Isolation and characterization of polymorphic microsatellite loci for the spiny-rat Clyomys bishopi (Rodentia: Echimyidae) Ana Carolina Ramos Arantes, Roberto Guilherme Trovati, Andréa Cristina Peripato & Fernando Pacheco Rodrigues Conservation Genetics Resources ISSN 1877-7252 Volume 4 Number 2 Conservation Genet Resour (2012) 4:335-337 DOI 10.1007/s12686-011-9541-1
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Author's personal copy Conservation Genet Resour (2012) 4:335–337 DOI 10.1007/s12686-011-9541-1
TECHNICAL NOTE
Isolation and characterization of polymorphic microsatellite loci for the spiny-rat Clyomys bishopi (Rodentia: Echimyidae) Ana Carolina Ramos Arantes • Roberto Guilherme Trovati Andre´a Cristina Peripato • Fernando Pacheco Rodrigues
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Received: 20 September 2011 / Accepted: 22 September 2011 / Published online: 7 October 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Ten microsatellite loci were isolated and characterized from the spiny-rat (Clyomy bishopi), a mediumsize Neotropical rodent that inhabits some of the last remaining savannas from Sa˜o Paulo state, Brazil. Between 5 and 17 alleles were detected per locus, with expected heterozygosity ranging from 0.605 to 0.921. All but one locus were found to be in Hardy–Weinberg equilibrium, and no linkage disequilibrium was detected among pairs of loci. These microsatellites should provide useful markers in genetic studies including parentage analyses and determining genetic and social structure of populations. Keywords Clyomys bishopi Microsatellite markers Primers Genetic diversity
The spiny-rat (Clyom bishopi, family Echimyidae) is a rodent whose described distribution is restricted to Cerrado A. C. R. Arantes (&) Laborato´rio de Biodiversidade Molecular e Conservac¸a˜o, Departamento de Gene´tica e Evoluc¸a˜o, Universidade Federal de Sa˜o Carlos–UFSCar, Via Washington Luı´s, Km 235, Sa˜o Carlos, SP, Brazil e-mail:
[email protected] R. G. Trovati Brası´lia, DF, Brazil A. C. Peripato Departamento de Biocieˆncias, Universidade Federal de Sa˜o Paulo—UNIFESP, Santos, SP, Brazil F. P. Rodrigues Laborato´rio de Gene´tica e Biodiversidade, Departamento de Gene´tica e Morfologia, Universidade de Brası´lia—UnB, Brası´lia, DF, Brazil e-mail:
[email protected]
fragments of Sa˜o Paulo state. First described in 1981 by ´ vila-Pires and Wutke (1981), it is an herbivorous rodent A with colonial, social and semi-fossorial habits (Vieira 1997; Burda et al. 2000; Bueno et al. 2004). Apparently it is an important dispersal agent of plant species, notably Attalea geraensis palms, the fruit of which it feeds on (Almeida and Galetti 2007). The rodents themselves are an important food for mammals, birds and reptiles (Bueno et al. 2002; Motta-Junior et al. 2004). Until this paper, no genetic study had been conducted on this species. Here we describe the characterization of ten microsatellite loci in C. bishopi. The loci were isolated from a genomic library enriched for repetitive sequences (Hamilton et al. 1999). For this we isolated the DNA from epithelial tissue of one male specimen, using a phenol:chloroform protocol (Sambrook and Russell 2001). A volume of 20 ll of DNA (200 ng/ll) was digested with restriction enzyme RsaI and subjected to electrophoresis on agarose gel. Digested fragments between 200 and 1,000 pb were eluted and purified from the gel and then linked to adapters (SuperSNX24). These fragments were then amplified by polymerase chain reaction (PCR) using primers complementary to the adapters. Enrichment was then performed by hybridization of the amplified fragments with a set of eight biotinylated tetranucleotide probes ([AAAC]6, [AAAG]6, [AATC]6, [AATG]6, [ACCT]6, [ACAG]6, [ACTC]6, [AATC]6). The fragments hybridized to the probes were recovered by magnetic particles linked to streptavidin (Promega). The recovered fragments were cloned using pGEM-T Easy Vector System (Promega) and recombinant plasmids transformed into Escherichia coli DH5a. The cloned inserts from 96 recombinant plasmids were sequenced on an ABI3730XL by the Sanger dideoxi-terminator method by Macrogen (South Korea). Microsatellites were localized using the software CID (Freitas et al. 2008). Identification and
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removal of sequences related to the adapters and cloning vector was done using VecScreen (http://www.ncbi.nlm. nih.gov/VecScreen/VecScreen.html). Design of primers pairs for the selected loci was performed using Gene Runner 3.05 (Hastings Software, Inc; http://www.generunner.net/). Synthesis of the primer pairs was done with the addition of an 18 bp M13 tail (50 -TGTAAAACGACGGCCAGT-30 ) at the 50 -end of one of the primers, allowing the realization of the economic genotyping protocol described by Schuelke (2000). All available 40 samples were genotyped using the primer pairs. PCRs were performed in 12 ll, containing 30 ng DNA, 19 buffer, 0.2 mM dNTPs, 2 mM MgCl2, 0.5 U of Taq DNA polymerase (Fermentas), 0.2 mM of primer with M13 tail, 0.5 mM primer untailed and 0.5 mM M13 primer with fluorescence FAM, HEX or TET. Gradient temperature tests were made to establish the best annealing temperatures. PCR cycles started with a denaturation step at 94°C for 10 min, followed by 35 cycles of one 94°C for 45 s, 45 s annealing (temperature according Table 1) and an extension step at 72°C for 1 min. We then carried out 10 additional cycles using the optimum annealing temperature for M13, consisting of 94°C for 30 s, 53°C for 45 s and 72°C for 45 s, and finished with an extension step at 72°C
for 10 min. The amplified loci were genotyped in automated sequencer and analyzed with the MegaBACE Fragment Profiler MegaBaceTM (GE Healthcare Life Science). Linkage disequilibrium between pairs of loci was tested using the exact test in Genepop 4.1 (Rousset 2008). This program was also used to obtain genetic diversity indices as the number of alleles per locus (NA), observed heterozygosity (HO) and gene diversity (HE) (Nei 1987). We also estimated the Polymorphic Information Content (PIC) (Botstein et al. 1980) for each of the loci using the program Cervus 3.0.3 (Marshall et al. 1998; Kalinowski et al. 2007). Deviations from Hardy–Weinberg equilibrium were evaluated for each locus using the exact test for deficiency of heterozygotes performed in the software Genepop. The statistical significance of the results was evaluated after performing the Bonferroni correction (Rice 1989). The program MicroChecker (Oosterhout et al. 2004) was used to assess the presence of genotyping errors due to the occurrence of null alleles, allele dropout and/or stutter peaks in electropherograms. The frequency of null alleles (FNA) was calculated for each locus using Freena (Chapuis and Estoup 2007), with the maximum-likelihood estimation proposed by Dempster et al. (1977).
Table 1 Description of 10 microsatellite loci isolated for the spiny-rat (Clyomys bishopi) Locus
GenBank accession no.
Primer sequence (50 –30 )
Repeat motif
Allele size range (pb)
Ta (oC)
N
NA
PIC
HO
HE
PEHW
Cbi 06
JN683834
F: TGCTGTCTGCCTCCTAAG
(TC)21
255–287
60
40
17
0.902
0.875
0.921
0.0971
(CTTT)17
278–314
64
38
9
0.805
0.789
0.839
0.0864
(TG)16
196–232
54
39
9
0.718
0.846
0.754
0.8754
(TGTC)10
149–189
60
40
9
0.777
0.675
0.816
0.0155
(GATT)6
94–130
58
40
5
0.541
0.675
0.604
0.8857
(GC)11(AC)15
166–184
54
39
7
0.717
0.615
0.759
0.0222
R: GGAGCAGGAAATAGGACG Cbi 07
JN683835
F: GGTGCTTATGCTGCTGAG R: AGGGACCGCAGTTTGAT
Cbi 08
JN683836
F: GCCTGTGTTATTGGTCTG
Cbi 11
JN683839
F: GTAGTTTGTCTCCCTGGC
R: CAGCAGGGTGACAAGAG R: CAGAAGTGGGCTCAAGG Cbi 12
JN683840
F: CAGGGATTTTCTTTGATGC R: GCAAGTGCAAGGTCATGAG
Cbi 14
JN683841
F: CTCCTACTGTCCTCCTGC
Cbi 15
JN683832
F: GTAACCACAAGGTTGACG R: CAACAGACAGACGGACAG
(TC)17
129–175
58
39
17
0.893
0.744
0.915
0.0007
Cbi 16
JN683833
F: TGCATAGTGAGTTCAAGTCC
(AAGC)18
212–284
56
40
13
0.848
0.850
0.874
0.0106
(AGAC)5(AGGC)7
185–201
58
40
5
0.660
0.750
0.714
0.6333
(CA)16
246–262
56
39
9
0.767
0.769
0.804
0.4013
R: GAAGTCCTGAGAAGTGCAC
R: GCTTCCTCTTCTCTCCAA Cbi 17
JN683837
F: CTTTCAGCTTCCACACAC R: CACCATTGAGTTATCTTCC
Cbi 18
JN683838
F: GGCAAACTTAGGCAAATG R: CACTTCTTTTCCATTCCAG
Ta annealing temperature, N number of individuals genotyped, NA number of alleles, PIC polymorphic information content, HO observed heterozygosities, HE expected heterozygosities, PEHW P value for Hardy–Weinberg test
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Of the 96 clones sequenced, 54 (56.25%) had inserts containing microsatellite regions, of which 32 (33.33%) had flanking regions suitable for the construction of primers. From these, 18 were selected to develop locusspecific primers, according to the methodology described above. From the 18 primer pairs designed, 10 showed satisfactory patterns of amplification for the desired analysis. The number of alleles per locus varied from 5 to 17, with an average of 10. Observed heterozygosity ranged from 0.615 to 0.875 and expected heterozygosity ranged from 0.604 to 0.921. All loci were in Hardy–Weinberg equilibrium, with the exception of locus Cbi 15, for which there was a deficiency of heterozygotes (P = 0.0007) (Table 1). Software Microchecker indicated, for this locus, the potential occurrence of null alleles, calculated as 0.083. There was no evidence of linkage disequilibrium between pairs of loci analyzed. The microsatellite markers described here will be useful for the study of various aspects of the life history of C. bishopi, including analysis of social and genetic structure of their populations. Acknowledgments The authors thank Instituto Florestal de Sa˜o Paulo (IF process no. 260108-014.277/2009) and Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade (ICMBio process no. 21075-1) for collecting permits; to Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for fellowship to ACRA and to Prof Pedro M. Galetti-Ju´nior for allowing the use of his laboratory facilities at UFSCar.
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