Isolation and characterization of microsatellite markers from the greater ani. Crotophaga major (Aves: Cuculidae)

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1 Page 1 of 9 Isolation and characterization of microsatellite markers from the greater ani Crotophaga major (Aves: Cuculidae) C. RIEHL * and S. M. BOGDANOWICZ * Department of Ecology and Evolutionary Biology, Princeton University, 106A Guyot Hall, Princeton, NJ 08544, USA Department of Migration and Immuno-ecology, Max Planck Institute for Ornithology, Schlossallee 2, Radolfzell, Germany Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY 14853, USA Keywords: Crotophaga, Crotophaga major, greater ani, microsatellite Correspondence: Christina Riehl, Fax: criehl@princeton.edu

2 Page 2 of Abstract The greater ani (Crotophaga major) is a communally breeding cuckoo found in Panama and northern South America. We developed 12 polymorphic microsatellite loci in 227 individuals, with a mean of 6.2 alleles per locus (range 4 10) and a mean observed heterozygosity of 0.58 (range ). These markers will allow determination of mating patterns and reproductive success of individuals within communally breeding groups The greater ani (Crotophaga major) practices a rare form of cooperative breeding in which several unrelated females lay eggs in one nest. Breeding groups are composed of socially monogamous pairs; however, the genetic mating system of this species has not been determined. Microsatellite loci have previously been developed for two related species (C. ani, Blanchard & Quinn 2001; and Guira guira, Muniz et al. 2003), but these loci are monomorphic in C. major. Here we describe the development of twelve polymorphic microsatellite loci for C. major, which will be used to determine mating patterns and reproductive success of individuals in nesting groups. Genomic DNA from a nestling greater ani from Barro Colorado Island, Panama, was used to create a DNA library enriched for microsatellites using the ligation procedure of Hamilton et al. (1999), with modifications following Grant & Bogdanowicz (2006). Briefly, blunt-ended DNA fragments were created by digestion with BsaA I and Hinc II, then ligated to SNX linker sequences and enriched for microsatellite sequences by a biotin-capture method using streptavidin-coated magnetic beads. Three libraries were

3 Page 3 of enriched in parallel using the dimeric, trimeric, and tetrameric biotinylated oligonucleotide repeat units described in Barnett et al. (2007). Captured fragments were amplified by polymerase chain reaction (PCR) using a primer complementary to the SNX linker (DYAD thermal cycler, Bio-rad), digested with Nhe I to remove the linker, and ligated into Xba I -digested puc 19 plasmids. Recombinant molecules were electroporated into competent Escherichia coli cells and transformed bacteria were screened with a 33 P-labelled probe of the oligonucleotide sequences used in the enrichment (Grant & Bogdanowicz 2006). We used PCR with universal M13 primers to amplify plasmid DNA from positive clones, then sequenced the products with BigDye Terminator version 3.1 Cycle Sequencing chemistry and an automated 3730xl DNA Analyser (Applied Biosystems). We sequenced 272 clones and designed primer pairs to amplify 30 microsatellite loci using the program Primer3 (Rozen & Skaletsky 2000). Primer pairs from 25 loci were successfully optimized and tested for variability on a panel of 16 unrelated greater ani nestlings from different nesting groups from Gatún Lake, Panama. We then selected twelve polymorphic loci to genotype 227 individuals from the same population (Table 1). For the initial screening for polymorphism, fluorescently labeled PCR products were created using the universal tag method of Schuelke (2000) with the conditions described in Makarewich et al. (2009). A pigtail (5 -GTTTCT) was attached to the reverse primer to ensure complete adenylation of the amplification products (Brownstein et al. 1996). Each PCR reaction contained ng DNA, 10 mm Tris-HCl, 50 mm KCl, 1.5 mm MgCl 2, 0.12 pm of the locus-specific forward primer, 1.2 pm of the universal forward primer, 1.2 pm of the locus-specific reverse primer, 0.2 U Taq polymerase

4 Page 4 of (Sigma), 200 µm dntps (Invitrogen), and H 2 O to bring the final volume to10 µl. Amplifications consisted of an initial denaturation (95 C, 4 min); 35 cycles of denaturation (95 C, 45 s), annealing (locus-specific annealing temperature, 60 s), and extension (72 C, 60 s); and a final extension of 5 min at 72 C. For the expanded genotyping, we amplified multiple loci in the same PCR reaction ( multiplexing ). Each locus-specific forward primer was fluorescently labeled at the 5 end (6-FAM, PET, NED, or VIC; Applied Biosystems) and was used with the pigtailed locus-specific reverse primer as described above. PCR conditions were as described above, with the following changes: i) 0.25 U Taq polymerase; ii) 3.25 mm MgCl 2 ; and iii) primer concentrations varied from 1 to 3 pm to yield similar fluorescent signals. PCR products were sized on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) with a GeneScan-500 LIZ molecular weight standard (Applied Biosystems) and GeneMapper 3.7 software (Applied Biosystems). We used GenePop 3.4 (Raymond and Rousset 1995) and Cervus 3.0 (Kalinowski et al. 2007) to determine observed and expected heterozygosity levels, conformation to Hardy-Weinberg proportions, null allele frequencies, and gametic disequilibrium between locus pairs. The number of alleles per locus ranged from 4 to 10 (mean = 6.2) with observed heterozygosities ranging from 0.22 to 0.8 (mean = 0.58; Table 2). Tests for gametic disequilibrium were not statistically significant after a Bonferroni correction for multiple comparisons. One locus, CrMa4-1, was not in HWE and had an estimated null allele frequency of 0.3. This may be due to the presence of alleles larger than 500 base pairs and therefore undetectable using a 500-bp size standard; however, it is possible that this locus could still be useful in genotyping analyses when used with a larger size standard.

5 Page 5 of The markers developed here will be used to determine mating patterns and reproductive success of individuals within communal groups, and will allow comparison of extra-pair copulation rates and reproductive skew between C. major and its congeners References Barnett JR, Stenzler LM, Ruiz-Gutierrez V, Bogdanowicz SM, Lovette IJ (2007) Isolation and characterization of microsatellite markers from the white-ruffed manakin Corapipo altera (Aves, Pipridae). Molecular Ecology Resources, 8, Blanchard L, Quinn JS (2001) The characterization of microsatellite loci in the communally breeding smooth-billed ani (Crotophaga ani). Molecular Ecology Notes, 1, Brownstein MH, Carpten JD, Smith JR (1996) Modulation of nontemplated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. BioTechniques, 20, Grant JB, Bogdanowicz SM (2006) Isolation and characterization of microsatellite markers from the panic moth, Saucrobotys futilalis L. (Lepidoptera: Pyralidae: Pyraustinae). Molecular Ecology Notes, 6, Hamilton MB, Pincus EL, Difiore A, Fleischer RC (1999) Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. BioTechniques, 27, Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program

6 Page 6 of CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology, 16, Makarewich CA, Stenzler LM, Ferretti V, Winkler DW, Lovette IJ (2009) Isolation and characterization of microsatellite markers from three species of swallows in the genus Tachycineta: T. albilinea, T. bicolor and T. leucorrhoa. Molecular Ecology Resources, 9, Muniz LSB, Macedo RHF, Graves J (2003) Isolation and characterization of dinucleotide microsatellite loci in communally breeding Guira cuckoos (Aves: Cuculidae). Molecular Ecology Notes, 3, Raymond M, Rousset F (2004) GenePop Version Updated from Raymond & Rousset (1995) GenePop version 1.2: population genetics software for exact tests and ecumenicism. Journal of Heredity, 83, Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology (eds. Krawetz S, Misener S), pp Humana Press, Totowa, NJ. Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology, 18, Acknowledgments We thank L. M. Stenzler, I. J. Lovette, and the Cornell Laboratory of Ornithology for assistance in microsatellite development and genotyping, and L. Jara for field assistance.

7 Page 7 of This work was supported by the Max Planck Institute for Ornithology, Princeton University, and a NSF Graduate Research Fellowship to C. Riehl

8 Page 8 of 9 Locus Table 1 Microsatellite loci developed in Crotophaga major Fluorescent Dye Label; Multiplex Combination Repeat Motif Primer Sequence (5 3 ) GenBank Accession no. CrMa3-27 VIC; 1 (CAA) 11 (TAA) 7 (CAAA) 5 F: GGTGCTTCCCAAACCTTAT R: TGCTTGTCATCCTCAAGTGG CrMa3-95 FAM; 1 (GTTT) 8 F: CATGCCTTGAACCCTGACTT R: GGGAGAATTCTTTTCCTTGGAC CrMa2-23 FAM; 2 (TC) 43 F: CAGTCTGAGGGAAGCCAAAT R: AAAACCAAACCAACACGAGAA CrMa2-24 PET; 2 (CA) 12 F: AGCAGAACTTTGCCCCTCTT R: ATGGCTGTCAAAATGCTGTG CrMa4-1 FAM; 3 (GAAA) 36 F: TTGCACAGGAAGAGTGGATG R: GTTGCACAGGAGGAAAAAGC CrMa4-6 FAM; 3 (GATA) 10 F: TTGCATGTATGGCATGAGGT R: TCTTCTGGCTGTGTGGATTTC CrMa3-25 VIC; 3 (CTT) 34 F: GTGGGAAAGCTTGAAGAGCA R: CAGAGGTTAAGCTCAAGGAAAA CrMa3-142 NED; 4 (GTT) 18 F: TCCAGTTCCACCTCCCACTA R: GGAAAGAATCTCAGCTTTTGCT CrMa3-37 PET; 4 (GAA) 14 (CAA) 6 (GAA) 15 F: TTCTTACTCTGCGACTGAGCAC R: GTGGGAAAGCTTGAAGAGCA CrMa3-45 VIC; 4 (GTT) 16 (GTTT) 4 F: AATGGTTCGGGTGTGAAGAG R: AGGGTCCAATTTCTGCAGTG CrMa3-8 PET; 5 (CAA) 17 F: AAGTGCCATACATGCTGCTG R: GCAGGTTCCAGTCATTGCTC CrMa3-104 NED; 5 (TAG) 5 CTG(TAG) 3 (GTT) 11 F: TCATGCTCTGGTCCATCATC R: TTGGACTTCTGGTACTGTGTCC GQ GQ GQ GQ GQ GQ GQ GQ GQ GQ GQ GQ144426

9 Page 9 of 9 Table 2 Characteristics of microsatellite loci amplified in Crotophaga major. Ta, optimized annealing temperature; MgCl 2, optimized concentration; n, number of individuals genotyped; N A, number of alleles; H O, observed heterozygosity; H E, expected heterozygosity. Locus T a ( C) MgCl 2 (m) Allele size n N A H O H E range (bp) CrMa CrMa CrMa CrMa CrMa4-1* CrMa CrMa CrMa CrMa CrMa CrMa CrMa * Indicates locus not in HWE