co, (EJM) ejmarque@unal.edu.co

https://orcid.org/0000-0002-9430-4683

Edna J. Márquez

https://orcid.org/0000-0003-0760-3747

Edited by

Guillermo Orti

Correspondence

Edna J. Márquez ejmarque@unal.edu.co

AUTHOR CONTRIBUTIONS

Natalia Restrepo-Escobar: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing (original draft), Writing (review & editing).

Edna J. Márquez: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing (original draft), Writing (review & editing).

ETHICAL STATEMENTS

Samples were collected under the environmental license # 0155 of January 30, 2009 from Ministerio de Ambiente, Vivienda y Desarrollo Territorial.

COMPETING INTERESTS

Not applicable.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (1)
Tables (3)

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FIGURE 1 |
Frequency of identified microsatellite loci and potentially amplifiable loci (PAL) obtained from 454 FLX sequencing for Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus.

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TABLE 1 |
Primer sequences and characteristics of 21 polymorphic microsatellite loci identified in Ageneiosus pardalis. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content. Annealing temperature: 56.5 °C, for all primers.

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TABLE 2 |
Primer sequences and characteristics of 18 polymorphic microsatellite loci identified in Pimelodus grosskopfii. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content.^1,2,3 Annealing temperatures: 56.5 °C, 59 °C, 60 °C, respectively.

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TABLE 3
| Primer sequences and characteristics of 24 polymorphic microsatellite loci identified in Sorubim cuspicaudus. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content. Annealing temperature: 56.5 °C, for all primers.

FIGURE 1 | Frequency of identified microsatellite loci and potentially amplifiable loci (PAL) obtained from 454 FLX sequencing for Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus.

TABLE 1 | Primer sequences and characteristics of 21 polymorphic microsatellite loci identified in Ageneiosus pardalis. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content. Annealing temperature: 56.5 °C, for all primers.

Locus	Repeat motif	Primer sequence for forward (F) and reverse (R) (5’→3’)	Ra	Na	Ho	He	P	PIC
Apar03	(ATT)_n	F: TTTAGAAGCAGCCTGGATGG R: CTTGACTTTGGGAAATGGGC	186-219	11	0.920	0.882	0.965	0.870
Apar25	(TCTG)_n	F: CCGGCTGTTCAATTTGTGG R: CACAGAGTAGAAGAGCTCACTTTGG	256-316	9	0.700	0.622	0.909	0.601
Apar11	(ATGG)_n	F: CACCAATGTATCCCCATCCC R: TTCAAGTACTGGCTACAAGCTGC	208-268	15	0.920	0.895	0.904	0.886
Apar36	(AATAG)_n	F: GAAGGAAATCACAGCCCTCG R: TCTGCTGGAAGGTGGAGAGG	230-280	8	0.800	0.825	0.832	0.801
Apar18	(ATCT)_n	F: GCATCTGCACCTCATATTTGC R: TGAGCTATTTCTGATTGGCTGG	206-266	13	0.860	0.871	0.816	0.858
Apar05	(ATT)_n	F: GGGAGGGAGAGGAGGATAGC R: GAAGGTAGGTGTTGGCAATGG	192-234	15	0.820	0.798	0.799	0.783
Apar35	(AATAG)_n	F: CCCTTTAAGCAAATTGTCTTCAGC R: CACCATCTGTCCTGTCCTGC	179-209	7	0.780	0.752	0.797	0.715
Apar22	(ATCT)_n	F: CAGAGGCTATTGTTCAGGCG R: TCATTGGGGTGGTGATATGC	235-271	10	0.800	0.850	0.612	0.833
Apar20	(ATCT)_n	F: TCCAACCATTACTGCATCCC R: GGTGATCGTTGGATGATTTGC	179-226	11	0.820	0.871	0.591	0.857
Apar21	(ATCT)_n	F: CACTGTGTGATCTCTCTGTGTTGG R: GAGATATTTCTTGAATTTCCTTCGG	173-225	13	0.920	0.891	0.494	0.881
Apar27	(AATG)_n	F: TCACCAAAGTGAAGCTGTTGC R: CAAACCAAGGACCTTCTCGC	146-178	9	0.900	0.827	0.416	0.806
Apar30	(ATATC)_n	F: GAACCTTGATTTTGCCCACG R: GCCAACATCAGGAAAGGGG	172-222	11	0.820	0.844	0.414	0.827
Apar04	(ATC)_n	F: CCTGCACTTCAACACTTCACC R: GAGAGAGTAAATGAGGGAAAGCG	256-298	10	0.880	0.844	0.401	0.826
Apar34	(ATCAC)_n	F: ACACTGCATCCCATCACACC R: TCCTCCTGGTCTTCTCACAGG	191-251	12	0.800	0.809	0.386	0.786
Apar23	(ATCT)_n	F: TCTTTGGTAACCCACCACCC R: ATGTGCAATGGGGATCTGC	241-269	8	0.860	0.814	0.295	0.791
Apar28	(ATGG)_n	F: AACTCCATGTCTGCTTCGGC R: CTGCTATTGCAGCCCATCC	273-301	8	0.600	0.698	0.179	0.666
Apar12	(ATCT)_n	F: GATTCCAGGATGCAGTTGAGG R: AGCGATTGCACACCATAACC	247-299	14	0.780	0.886	0.129	0.876
Apar31	(AATAG)_n	F: CTGGAGGGATGCAAACTGC R: GGGAATTCCGTTATTTTCAAGC	151-236	10	0.560	0.843	0.000	0.825
Apar14	(ATCT)_n	F: TTCCGGTATCGTGCATTTCC R: AACAATTGGGTGCCTAAGACG	165-221	15	0.500	0.910	0.000	0.903
Apar32	(ATTAG)_n	F: TTGCACTTCTGGTTGGATGC R: TCCAAAGCAAATGGTCATGG	151-216	13	0.540	0.900	0.000	0.892
Apar19	(ATCT)_n	F: TCAGCTAAGGCAAGTTGTTTGC R: GGGATTTCCTATATCGGCAGC	190-250	15	0.320	0.885	0.000	0.874
Across loci				11.286±0.574	0.757± 0.035	0.834± 0.015	0.000	0.817

TABLE 2 | Primer sequences and characteristics of 18 polymorphic microsatellite loci identified in Pimelodus grosskopfii. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content.^1,2,3 Annealing temperatures: 56.5 °C, 59 °C, 60 °C, respectively.

Locus	Repeat motif	Primer sequence for forward (F) and reverse (R) (5'→3')	Ra	Na	Ho	He	P	PIC
¹ Pgrk40	(ATCT)_n	F: TTTGGTAACATTCAAGGTTTACTTGC R: AAAATAGCAACGTTCTAACTAGGGG	159-219	15	0.920	0.916	0.743	0.910
¹ Pgrk27	(ATATT)_n	F: CTTCCAAGCATGACAAGCCC R: CTCTCCACCACATCTTCCCC	264-359	16	0.920	0.866	0.517	0.852
¹ Pgrk15	(ATCT)_n	F: TGCCATCAGTGGTCTTCACC R: CCCCATCACCCTGTTCACC	268-328	14	0.760	0.852	0.225	0.840
² Pgrk14	(ATCT)_n	F: TCATGGCCTTGTACTTGTACCG R: CACCAGTTCATGCTTCTGCC	169-261	15	0.800	0.903	0.170	0.895
² Pgrk03	(ATCT)_n	F: GGATGAAAGAAAAGGATTTGGC R: GATAAGACGCGCTTACACTTGC	174-246	16	0.740	0.909	0.083	0.902
² Pgrk24	(AAAT)_n	F: ACACGCATGTCTCTTGCCC R: ACTTGTACTGCGGATGCGG	188-232	11	0.660	0.866	0.016	0.852
² Pgrk01	(ATCT)_n	F: GTCCCAGTCCTGCTTCACG R: GCTAATGGTACAACATCGCCC	156-292	30	0.760	0.948	0.002	0.946
² Pgrk19	(AAAT)_n	F: TAGTCGGTGCTAATTCGCCG R: ACTGACTGGAGACCACAGCG	201-237	10	0.520	0.810	0.000	0.787
² Pgrk12	(ATCT)_n	F: TGAGATTGGTTTATAACACAGACCG R: TGCAGCTGAAGAACTCAGGG	192-292	22	0.380	0.913	0.000	0.908
¹ Pgrk18	(ATCT)_n	F: GGATAAATATGGGTGGGTGGC R: GGAGCTGTGGAAGAGACATCG	210-282	15	0.580	0.907	0.000	0.900
² Pgrk07	(ATCT)_n	F: TGGCACATGAGTTGAAGACG R: AAACACATTGACTGATAGCCTTCC	197-265	17	0.400	0.907	0.000	0.899
³ Pgrk06	(ATCT)_n	F: GCGGGAAAAGTACAGGAAGG R: CGACGCAGCTCAGAATAAAGC	180-264	13	0.520	0.899	0.000	0.890
¹ Pgrk31	(ATT)_n	F: TGGAATTGTGCATTTCTTTGC R: GGAGGTTGAATTCCTCTGTGG	154-208	15	0.580	0.888	0.000	0.878
² Pgrk02	(ATCT)_n	F: ATCTGTCTGGCCATCCACC R: CAGATAGACGGACGCACG	77-157	15	0.400	0.879	0.000	0.867
¹ Pgrk20	(ATCT)_n	F: CAAGCTGCCTCCTGAAAACC R: GCTCATCTGTGTGAGTGGTGC	124-216	14	0.600	0.868	0.000	0.855
¹ Pgrk10	(ATCT)_n	F: TGTTGCGTAATATTCCCAGGC R: GACACAAACTGGTGAAAATCTGC	230-282	11	0.480	0.864	0.000	0.849
² Pgrk28	(AATAG)_n	F: ACTGTCTTGTGGCTTTGTGC R: TACGGGAAAATAATCCTGGG	116-226	11	0.660	0.843	0.000	0.825
² Pgrk08	(AAAT)_n	F: TTGGCGTGCAAATGAATCC R: ATGGGACCACAATCAGAGGC	261-301	9	0.340	0.771	0.000	0.748
Across loci				14.944±1.136	0.612±0.042	0.878± 0.010	0.000	0.867

TABLE 3 | Primer sequences and characteristics of 24 polymorphic microsatellite loci identified in Sorubim cuspicaudus. Ra: Allelic size range; Na: Average number of alleles per locus; Ho and He: observed and expected heterozygosity, respectively; P: Statistical significance for tests of departure of Hardy-Weinberg equilibrium; PIC: Polymorphic information content. Annealing temperature: 56.5 °C, for all primers.

Locus	Repeat motif	Primer sequence for forward (F) and reverse (R) (5’→3’)	Ra	Na	Ho	He	P	PIC
Scus22	(ATCT)_n	F: CGCCACCTTAGGAACCTACC R: GTGGTATGGTGGTGTCGAGG	180-208	8	0.800	0.808	0.932	0.784
Scus15	(TTC)_n	F: GCCTTCATATGCTTGGACCC R: TGAAGGTCCCTTTCAATGGC	201-216	6	0.620	0.637	0.913	0.599
Scus41	(ATTAC)_n	F: CAGATAAAGGACCATGCTGAGG R: CTGGATGCTCTGTGTGAGGC	161-186	6	0.780	0.689	0.895	0.636
Scus24	(ATCT)_n	F: CCTTGAGATACCTGCAGCCC R: GTTCCCTCCCTTCGTCTTCC	257-289	9	0.820	0.831	0.847	0.809
Scus17	(ATCT)_n	F: TGATAGAGGCACAGAGTGAACG R: CCTGTGTGCCTTGATGTCG	181-229	12	0.900	0.862	0.809	0.847
Scus43	(ATTAG)_n	F: TGACTCTGTGACTCTAATTCCTGC R: TCAGTTTCCCTGAGGAACCC	141-166	6	0.720	0.629	0.809	0.568
Scus28	(ATCT)_n	F: TCGGATGCAAAGTTAGACGG R: AGGATTTGTTGCCTCATGCC	252-280	8	0.820	0.803	0.794	0.774
Scus25	(ATCT)_n	F: TAAAACTGCAAGCCCCTTCG R: CAGAGTCCAAACCCTGTGGG	173-237	14	0.920	0.867	0.746	0.853
Scus23	(ATCT)_n	F: GATCTTTGTTCAGTCCACGGC R: GACGTTGTTAGCCCAGATGC	275-309	11	0.780	0.838	0.523	0.818
Scus12	(ATT)_n	F: TCTACACAAGCAGAAATGCAGC R: TCACATTGCTTGACAGATGCC	204-258	11	0.680	0.695	0.514	0.670
Scus35	(ATATT)_n	F: GTCAAAACGCATTCTGTAAATGG R: GTAGTGCTCAGCTCCAGGGC	209-249	9	0.880	0.832	0.479	0.812
Scus40	(ATCTT)_n	F: TGGAAGTAGTTGCTGCTGTTGC R: CAGAAGGCATTTACTGTCTGGC	241-276	7	0.660	0.641	0.438	0.594
Scus20	(ATCT)_n	F: GTCACATCTGGGTGTGCTGG R: GCCTGAGCTTTGGATTGAGC	249-317	14	0.940	0.887	0.321	0.876
Scus39	(ATCTT)_n	F: GGGACACTATGATTTTCCTTCAGC R: CTGTCATCCCCAGCAGTAGC	128-168	9	0.820	0.812	0.199	0.785
Scus32	(AGGGC)_n	F: AACGAGCCATGCAAGAACG R: CCCAGAAGCAGTAAGGTGTGC	222-287	14	0.820	0.861	0.122	0.848
Scus21	(ATCT)_n	F: CTACCCAATCTAGCTGCCCC R: CGACCTGGTGGGTACGTAGG	117-221	11	0.900	0.820	0.080	0.798
Scus03	(ATC)_n	F: GGATCGTTGGTGTAAATCTCTGC R: TGACTCATGGTGTGTGGTGC	114-135	7	0.520	0.575	0.052	0.536
Scus11	(ATT)_n	F: CACTTCCACCTGAACCATGC R: GCATCAGGGTTAGCACAGGC	276-288	5	0.300	0.419	0.037	0.383
Scus16	(AATG)_n	F: ACGTCATCTGGTGTCGTTGG R: CCAGAAGATCTGTGTGAACCG	104-136	9	0.620	0.780	0.026	0.748
Scus13	(ATT)_n	F: AGACGAGAGGGGTGACATGC R: AAGGTGTTGGCAGGATGAGG	272-314	9	0.740	0.809	0.024	0.783
Scus10	(ATT)_n	F: CCTGAAGAAATCCAGCCACC R: ACCACAACTCCGACTCCAGG	168-213	13	0.820	0.865	0.020	0.851
Scus18	(ATCT)_n	F: CAAGCATCAAAGCAGAAAGCC R: ATCACCATCTTGCGTTCTATCC	257-305	12	0.640	0.867	0.005	0.853
Scus37	(ATCAG)_n	F: GATCTGGGCTCTGCTTTTGG R: GCAGCACAATGAGTACTGCG	265-330	9	0.540	0.597	0.002	0.564
Scus07	(ATT)_n	F: CAACATCTGCAACCAAATGC R: GATTTTGCACCAGGACAACG	146-176	9	0.880	0.753	0.000	0.713
Across loci				9.500±0.552	0.747±0.031	0.757±0.025	0.000	0.729

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Sociedade Brasileira de Ictiologia Neotropical Ichthyology, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá., Av. Colombo, 5790, 87020-900, Phone number: +55 44-3011-4632 - Maringá - PR - Brazil
E-mail: neoichth@nupelia.uem.br

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Microsatellite loci development for three catfish species from northwestern South America

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[1] Edited by

Guillermo Orti