Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing

Vianna, Juliana A.; Noll, Daly; Mura-Jornet, Isidora; Valenzuela-Guerra, Paulina; González-Acuña, Daniel; Navarro, Cristell; Loyola, David E.; Dantas, Gisele P. M.

doi:10.1590/1678-4685-GMB-2016-0224

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Evolutionary Genetics • Genet. Mol. Biol. 40 (3) • Sept 2017 • https://doi.org/10.1590/1678-4685-GMB-2016-0224 copy

Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Microsatellites are valuable molecular markers for evolutionary and ecological studies. Next generation sequencing is responsible for the increasing number of microsatellites for non-model species. Penguins of the Pygoscelis genus are comprised of three species: Adélie (P. adeliae), Chinstrap (P. antarcticus) and Gentoo penguin (P. papua), all distributed around Antarctica and the sub-Antarctic. The species have been affected differently by climate change, and the use of microsatellite markers will be crucial to monitor population dynamics. We characterized a large set of genome-wide microsatellites and evaluated polymorphisms in all three species. SOLiD reads were generated from the libraries of each species, identifying a large amount of microsatellite loci: 33,677, 35,265 and 42,057 for P. adeliae, P. antarcticus and P. papua, respectively. A large number of dinucleotide (66,139), trinucleotide (29,490) and tetranucleotide (11,849) microsatellites are described. Microsatellite abundance, diversity and orthology were characterized in penguin genomes. We evaluated polymorphisms in 170 tetranucleotide loci, obtaining 34 polymorphic loci in at least one species and 15 polymorphic loci in all three species, which allow to perform comparative studies. Polymorphic markers presented here enable a number of ecological, population, individual identification, parentage and evolutionary studies of Pygoscelis, with potential use in other penguin species.

Keywords:
microsatellite markers; penguin; genome; polymorphism; tetranucleotide

	P. papua	P. antarcticus	P. adeliae
Filtered reads	186,606,554	94,746,776	59,889,052
Aligned reads	158,565,602	80,496,366	52,997,551
No of loci	42,057	35,265	33,677
No of loci with primers	29,618	17,777	13,492
Total of Dinucleotide	24,528	21,250	20,361
Total of Trinucleotide	11,667	9216	8,607
Total of Tetranucleotide	4,370	3,788	3,691
Dinucleotide with primers	18,639	11,449	8,673
Trinucleotide with primers	7,385	4,314	3,198
Tetranucleotide with primers	2,699	1,594	1,287

					Pygoscelis adeliae				Pygoscelis papua				Pygoscelis antarcticus
	Scaffold	Locus	Primer sequence (5′-3′)	Motif	Size (bp)	N	A	Ho	Size (bp)	N	A	Ho	Size (bp)	N	A	Ho
P3	51	AP-26	F: TGGGGAGCAGAGTATTTTTGTT	(GATA)7	205-245	6	8	0.67	217-241	9	6	0.78	231-263	9	5	0.56
			R: AGCCACGAAAAGTGAGCCTA
	46	AP-61	F: GCTCTCAGTTGAGCAAACCC	(TTTG)6	230-242	8	4	0.63	216-228	9	2	0.67	230-250	9	6	0.78
			R: TTCGTGCTTCTTGCTTTCCT
	181	AP-78	F: CCACTGCAGTATCCCATTTTT	(TATC)6	188-200	8	4	0.50	188-208	9	5	0.56	188-208	9	3	0.22
			R: GGTCAAAGCTTACCATCCCA
	27	AP-85	F: CAAAACAGTCACTAGTGTGCCA	(ATCT)9	212-232	8	6	1.00	188-204	9	4	0.44	204-210	9	4	0.44
			R: TACGCCAATGAAAAGCACTG
	44	AP-90	F: TGCATGTTGGAACATCAAAA	(GATA)7	162-178	8	5	0.88	194-206	9	3	0.78	162-186	9	7	0.44
			R: AACACACACACGTGCACTCTT
	18	GP-4	F: TCATCACCAATGGTTCAGAAA	(ATGA)5	212-232	8	6	1.00	256-264	9	3	0.44	268-284	9	5	0.78
			R: CCATGTGGTATTCATTCtGGTG
	100	GP-6	F: AACATCTCATGAAGGCACAGC	(CATC)5	234-246	8	4	0.63	230-238	9	3	0.67	234-238	9	2	0.11
			R: GTACCATGCCCTTTCTGATGA
	57	GP-13	F: CACATCCTTTCCCTCTCTTCC	(AGAT)11	182-202	7	4	0.29	194-202	9	3	0.67	190-202	6	3	0.33
			R: AAGGAGYGTGCCTAGTTTTGG
	86	GP15	F: CTGTATTGAGATGGCCGTTGT	(CATC)7	250-274	8	7	0.38	254-266	9	5	0.44	250-274	9	4	0.22
			R: GTACCCACCGACTTCTCTTCC
	101	GP-18	F: GGTCACCATGAGCAGTCAGTT	(ATAG)10	194-206	8	4	0.63	190-202	9	3	0.22	194-202	9	2	0.11
			R: TGAAAAGCTCTCCCCACAGTA
	356	GP-19	F: TTGGGGAAATGACAACCCTAT	(TATC)5	223-239	7	4	0.71	227-235	9	3	0.67	221-227	9	3	0.22
			R: CCCTCACTGCTCAAGTCTGTC
	241	GP-36	F: CTGTAAGTCACAGCGTGCAAA	(ATAC)5	248-256	8	3	0.25	248-268	8	5	0.50	252-264	6	4	0.17
			R: TGTGAGAACCATTGGACTTGT
	240	CP-6	F: AGGCTTTCTCACACTGTGCTC	(CTAC)5	212-232	8	5	0.88	212-216	9	2	0.22	204-228	9	6	0.67
			R: AATGAGCAATTCAGGATGGTG
	174	CP-25	F: GTCAAAGCCTGCRTCAACTCT	(CATC)13	196-224	7	4	0.43	200-216	9	5	0.78	208-242	8	7	0.75
			R: ATGACACTGGCAAAGGAGATG
	189	CP-29	F: GCATCAGATCCCAGAATACCA	(AGAC)5	184-216	7	5	0.57	180-188	9	2	0.11	188-192	8	2	0.13
			R: TCTGGCAGTATGGGAAAACAC
P2	108	AP-3	F: AAGCAGGGAAACCATACAAAGA	(ATAA)7	174-186	8	3	0.38	182-190	9	2	0.78	178	9	M	–
			R: GGTCTGAGTAAGGCTCTTCAGG
	197	AP-12	F: AGGACAACGAGGCGAGAGT	(GAAG)5	212-216	8	2	0.25	212	9	M	–	212-216	9	2	0.33
			R: CCCTCCACCCTTTCTCTCTT
	218	AP-14	F: TGGTAAACAGTCACACGGGA	(AAAC)5	224-228	8	2	0.75	228	9	M	–	220-224	9	2	0.22
			R: CCAAAACTGAGAAGCAACCC
	37	AP-19	F: CCAGTGTTTGACCACCCTCT	(ATAA)7	250-258	8	3	0.5	246	9	M	–	250-262	7	4	0.71
			R: TGCATTTTTCCATTCCATTTC
	590	AP-29	F: CACATCCGTGTGTTGGAAAG	(CATC)5	256-260	8	2	0.13	256-260	9	2	0.22	256	9	M	–
			R: GTTGGCGTTAACTGGGAAGA
	18	AP-79	F: TTTTTCAAGGTAGAGGGCTCA	(TAGA)8	174-190	8	4	0.75	170-174	9	2	0.67	178	9	M	–
			R: GGAAGATAATATTTTGCATTTTCA
	30	AP-87	F: GCCTTCCCATCTGTAGAAGC	(TATC)9	170-224	8	9	0.75	174	9	M	–	162-166	9	2	0.33
			R: TTTTCCAAAGTACGCAAGCA
	9	GP-2	F: AGCACCACTCTCTCTTCCTCC	(AGTA)5	234-242	7	3	0.57	234-242	9	2	0.11	234	9	M	–
			R: GCAATTTCTTTTGAAACCCCt
	349	GP-30	F: TTCCTCTACCYGCCTCAATTT	(CAGA)7	158-174	7	4	0.71	170-182	9	4	0.33	170	9	M	–
			R: TTACCTTTTCCTGTGCCCTTT
	21	CP-5	F: CCAAATAGTCCCCCAAACCTA	(CATC)8	226-238	7	2	0.29	218	9	M	–	225-234	9	3	0.56
			R: GGGATAAAAATGGATGGATGAA
	38	CP-17	F: TGAGTCATTTTGCAACTGGTG	(TAGA)5	255-267	8	4	0.75	255-263	9	3	0.67	259	9	M	–
			R: GAATGCACGCTGAAAAGAAAG
	203	CP-22	F: AAAGAAGCTGGAGCCAAACT	(GATG)5	206-210	7	2	0.29	206-214	9	3	0.66	206	9	M	–
			R: ACCCCAGTGCTTCCATGTATT
	175	CP-27	F: CACACTCCATATTGCCACACA	(TATG)5	170	8	M	–	158-170	9	2	0.11	170-174	9	2	0.44
			R: TTCAGAAAGTGGCTCCAAGA
P1	367	AP-52	F: GCCTCAAACAGGACAGAAGC	(AAAC)5	224	8	M	–	216-228	9	3	0.67	224	9	M	–
			R: GGAATGGCTTCTGGTTGAAA
	44	AP-57	F: CTGCTAGCCTTAGGCATTGG	(CAAA)6	230	8	M	–	228	9	M	–	222-234	9	4	0.44
			R: CTTTTGCCTTCTGCCTTGTC
	92	GP-1	F: ATACCTGTGCTCATCTGTGGG	(AGCA)5	240-244	8	2	0.38	240	9	M	–	240	9	M	–
			R: AATGAGGTAGCACCCTGGACT
	54	GP-24	F: ACGCATGAAGTAGGCAAGAGA	(GATA)10	233-237	8	2	0.25	233	9	M	–	233	9	M	–
			R: TACGAAGTGGTGGTGAAGAC
	27	CP-4	F: GGAAACCAAAATCATCCATCC	(CATC)6	252	8	M	–	248-252	9	2	0.11	248	9	M	–
			R: AGGACCTGCATCTTTTCCAGT
	7	CP-21	F: AGATGACAGTCTGGGGAAAGG	(ATAG)6	162-190	7	7	0.43	158	9	M	–	158	9	M	–
			R: CTCCCAAGGAAAACCTACCAG

Microsatellite loci	P. adeliae		P. papua		P. antarcticus
Microsatellite loci	N_A	H_O	N_A	H_O	N_A	H_O
AP	3.85	0.52	2.43	0.33	3.07	0.9
GP	3.91	0.53	2.91	0.40	2.82	0.25
CP	3.56	0.23	2.33	0.22	2.67	0.17

	Pygoscelis papua						Pygoscelis antarcticus
Locus	Size (bp)	N	A	Ho	He	F _IS	Size (bp)	N	A	Ho	He	F _IS
AP-3	178-186	30	2	0.37	0.49	0.261^* * Significant FIS values, p < 0.05.	174-178	30	2	0.067	0.065	-0.018
AP-19	246	30	1	0	0		250-290	28	8	0.82	0.79	-0.034
AP-26	225-241	30	6	0.70	0.77	0.100	231-255	30	6	0.8	0.8	0
AP-61							218-250	28	8	0.643	0.755	0.151^* * Significant FIS values, p < 0.05.
AP-78	188-212	30	7	0.80	0.69	-0.169	188-208	30	4	0.266	0.38	0.309^* * Significant FIS values, p < 0.05.
AP-85	188-208	30	5	0.67	0.70	0.051	200-224	30	7	0.700	0.763	0.084
AP-90	194-214	30	6	0.53	0.56	0.045	158-190	30	9	0.900	0.840	-0.072
GP-6	230-234	29	2	0.28	0.29	0.051	234	28	1	0	0
GP-13	190-206	30	5	0.90	0.62	-0.479
GP15							250-286	30	10	0.767	0.806	0.049
GP-36	240-260	30	5	0.13	0.16	0.171^* * Significant FIS values, p < 0.05.	244-260	30	5	0.333	0.473	0.30^* * Significant FIS values, p < 0.05.
CP-6							228-236	30	3	0.600	0.524	-0.146
CP-17	255-259	29	2	0.10	0.09	-0.037
CP-22	190-234	30	7	0.73	0.76	0.311
CP-25	192-212	30	6	0.63	0.55	-0.152	204-268	30	10	0.866	0.826	-0.05

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[1] Send correspondence to Juliana A. Vianna. Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile. Av. Vicuña Mackenna 4860, Santiago, Chile. E-mail: jvianna@uc.cl.