Conservation Genet Resour DOI 10.1007/s12686-009-9094-8

TECHNICAL NOTE

Isolation, characterization and cross-species amplification of new microsatellite markers for three species of the Didelphidae family

Isabela M. G. Dias Æ George Amato Æ Heitor M. Cunha Æ Rob DeSalle Æ Adriano P. Paglia Æ Jennifer K. Peterson Æ Cleusa G. Fonseca

Received: 23 August 2009 / Accepted: 29 August 2009 Ó Springer Science+Business Media B.V. 2009

Abstract The American from the Didelphidae Keywords Didelphidae Microsatellite Caluromys family (Order: Didelphimorphia) are on the IUCN Red Metachirus MicoureÁ us OpossumÁ Á Á Á Á List, and their threatened status varies from extinct to data deficient. Although microsatellites have already been developed for some species, only a small amount of pop- The American opossums from the Didelphidae family ulation genetic analysis of the didelphid wild populations (Order: Didelphimorphia) include 19 genera and 95 cur- has been made using them. In this work, we developed new rently recognized species, most of them occurring in South microsatellites and tested for cross-species amplification America. They are divided into two subfamilies: Calur- for one species from the subfamily , Cal- omyinae, which includes five species in three genera, and uromys philander, and two from the subfamily Didelphi- Didelphinae, which includes 90 species in 14 genera. The nae, Didelphis aurita and Metachirus nudicaudatus, to members of this family are small- to medium-sized mam- determine the degree of gene flow and isolation of popu- mals, and most of them are nocturnal and insectivorous– lations of those species in some fragments of the Atlantic frugivorous or omnivorous (Gardner 2007). All of them are in Minas Gerais state, . Among all microsat- on the IUCN Red List, and their threatened status varies ellite loci amplified, 18 from C. philander, 9 from from extinct to data deficient (IUCN 2008). Although D. aurita, and 17 from M. nudicaudatus were polymorphic. microsatellites have already been developed for Didelphis Cross-species amplification of these polymorphic micro- virginiana (43 loci; Faircloth et al. 2008), Didelphis mar- satellite loci were performed in these three species and in supialis (10 loci; Lavergne et al. 1999), Monodelphis one additional related species Micoureus paraguayanus. domestica (122 loci; Samollow et al. 2004, 2007), Micou- Our data showed that microsatellites were not highly reus demerarae (8 loci; Dias et al. 2008), Micoureus par- conserved among Didelphidae species. aguayanus (5 loci; Rodrigues et al. 2006), and Philander opossum (4 loci; Guillemin et al. 2000), only a small amount of population genetic analysis of the didelphid wild populations has been made using those microsatellites (Rodrigues et al. 2006; Dias et al. 2008). A lot of work still I. M. G. Dias H. M. Cunha C. G. Fonseca Departamento Áde Biologia Geral,Á ICB, Universidade Federal needs to be done regarding the real threat that those species de Minas Gerais, Belo Horizonte, MG 31270-010, Brazil are facing in the Atlantic Forest in Brazil. In this study, we developed microsatellites and tested for cross-species I. M. G. Dias (&) G. Amato R. DeSalle J. K. Peterson amplification for one species from the subfamily Calur- Sackler Institute forÁComparativeÁ Genomics, ÁAmerican Museum of Natural History, 79th Street at Central Park West, omyinae, Caluromys philander, and two from the sub- 10024 New York, NY, USA family Didelphinae, Didelphis aurita and Metachirus e-mail: [email protected]; [email protected] nudicaudatus, all of which coexist in the Atlantic Forest. Our goal is to determine the degree of gene flow and iso- A. P. Paglia Conservation International Brazil, Belo Horizonte, lation of populations of those species in some fragments of MG 30112-021, Brazil the Atlantic Forest in Minas Gerais state, Brazil.

123 Conservation Genet Resour

Table 1 Characteristics of 44 microsatellite loci in three species of mouse opossums from Didelphidae family: C. philander, D. aurita, and M. nudicaudatus

Locus/ Primer sequence (50-30) Repeat motif Ta Size Na HO HE PIC accession (°C) range (bp)

Caph01 CAGTCGGGCGTCATCATCCCTCCTAAGGACCAGTCT (GT)22 53 194– 11 0.842 0.910 0.876 236 GQ465285 GTTTATTTCCCTCCACTTCAATCC

Caph05 CAGTCGGGCGTCATCAGGCTGATCCCAGAGAAGTTG (CA)12 58 207– 3 0.105 0.199 0.185 217 GQ465286 GTTTCTCGGGCTGTCTCTGTCTT

Caph07 CAGTCGGGCGTCATCAGACTTCCTAGCCATGTGACC (AG)20 59 170– 11 0.842 0.883 0.846 208 GQ465288 GTTTCCAATCTCTTCCCTTTAGCC

Caph08 CAGTCGGGCGTCATCACTCTCTTCTCCCCATCCATT (TC)25(AC)4 54 196– 10 0.526 0.526 0.526 240 GQ465289 GTTTATTCCAAAGGACTCATCACG

Caph10 CAGTCGGGCGTCATCAGGGTCTTTGCTCCAGAAATC (TC)27 57 209– 6 0.150 0.319 0.300 271 GQ465290 GTTTCACTTAACCCCAGTTGCCTA

Caph11 CAGTCGGGCGTCATCAGGACAAGGGGGTTTTGTTAG (GT)10GA(GT)9 56 235– 9 0.650 0.758 0.701 287 GQ465291 GTTTAAGAGGGCAAAGAAGCATTT

Caph13 CAGTCGGGCGTCATCATTGGGGTATTTCAAAGGACA (TG)12 55 265– 4 0.444 0.559 0.480 281 GQ465292 GTTTCCTGCTCTTCTTAGCTTGC

Caph16 CAGTCGGGCGTCATCACAAGCCTGACTTGAACAGG (GA)13 54 215– 7 0.222 0.397 0.375 243 GQ465293 GTTTCTGCTTGGCTAATCATCTCC

Caph17 CAGTCGGGCGTCATCAGATTGCAGCCTTTTGAGAAA (AC)13 54 242– 5 0.526 0.676 0.619 260 GQ465294 GTTTAGCACTCCAGTGTGGAGAAA

Caph18 CAGTCGGGCGTCATCAATTGATGGGTGCCTATT (AC)21 54 148– 8 0.650 0.753 0.706 178 GQ465295 GTTTGACCCAGAGATCAAATCAAA

Caph19 CAGTCGGGCGTCATCAGAGCAAAAGGGACTGAGA (GT)16 53 222– 4 0.154 0.502 0.445 234 GQ465296 GTTTCCCTTGATTCCATCTCCTCT

Caph21 CAGTCGGGCGTCATCAGACGCTCATCTGCCAGTAGT (AC)16 53 224– 4 0.333 0.576 0.491 242 GQ465297 GTTTACCAAAAGATTGGGGAAGAA

Caph22 CAGTCGGGCGTCATCAAAGGTAAGCTCAATGGTCT (CT)22 54 197– 14 0.833 0.922 0.888 231 GQ465298 GTTTAGGCAACACAAATTTGAAGC

Caph27 CAGTCGGGCGTCATCAGGGGTATCAAACTCCATCC (CT)12 54 233– 6 0.737 0.711 0.638 255 GQ465299 GTTTCAGCATGAAAACATCTATGAAGA

Caph28 CAGTCGGGCGTCATCAGCATGAAAACATCTATGAAGA (AC)12 54 233– 6 0.700 0.724 0.651 251 GQ465300 GTTTAGGGGTATCAAACTCCATCC

Caph29 CAGTCGGGCGTCATCATCCATTTGGAACGTGATTCT (AC)22 54 149– 9 0.737 0.853 0.811 179 GQ465301 GTTTAGGGAGGAGAAAACCGAGT

Caph31 CAGTCGGGCGTCATCATGCACACATAGAGCGAGAAA (CA)8 54 238– 3 0.000 0.190 0.177 254

123 Conservation Genet Resour

Table 1 continued

Locus/ Primer sequence (50-30) Repeat motif Ta Size Na HO HE PIC accession (°C) range (bp)

GQ465302 GTTTGTATCGACTGACACGGAGA

Caph33 CAGTCGGGCGTCATCATGAGCACATATTCCCGAAA (CA)8 54 172– 5 0.389 0.459 0.412 186 GQ465303 GTTTATCACAGGAGTGGAGCTCTG

Daur02 CAGTCGGGCGTCATCACCTTGGAACCAACACACAGT (AG)6AA(AG)5 65 263– 3 0.840 0.513 0.395 AT(AG)5 273 GQ465304 GTTTGAGTATCTTTTTCACCTTCATTT

Daur05 CAGTCGGGCGTCATCATCTGTATTCATGGAACCTGGA (TTCC)9T(TTTC)18 62 242– 9 0.320 0.843 0.805 (TTCC)9 336 GQ465305 GTTTCACCACCCTGGTCTAAC

Daur06 CAGTCGGGCGTCATCAGCTCTTTCTATAGATGCCAGCTC (GTA)10(GCA)7 54 254– 4 0.680 0.509 0.423 275 GQ465306 GTTTGAGCCCCTCTCTTGAGGT

Daur08 CAGTCGGGCGTCATCACCAATCAAAAATGCAGGAAC (AC)14 56 193– 8 0.760 0.830 0.789 211 GQ465307 GTTTCCCTCCAACTTTTCACTT

Daur09 CAGTCGGGCGTCATCATGCCTGGCATTATTACTGGT (GT)20 54 212– 9 0.720 0.833 0.795 260 GQ465308 GTTTCAGCAGGTTCAGCAAGAGAT

Daur10 CAGTCGGGCGTCATCATAGGTTCAAATCTGGCCTCA (AAC)7 60 197– 4 0.080 0.118 0.113 263 GQ465309 GTTTAAATCACATCTGTCCATCCA

Daur11 CAGTCGGGCGTCATCAGCTGATGCAGACACACCTAA (AC)17 54 220– 12 0.920 0.851 0.815 274 GQ465310 GTTTAATAAGGCTCATGCCTGTGT

Daur12 CAGTCGGGCGTCATCACCTACTGGTGGGTCTGTTTG (CA)18 60 199– 10 0.560 0.766 0.717 225 GQ465311 GTTTACTGGAGAGGGTGGGAAGT

Daur13 CAGTCGGGCGTCATCACCTTTCCAGGGAGAGAGAAG (TG)18 56 258– 14 0.800 0.852 0.820 294 GQ465312

Mnud20 CAGTCGGGCGTCATCAGCTCTTTCAGATGGACAGC (GT)11 54 161– 3 0.021 0.063 0.061 221 GQ465313 GTTTGGCCGTATACAAGCACAGAA

Mnud21 CAGTCGGGCGTCATCAGGCTGCACAGTTTTTCAAAT (AC)9 54 187– 2 0.362 0.491 0.368 217 GQ465314 GTTTCATCCTCTCTAGGAAGCAAA

Mnud23 CAGTCGGGCGTCATCATGGATAAAAGGAAACACAGACA (AC)16T(CA)5 54 228– 10 0.660 0.805 0.774 250 GQ465315 GTTTCACTCTGACTGCTCCCAAT

Mnud24 CAGTCGGGCGTCATCATTGCTTTGTCCCAAAAACTT (CA)21 54 187– 7 0.638 0.571 0.501 207 GQ465316 GTTTTATTCTTCCCAATGAATGC

Mnud33 CAGTCGGGCGTCATCAGACAGAAGAATGGCCAAGAA (TG)17 56 232– 10 0.809 0.834 0.803 250 GQ465317 GTTTCCCCCATTAAGAAAGGAAGA

Mnud37 CAGTCGGGCGTCATCATTTCTTCACCACTTCCAAGC (GT)24 54 201– 13 0.809 0.835 0.812 239 GQ465318 GTTTAGGCCCTGTCACATACCTTA

Mnud38 CAGTCGGGCGTCATCAGAAAGAGTGGGACATGATGC (ATG)8 55 217– 4 0.319 0.280 0.253 247

123 Conservation Genet Resour

Table 1 continued

Locus/ Primer sequence (50-30) Repeat motif Ta Size Na HO HE PIC accession (°C) range (bp)

GQ465319 GTTTCTTTGCAGGCCTTTGAAAT

Mnud40 CAGTCGGGCGTCATCACCACTCTTTGATAAGTATGGGACT (TCTG)2(TCTA)12 56 213– 11 0.894 0.863 0.838 239 GQ465320 GTTTGAGCTGAGTATGGAAGGAAGC

Mnud41 CAGTCGGGCGTCATCATTGTTTCCCATCCATTAGGA (GA)5 GT (GA)22 56 175– 19 0.915 0.925 0.909 237 GQ465321 GTTTCTGGTGCAAAATAGGTGCTT

Mnud42 CAGTCGGGCGTCATCAGAGGTAGGAAGGGACAAAGC (TG)10 56 263– 16 0.830 0.892 0.872 313 GQ465322 GTTTAGCCTCTTAACCTTGGAGGA

Mnud43 CAGTCGGGCGTCATCAGCATGCATTTGTATTGCAC (TGC)5 (TAC)6 56 203– 6 0.128 0.163 0.159 269 GQ465323 GTTTCTATGCGACTCTGGACAAGC

Mnud47 CAGTCGGGCGTCATCATGAGAAACCACGTGTGACAG (AC)8AAA(AC)7 54 182– 8 0.787 0.755 0.709 236 GQ465324 GTTTCCCCTCCACTTACTCCATTT

Mnud49 CAGTCGGGCGTCATCAAGTCTTGCCAAGTGTGGAG (AC)20 54 194– 10 0.766 0.789 0.751 216 GQ465325 GTTTGGCCTTTTAGTTCTGTTCG

Mnud50 CAGTCGGGCGTCATCACTCCAGAAATTTGCCAAGAA (ATG)15 54 135– 7 0.617 0.677 0.615 207 GQ465326 GTTTGAGACCATCTACTTTAACCTTCTCA

Mnud52 CAGTCGGGCGTCATCAGACCTTGGAGCATCTCTTCA (GT)18 58 126– 14 0.489 0.873 0.850 214 GQ465327 GTTTGTAAGGGGCAATCCATTCTT

Mnud53 CAGTCGGGCGTCATCAGGGGGAGGTACATCTAGTCA (GT)16 54 213– 17 0.894 0.920 0.903 259 GQ465328 GTTTGGTTCATTCATGCAACCAGT

Mnud56 CAGTCGGGCGTCATCAGGGAATGAAGCTAATGCAAA (AC)16 G (CA)3 54 230– 5 0.681 0.734 0.679 250 GQ465329 GTTTACCCCAACAGACAAAAACAT

Locus name and GenBank accession number; primer sequences; repeat motifs; Ta (°C), annealing temperature; size range (bp); Na, number of alleles in each locus; HO, observed heterozygosity; HE, expected heterozygosity; PIC polymorphism information content

Two samples were obtained from the tissue collection SuperSNX linkers, PCR reactions were performed with the of the Mammalogy Laboratory in ICB/UFMG—Brazil DNA fragments of each sample to ensure successful liga- (D. aurita, ID: HMC019 and C. philander, ID: HMC023, tion (Glenn and Schable 2005). The PCR products were both from RPPN Mata Samuel de Paula, Nova Lima, enriched with a mix of 12 biotinylated oligonucleotide MG, Brazil), and one was accessioned in the Ambrose from Dias et al. (2008), using Dynabeads (Dynal, Oslo, Monell Cryo Collection at AMNH (M. nudicaudatus, ID: Norway). PCR products were used for cloning with Topo AMCC109511, from Rio Galvez, Loreto, ). The TA Cloning Kit 45-0641 pCRÒ 2.1, One Shot TOP 10F genomic DNA was extracted using QIAGEN DNeasyÒ Chemically Competent cells and imMediaTM agar plates Tissue kit according to the manufacturer’s instructions. following manufacturer’s protocols (Invitrogen). A total of DNA concentration was estimated by electrophoresis on a 576 positive colonies were selected for each species. After 0.8% agarose gel stained with Invitrogen SYBRÒ Safe. incubating at 95°C for 10 min in 200 ll of TE (10 mM Our libraries were constructed according to the Glenn and Tris–HCl, 0.1 mM EDTA, pH = 8.0), PCRs were per- Schable (2005) protocols and some modifications described formed using M13 forward and reverse primers (Glenn and in Dias et al. (2008). Concisely, after digested with RsaI Schable 2005). PCR products were checked for inserts of enzime (New England BioLabs, Inc.) and ligated to desired size range (400–800 bp) by electrophoresis of 1.0%

123 Conservation Genet Resour agarose gel with Invitrogen SYBRÒ Safe DNA gel stain, Beads according to manufacturer’s instructions (GE Heal- and were sequenced using BigDyeÒ Terminator method thy Care, Amersham Bioscience, 2004), 25 from C. phi- (Applied Biosystems Inc.) on an ABI 3730xl sequence lander, 15 from D. aurita, and 24 from M. nudicaudatus analyzer. After edited in Sequencher 4.1 (Gene Codes had successful amplification. To identify which ones were Corp.), the sequences were run using Simple Sequence polymorfic, the CAG-tag (50-CAGTCGGGCGTCATCA- Repeat Identification Tool (Temnykh et al. 2001). Primers 30) and the GTTT-tail (50-GTTT-30) were added to the 50 were designed for the promising loci from each species end of each shorter and longer primer, respectively, omit- using Primer 3 (Rozen and Skaletsky 2000): 46 loci from ting bases that were identical to the 30 end of the CAG-tag C. philander, 15 loci from D. aurita, and 57 loci from or GTTT-tail (see Table 1). Inclusion of the 50-tag allowed M. nudicaudatus. the use of a third primer in the PCR (CAG-tag: 50-CAGT Primers pairs of each locus were tested, optimized, and CGGGCGTCATCA-30) that was FAM fluorescently evaluated for polymorphic content using DNA samples labeled. This primer is used to decrease the cost of indi- from 20 individuals from C. philander, 25 individuals from vidual fluorescent-labeled primers (Brownstein et al. 1996; D. aurita, and 50 individuals from M. nudicaudatus. All Shuelke 2000). PCRs were conducted in Eppendorf individuals were sampled in two small fragments of the Gradient S—Mastercycle machines and standard cycling Atlantic Forest in Minas Gerais, Brazil, using live traps, as conditions were: a denaturation step of 95°C for 2 min; part of a Long-Term Ecological Research Project (Atlantic 4 cycles of 95°C/30 s, Ann. temp. ?1°C/1 min and 72°C/ Forest and Lacustrine Ecosystems of the Middle Rio Doce 1 min; 4 cycles of 95°C/30 s, Ann. temp. -2°C/1 min and River/PELD). Genomic DNA was isolated from blood or 72°C/1 min; 8 cycles of 95°C/30 s, Ann. temp. -5°C/ mouth-swab samples using simplified Proteinase K-saline 1 min and 72°C/1 min; 10 cycles of 95°C/30 s, Ann. temp. precipitation protocol (Sambrook and Russel 2001). To test -7°C/1 min and 72°C/1 min; 12 cycles of 95°C/30 s, Ann. for cross-species amplification, additional eight Micoureus temp. -9°C/1 min and 72°C/1 min; and a final extension paraguayanus DNA samples from Dias et al. (2008) were step at 72°C for 20 min. Genotyping with fluorescent used. labeled CAG-tag primer on an ABI 3730xl sequence ana- Among all microsatellite loci amplified in a 25 ll lyzer and GeneMapper 3.5 software (Applied Biosystems reaction volume using PuReTaqTMReady-To-GoTM PCR Inc.) resulted in some informative loci, which conditions

Table 2 Cross-species amplification of 43 microsatellite loci in four mouse opossum species from Didelphidae family, using eight samples for each species Caph01 Caph05 Caph07 Caph08 Caph10 Caph11 Caph13 Caph16 Caph17 Caph18 Caph19

C. phylander ?* ?* ?* ?* ?* ?* ?* ?* ?* ?* ?* D. aurita ? ? ? ? ? ?* ? ?* - ?* - M. nudicaudatus - - ? ? - ?* ?* - - ?* - M. paraguayanus ?* - ? ?* - ?* ?* - - ?* - Caph21 Caph22 Caph27 Caph28 Caph29 Caph31 Caph33 Daur02 Daur05 Daur06 Daur08 C. phylander ?* ?* ?* ?* ?* ?* ?* ? - - ?* D. aurita - ? - - - ? - ?* ?* ?* ?* M. nudicaudatus - ? - - - - - ?* - - - M. paraguayanus ------?* - Daur09 Daur11 Daur12 Daur13 Mnud20 Mnud21 Mnud23 Mnud24 Mnud33 Mnud37 Mnud38 C. phylander - - - ? ? ?* - - ? ?* ? D. aurita ?* ?* ?* ? ? ?* - ? ?* - ? M. nudicaudatus ?* ? ? ?* ?* ?* ?* ?* ?* ?* ?* M. paraguayanus - - - ?* ?* ?* - - - ?* - Mnud40 Mnud41 Mnud42 Mnud43 Mnud47 Mnud49 Mnud50 Mnud52 Mnud53 Mnud56 C. phylander - ?* ?* ?* ?* ? - - - - D. aurita ? ? - - - ?* - - ?* ? M. nudicaudatus ?* ?* ?* ?* ?* ?* ?* ?* ?* ?* M. paraguayanus - ?* ?* ?* - ?* ? - - ?* ?* amplification, not variable; ? amplification, variable (two or more alleles); - no amplification

123 Conservation Genet Resour and characteristics were given in Table 1. CERVUS loci in the woolly mouse opossum, Micoureus paraguayanus version 3.0 (Kalinowski et al. 2007) was used to estimate isolated from Micoureus demerarae. Mol Ecol Resour 8:345–347 Faircloth BC, Shamblin B, Eo SH et al (2008) Microsatellites from the number of alleles per locus, observed and expected the Virginia opossum (Didelphis virginiana). In: NCBI Entrez heterozygosity (HO and HE) and polymorphic information Nucleotide. http://www.ncbi.nlm.nih.gov/sites/entrez of subor- content (PIC) for each locus, after Bonferroni correction dinate document. Accessed Aug 12, 2009 for all analysis performed (Table 1). Some loci showed Gardner LA (2007) of volume 1: Marsu- pials xenarthrans, shrews, and bats. The University of Chicago significant deviation from Hardy–Weinberg equilibrium Press, Chicago, IL (P \ 0.01): Caph05, Caph19, Daur02, Daur05, Daur09, Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. Daur12, Daur13, Mnud20, Mnud21, Mnud23, Mnud42, Methods Enzymol 395:202–222 and Mnud52. The deviations from HWE are not unex- Guillemin ML, Lavergne A, Catzeflis F (2000) Microsatellite markers in the common grey four-eyed opossum (Philander opossum pected given that our samples did not come from a single Didelphidae, Marsupialia). Mol Ecol 9(9):1440–1442 breeding population. Linkage disequilibrium was detected IUCN (2008) 2008 IUCN Red list of threatened species. http://www. between loci Daur05 and Daur13, and between loci iucnredlist.org. Accessed Aug 12, 2009 Mnud41 and Mnud49 (P \ 0.01), using Genepop 4.0 Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error (Rousset 2008). increases success in paternity assignment. Mol Ecol 16:1006–1099 Cross-species amplification of all reliable microsatellites Lavergne A, Douady C, Catzeflis FM (1999) Isolation and characteriza- among those three species and in M. paraguayanus tion or microsatellite loci in Didelphis marsupialis (Marsupialia: revealed that some microsatellites amplified specific and Didelphidae). Mol Ecol 8(3):517–518 Rodrigues FP, Rocha FS, Garcia JE et al (2006) Isolation and polymorphic PCR products among species (see Table 2). characterization of microsatellite loci in the woolly mouse opossum, Our data showed that microsatellites were not highly Micoureus paraguayanus (Marsupialia : Didelphimorphia). Mol conserved among Didelphidae species. These microsatel- Ecol Notes 6:686–688 lites will be used in a broad study about the genetic Rousset F (2008) Genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour 8(1): diversity and threatened status of the that occur 103–106 in the Rio Doce State Park, a small fragment of the Atlantic Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users Forest in Minas Gerais state in Brazil, which seems com- and for biologist programmers. In: Krawetz S, Misener S (eds) pletely isolated from other ’ fragments in the region. Bioinformatics methods and protocols. Humana Press, Totowa, NJ, pp 365–386 Sambrook J, Russel DW (2001) Molecular cloning: a laboratory Acknowledgments We would like to thank our colleagues from the manual, 3rd edn. CSHL Press, New York Sackler Institute of Comparative Genomics, especially Matt Leslie; Samollow PB, Kammerer CM, Mahaney SMetal (2004) First-generation colleagues from the Mammalogy Lab of the General Biology linkage map of the gray, short-tailed opossum, Monodelphis Department of ICB-UFMG, who helped us with the field work and domestica, reveals genome-wide reduction in female recombination who furnished samples, especially Raquel Moura. This study is fun- rates. Genetics 166(1):307–329 ded by the CNPq Project PELD-PERD, the CAPES Foundation Samollow PB, Gouin N, Miethke P et al (2007) A microsatellite- (Ph.D. student fellowship), and the Sackler Institute for Comparative based, physically anchored linkage map for the gray, short-tailed Genomics—AMNH. Opossum (Monodelphis domestica). Chromosome Res 15:269– 281 Shuelke M (2000) An economic method for fluorescent labeling of References PCR fragments. Nat Biotechnol 18:233–234 Temnykh S, DeClerck G, Lukashova A (2001) Computational and Brownstein M, Carpenter J, Smith J (1996) Modulation of nontem- experimental analysis of microsatellites in rice (Oryza sativa L.): plated nucleotide addition by Taq polymerase: primer modifica- frequency, length variation, transposon associations, and genetic tions that facilitate genotyping. BioTechniques 20:1004–1010 marker potential. Genome Res 11:1441–1452 Dias IMG, Amato G, Carvalho MRS, Cunha HM, Paglia AP, DeSalle R, Fonseca CG (2008) Characterization of eight microsatellite

123