Development of polymorphic microsatellite markers for the North American porcupine, Erethizon dorsatum, using paired-end Illumina sequencing Erika L. Barthelmess, Cara N. Love, Kenneth L. Jones & Stacey L. Lance
Conservation Genetics Resources ISSN 1877-7252 Conservation Genet Resour DOI 10.1007/s12686-013-9933-5
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Author's personal copy Conservation Genet Resour DOI 10.1007/s12686-013-9933-5
TECHNICAL NOTE
Development of polymorphic microsatellite markers for the North American porcupine, Erethizon dorsatum, using paired-end Illumina sequencing Erika L. Barthelmess • Cara N. Love Kenneth L. Jones • Stacey L. Lance
•
Received: 10 April 2013 / Accepted: 12 April 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We isolated and characterized a total of 19 microsatellite loci from the North American porcupine, Erethizon dorsatum. Loci were screened in 22 individuals from St. Lawrence and Franklin Counties in northern New York State. The number of alleles per locus ranged from 4 to 14, observed heterozygosity ranged from 0.316 to 1.000, and the probability of identity values ranged from 0.028 to 0.243. These new loci will provide tools for examination of the effects of habitat fragmentation, for instance by roads and other infrastructure, on gene flow and population subdivision in this species. Keywords Erethizon dorsatum Porcupine Illumina Microsatellite PAL_FINDER PCR primers SSR
North American porcupines occur across a wide geographic range that extends from Mexico in the south past the Arctic Circle in the north and from Alaska and California in the west through Maine and Labrador in the east, though the species is apparently absent from the Appalachian Mountains and southeastern United States. Despite this large range, little is known about population structure
E. L. Barthelmess (&) Biology Department, St. Lawrence University, Canton, NY 13617, USA e-mail:
[email protected] C. N. Love S. L. Lance Savannah River Ecology Laboratory, University of Georgia, Aiken, SC 29802, USA K. L. Jones Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA
in the species. North American porcupines are the most frequently detected mammalian casualties in wildlifevehicle collisions in northern New York (Barthelmess and Brooks 2010), yet, because of the difficulty of studying populations in the wild, little is known about the consequences of this mortality to populations. Use of a genetic approach would facilitate a greater understanding of porcupine population demographics and structure. However, as the only Hystricomorph rodent in the United States and Canada (Wilson and Reeder 2005), no microsatellite loci have been available to use from a closely related species. Therefore these new loci will provide tools for examination of genetic structure and demographics in porcupine populations and will enable examination of the effects of habitat fragmentation, for instance by roads and other infrastructure, on gene flow and population subdivision in this species. Total DNA was extracted from tissue collected from a road-killed individual of Erethizon dorsatum, following protocol 8 of the Invitrogen Easy-DNA kit, for use in isolation of microsatellite loci. An Illumina paired-end shotgun library was prepared by shearing 1 lg of DNA using a Covaris S220 and following the standard protocol of the Illumina TruSeq DNA Library Kit and using a multiplex identifier adaptor index. Illumina sequencing was conducted on the HiSeq with 100 bp paired-end reads. Five million of the resulting reads were analyzed with the program PAL_FINDER_v0.02.03 (Castoe et al. 2012) to extract those reads that contained di-, tri-, tetra-, penta-, and hexanucleotide microsatellites. Once positive reads were identified in PAL_FINDER_v0.02.03 they were batched to a local installation of the program Primer3 (version 2.0.0) for primer design. To avoid issues with copy number of the primer sequence in the genome, loci for which the primer sequences only occurred once in the 5 million reads
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Author's personal copy Conservation Genet Resour Table 1 Details for 19 polymorphic microsatellite loci developed for E. dorsatum Locus
Primer sequence 50 ? 30
Repeat motif
Size (bp)
N
Erdo7
F: TTGGAATGGTCATTTGTGTGGa
ATCT
322–346
22
AAAG
220–270
AAAG
K
Ho
He
PI
7
0.636
0.758
0.097
22
13
0.727
0.875
0.028
208–260
22
12
0.773
0.822
0.051
AAAT
124–152
21
7
0.762
0.788
0.074
AAAG
196–228
20
8
0.600
0.818
0.057
R: AGAGCTTTCATGTTGAGCTGC Erdo9
F: CACTGGGAGGTTGTTAGCCa
Erdo10
F: AACCACACGATACCAACCTGCa
R: AGGACACATCAACATAAGCACC R: AGTGGTGAAGTTGCTCTGGG Erdo11
F: TACATAGCAAGACCCGACCCa R: CAAGTGGTTTAGCACTGGGC
Erdo12
F: AAACAGAACCCAGAATTGTCATATACCa
Erdo14
F: AAATAAAGTTTACAAGCCAAAGCCa R: CATGACTAAGTACATCCCAATTTCC
ATCT
296–374
22
13
0.636
0.840
0.043
Erdo17
F: TTTGCACTCCTTACTACGAGGGa
AAAG
358–398
21
8
0.381
0.825
0.054
AAAG
196–228
21
9
0.810
0.833
0.048
AAAG
166–194
22
8
0.727
0.815
0.058
AAAG
220–260
22
10
0.864
0.835
0.048
AAAGC
201–276
21
12
0.619
0.851
0.039
AAAG
306–334
21
12
0.714
0.848
0.039
AAAG
176–236
22
10
0.682
0.868
0.032
R: CACACTGCTAGGATGTTGTCTCC
R: CCTGAGTAAATGTGGGCAAGG Erdo18
F: CATTGCAATCGTCAGAGAGGa R: CCTGTGTCTCCATCTTGGC
Erdo19
F: ATTACAGAGAAATGGAAATGTAATCCa R: CTATGTGTTTAATCTCCAGTACCACC
Erdo21
F: CCCATACCCACACACACGa
Erdo22
F: GGGAAGCACAATGTAGGATGCa
R: CTGAGACCCATTAGCTTGGC R: CTGTTCTGGTCTCAGCGTGG Erdo24
F: CCACTGCCTGGACTTGAAGCa R: GATGTCCTGAGCCTGGTGC
Erdo28
F: GAGAGTGGACAAATTTATGATTACATAGGa
Erdo29
F: TGTTCTGGGAAATTGATAAGTAGCCa R: GGGTCTTGCCTCAGTAAAGGG
ATCT
142–162
22
6
1.000
0.777
0.085
Erdo30
F: TTCCACTCCAGGACATTCCCa
AAAG
367–411
21
14
0.667
0.867
0.030
AAGAG
188–213
21
6
0.810
0.715
0.128
AAAG
204–236
21
8
0.762
0.842
0.044
AAAT
198–210
22
4
0.318
0.598
0.243
ATCT
220–244
19
7
0.316
0.749
0.092
R: GGCTGGGAGTGTAGCTGAGG
R: GTCACCATTAGAATCTCTGCTGC Erdo31
F: GGGTAGCATGAGGGTATAGAGCa R: GTGTAGCCACACAGGCAGG
Erdo32
F: GCTGCAATCAAAGACAAGCCa R: TAAGGGCCCATTAGCTGGC
Erdo40
F: CCAAAGCCTTCGGACAAAGCa R: TGTAATAAGGGACGGGATTAAATTATGG
Erdo42
F: TGTAGATACAGATCAGCCAATAGGGa R: TTCACCAACTGTGAACATTTCC
The size indicates the range of observed alleles in base pairs and includes the length of the CAG tag; number of individuals genotyped is N; k is number of alleles observed; Ho and He are observed and expected heterozygosity, respectively; PI is the probability of identity for each locus a
Indicates CAG tag (50 - CAGTCGGGCGTCATCA-30 ) label; Bonferroni corrections
indicates significant deviations from Hardy–Weinberg expectations after
were selected. Forty-eight loci of the 6,064 that met this criterion were chosen. One primer from each pair was modified on the 50 end with an engineered sequence (CAG tag 50 -CAGTCGGGCGTCATCA-30 ) to enable use of a third primer in the PCR (identical to the CAG tag) that was
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fluorescently labeled. The sequence GTTT was added to primers without the universal CAG tag addition. Forty-eight primer pairs were tested for amplification and polymorphism using DNA obtained from eight individuals. All methods for primer screening and genotyping
Author's personal copy Conservation Genet Resour
are as described in O’Bryhim et al. (2012). A touchdown thermal cycling program (Don et al. 1991) encompassing a 10 °C span of annealing temperatures ranging between 65 and 55 °C (TD65) was used for all loci. Nineteen of the tested primer pairs amplified high quality PCR product that exhibited polymorphism. We assessed the variability of the 19 polymorphic loci in 22 specimens from St. Lawrence and Franklin Counties, New York. Conditions and characteristics of the loci are provided in Table 1. We estimated the number of alleles per locus (k), observed and expected heterozygosity (Ho and He), and probability of identity (PI) using GenAlEx v6.5 (Peakall and Smouse 2006). Tests for deviations from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium were conducted using GENEPOP v4.0 (Rousset 2008). After Bonferroni correction for multiple comparisons 4 loci showed significant deviations from expectations under HWE and no linkage disequilibrium was detected for any of the 171 paired loci comparisons. These new loci will assist in examining population genetic structure in the North American porcupine. Acknowledgments Microsatellite development was supported by a Mellon Foundation grant to St. Lawrence University. M. Brooks, C. Neill, N. Panshin and T. Barthelmess assisted with tissue collection. Manuscript preparation was partially supported by the DOE under Award Number DE-FC09-07SR22506 to the University of Georgia Research Foundation.
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