Isolation and characterization of polymorphic ... - MacManes Lab

Molecular Ecology Resources (2009)

doi: 10.1111/j.1755-0998.2009.02536.x

PERMANENT GENETIC RESOURCES NOTE Blackwell Publishing Ltd

Isolation and characterization of polymorphic microsatellite loci from Octodon degus Y. F. Q U A N ,* M . D . M A C M A N E S ,† L . A . E B E N S P E R G E R ,‡ E . A . L A C E Y † and L . D . H AY E S * *Department of Biology, University of Louisiana at Monroe, Monroe, LA 71209, USA, †Museum of Vertebrate Zoology, University of California, Berkeley, 3101 Valley Life Sciences Building, Berkeley, CA 94720, USA, ‡Facultad de Ciencias Biológicas and Center for Advanced Studies in Ecology and Biodiversity, Pontificia Universidad Católica de Chile, 6513677 Santiago, Chile

Abstract Quantifying genetic kinship and parentage is critical to understanding the adaptive consequences of sociality. To measure fitness in a species with variable group structure, we isolated 14 microsatellite loci from Octodon degus, a semi-fossorial rodent endemic to Chile. The number of alleles per locus ranged from four to 14. Thirteen loci were in Hardy–Weinberg proportions, with values of observed heterozygosity ranging from 0.550 to 0.950. These markers provide the basis for future studies of the direct fitness consequences of sociality in O. degus. Keywords: fitness, microsatellite markers, Octodon degus, parentage Received 29 October 2008; revision accepted 3 December 2008

Degus (Octodon degus) are burrow-dwelling rodents (family Octodontidae) inhabiting areas of open, savannah habitat in central Chile (Woods & Kilpatrick 2005). Degus live in social groups of up to nine adults that include related and unrelated individuals (Ebensperger et al. 2004; Lacey & Ebensperger 2007). Current estimates of direct fitness based on the per capita production of offspring suggest that group living is costly to females (L. D. Hayes & L. A. Ebensperger, unpublished data), but such estimates may be misleading because they fail to detect important within-group differences in direct fitness (i.e. reproductive skew). Consequently, molecular data are required to provide more precise information regarding fitness in degus. The objective of this study was to develop microsatellite markers for degus that will allow accurate measures of parentage and direct fitness in this species. Microsatellite loci were isolated following a magnetic bead enrichment protocol (Glenn & Schable 2005). Genomic DNA was extracted from ear tissue collected from a single female using a salt extraction protocol developed by M.D.M. (MVZ EGL 2008). Approximately 2 μg of whole genomic DNA was digested using 10 U Rsa1 restriction enzyme (New England Biolabs), after which double-stranded SNX linkers were ligated to both ends of each fragment. Ten microlitres of linker-ligated digest was hybridized to each of following Correspondence: L. D. Hayes, Fax: 318 3421779; E-mail: [email protected]

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three biotinylated probe mixtures (1 μm): [(AG)12, (TG)12, (AAC)6, (AAG)8, (AAT)12, (ACT)12, (ATC)8], [(AAAC)6, (AAAG)6, (AATC)6, (AATG)6, (ACAG)6, (ACCT)6, (ACTC)6, (ACTG)6] and [(AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8]. Fragments containing microsatellite regions were captured with Streptavidin M-280 Dynabeads (Invitrogen) and recovered using the polymerase chain reaction (PCR) conditions described in Glenn & Schable (2005). Microsatellite-enriched PCR products were cloned using a TOPO TA cloning kit (Invitrogen). Two hundred and twenty positive (white) clones were amplified using M13 forward with either M13 reverse or T7 primers and sequenced with an ABI 3730 sequencer (Applied Biosystems). Sequence data were analysed using Sequencher 4.8 (Gene Codes). Unique microsatellite regions with sufficient (> 50 bp) flanking region sequences were found in 24 clones. Locusspecific PCR primers were designed using the program Primer 3 (Rozen & Skaletsky 2000). PCR amplification of loci was conducted in 15-μL reactions consisting of 1× PCR buffer [20 mm Tris-HCl (pH 8.4), 50 mm KCl], MgCl2, dNTPs, forward and reverse primer, Taq polymerase (NEB) and gDNA template (20 ng/μL); the final, locus-specific concentrations of these reagents are given in Table 1. Amplification was conducted using an iCycler (Bio-Rad) programmed for 5 min at 94 °C, followed by 35 cycles of 94 °C for 30 s, the locus-specific annealing temperature (Table 2) for 30 s, and 72 °C for 45 s, with a final extension of 72 °C for 5 min. Following amplification, 17 loci consistently yielded clear

2 PERMANENT GENETIC RESOURCES NOTE Table 1 Final concentrations of reagents used in the optimized PCRs for each microsatellite locus. In all cases, total reaction volume was 15 μL Final PCR concentrations

Locus

Mg2+ (mm)

dTNPs (mm)

Primer Taq polymerase F/R (μm) (unit)

DNA (ng)

OCDE1 OCDE2 OCDE3 OCDE4 OCDE5 OCDE6 OCDE7 OCDE8 OCDE9 OCDE10 OCDE11 OCDE12 OCDE13 OCDE14

2.00 2.00 2.00 2.00 1.75 1.75 1.75 1.25 1.50 1.25 1.75 1.75 1.50 1.75

0.8 0.8 0.8 0.8 0.4 0.4 0.4 0.2 0.2 0.2 0.4 0.4 0.2 0.4

0.80 0.80 0.80 0.80 0.53 0.53 0.53 0.40 0.53 0.53 0.53 0.53 0.53 0.40

30 30 30 30 30 30 30 50 30 40 30 50 20 50

0.75 0.75 0.75 0.75 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50

bands in the anticipated size range when electrophoresed on 1% agarose gels and visualized with ethidium bromide. To assess variability at these loci, whole genomic DNA from 20 degus belonging to five social groups at Rinconada de Maipú, Chile was amplified with fluorescently labelled primers (Applied Biosystems, Invitrogen and Integrated DNA Technologies) using the above PCR conditions. Genotyping was performed on an ABI 3730 using 0.5 μL of PCR product and 0.2 μL of GS500LIZ size standard (Applied Biosystems) per sequencing well. Fourteen loci produced consistent, distinct peaks in the resulting electropherograms. Allele sizes were determined using GeneMapper (Applied Biosystems). All 14 loci were polymorphic (0.95 criterion, Hartl & Clark 1997), with the number of alleles per locus ranging from four to 14 (mean = 8.4 ± 2.4; Table 2). Observed and expected heterozygosities were estimated and tests of Hardy–Weinberg proportions and gametic disequilibrium were conducted using GenePop (Raymond & Rousset 1995). Observed heterozygosities ranged from 0.200 to 0.950. One locus (OCDE4) deviated significantly from Hardy–Weinberg expectations (Table 2). Significant gametic disequilibrium

Table 2 Characteristics of 14 microsatellite loci isolated from Octodon degus including repeat motif, 5′–3′ primer sequence, annealing temperature (°C) and PCR product size (bp)

Locus

Repeat motif

Primer sequence (5′–3′)

OCDE1 OCDE2

(CTTT)7CTCT (CTTT)10 (CA)13

OCDE3

(AAGG)9(AAAG)19

OCDE4

(CTTC)12(CTTT)14

OCDE5 OCDE6

(GAAA)11GAGA (GAAA)7 (CA)15

OCDE7

(GAAA)15

OCDE8

(TG)19

OCDE9

(GA)23

F: HEX-CTAGGTGCCAGAGACCCTTG R: CAAAGACCCTGGGTTCAATC F: HEX-GTTCGAGCTGCCTAGTGAGG R: ACTGGACATGGTGGTGTGTG F: HEX-GACACCTGTCTGGAAAATACAAGAT R: TGCTTGTCATGTCATGCTGT F: HEX-GAGCATTGGAGGTAGCAAGC R: AGATGATCAATGACAGATGATGC F: PET-CAAAGACCCTGGGTTCAATC R: CATGATTGAGCTTGCCTCTG F: NED-TGGGGAAATGTAAATGTAGG R: GAGAAGAACAACGTGCAGAT F: PET-CAAGCTTGTCAAAGCACAGG R: GGCAGAAAATTCTGGACAGG F: FAM-CTTTGTTGCAGTGGTGTGAT R: GGCTCAGTGGAATAAGCACT F: HEX-CATGTAGTTTTCCAGGCACT R: TTCCTCCACTTTCTGACAAT F: FAM-AAGGCAGCAGTTGGGAGAACAA R: TGAGATTGTCCTTTGAGTCCACATGA F: NED-TAGGAAGGAAAGGAGCTGGA R: CAACAAGCTCGGGTGATTTA F: HEX-GCAGAGCTAAGGACTAAAGGTTCCA R: CCAAGTTGCTAAGAGGTCCCTTG F: FAM-TGCAAGAAGCGCCCAGATATT R: TTTTTCATGGCAAGTGGCAGAT F: FAM-GCTCTGGGGGCAATCAATATTCT R: AAACCACTACTTCTGCACTGTTCCA

OCDE10 (TG)13 OCDE11 (CA)5TATA(CA)4 GAGACAAATA(CA)20 OCDE12 (GT)15 OCDE13 (GT)18 OCDE14 (GT)20(TG)2

Ta PCR product (°C) size (bp)

NA

HO

HE

P

GenBank Accession no.

65

150–178

9

0.950 0.878

0.9805

FJ418930

64.5

205–214

7

0.800 0.745

0.0821

FJ418931

62

286–320

10

0.800 0.830

0.2857

FJ418932

64.5

228–274

14

0.200 0.900

0.0000*

FJ418933

58

199–227

10

0.850 0.877

0.5412

FJ418934

58

198–204

4

0.550 0.645

0.3421

FJ418935

64

201–229

9

0.850 0.836

0.7969

FJ418936

58.5

158–185

11

0.650 0.814

0.1193

FJ418937

61

172–188

7

0.900 0.800

0.0578

FJ418938

65

156–179

8

0.800 0.729

0.8304

FJ418939

58.5

165–176

6

0.800 0.726

0.8676

FJ418940

63

173–217

9

0.950 0.854

0.9683

FJ418941

58

149–163

7

0.800 0.796

0.8639

FJ418942

63.5

150–168

7

0.950 0.842

0. 1996

FJ418943

Number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), Hardy–Weinberg P value, and GenBank Accession number. Data on variability are based on analyses of 20 adult degus from Rinconada de Maipú, Chile. *indicates a significant deviation from Hardy–Weinberg expectations.


PERMANENT GENETIC RESOURCES NOTE 3 was detected between two pairs of loci (OCDE5 and OCDE7, OCDE10 and OCDE14) after sequential Bonferroni correction of P values (both P’s ≤ 0.0001). Applying MicroChecker (van Oosterhout et al. 2004) revealed no evidence of null alleles or allelic dropout for 13 loci, although the excess of homozygotes at OCDE4 may reflect the presence of null alleles. Even if OCDE4 and two of the four loci in gametic disequilibrium are excluded from subsequent analyses, the current study produced 11 highly polymorphic loci that should allow accurate determination of parentage and direct fitness in free-living degus.

Acknowledgements LAE was funded by FONDECYT grants 1020861 and 1060499. LDH was funded by NSF (grant #0553910) and the Louisiana Board of Regents (LINK-2006 and LEQSF 2007-09-RD-A-39).

References Ebensperger LA, Hurtado MJ, Soto-Gamboa M, Lacey EA, Chang AT (2004) Communal nesting and kinship in degus (Octodon degus). Naturwissenschaften, 91, 391–395.


Glenn TC, Schable NA (2005) Isolating microsatellite DNA Loci. Methods in Enzymology, 395, 202–222. Hartl DL, Clark AG (1997) Principles of Population Genetics, 3rd edn. Sinauer & Associates, Sunderland, Massachusetts. Lacey EA, Ebensperger LA (2007) Social structure in octodontid and ctenomyid rodents. In: Rodent Societies: An Ecological and Evolutionary Perspective (eds Wolff JO, Sherman PW), pp. 403– 415. University of Chicago Press, Chicago, Illinois. Museum of Vertebrate Zoology Evolutionary Genetics Lab (2008) MacManes Salt Extraction Protocol. http://mvz.berkeley.edu/egl/ protocols/extraction/MacManesSaltExtraction.pdf [Accessed 17 October 2008]. van Oosterhout CV, Hutchinson WF, Wills DPM, Shipley P (2004) Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology, 4, 535–538. Raymond M, Rousset F (1995) GenePop (version 1.2): population genetics software for exact tests and ecumenicism. Journal of Heredity, 86, 248–249. Rozen S, Skaletsky H (2000) Primer 3 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. 365–386. Humana Press, Totowa, New Jersey. Woods CA, Kilpatrick CW (2005) Infraorder Hystricognathi. In: Mammal Species of the World: A Taxonomic and Geographic Reference, 3rd edn (eds Wilson DE, Reeder DM), pp. 1538–1599. The Johns Hopkins University Press, Baltimore, Maryland.