Conservation Genet Resour (2014) 6:107–109 DOI 10.1007/s12686-013-0016-4
MICROSATELLITE LETTERS
Isolation and characterization of 23 novel polymorphic microsatellite markers from the endangered Puerto Rican boa (Chilabothrus inornatus) using paired-end Illumina shotgun sequencing R. Graham Reynolds • Alberto R. Puente-Rolo´n Karen Kolodzaike • Tiara Butler-Smith
•
Received: 18 July 2013 / Accepted: 24 July 2013 / Published online: 2 August 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We developed 23 microsatellite loci for the endangered Puerto Rican boa, Chilabothrus inornatus, from 100 bp reads obtained from a paired-end Illumina shotgun library. Importantly, these loci can be amplified using the same touchdown PCR program. All loci were highly polymorphic, with between 5 and 15 alleles found among 24 individuals genotyped from three populations. We anticipate that these loci will be extremely useful in conservation genetic studies of this species. Keywords Boidae Conservation Endangered species Epicrates Microsatellites Next generation DNA sequencing
The Puerto Rican boa (Chilabothrus inornatus), the largest native snake in Puerto Rico, was placed on the United States Endangered Species List in 1970 (Endangered Species Act 1973). Since that time, little progress has been made to clarify the conservation or recovery status of this species, much of the species biology and ecology is still unknown, and the species remains endangered. Puerto Rican boas still face a variety of threats, including habitat loss, predation by invasive vertebrates, persecution, road mortality, and competition with introduced species
R. G. Reynolds (&) K. Kolodzaike T. Butler-Smith Department of Biology, University of Massachusetts Boston, Boston, MA 02125, USA e-mail:
[email protected] URL: www.rgrahamreynolds.info A. R. Puente-Rolo´n Departamento de Ciencias y Tecnologı´a, Universidad Interamericana de Puerto Rico, Recinto de Arecibo, Arecibo, PR 00614, USA
(Puente-Rolo´n et al. 2013). Any recovery plan for the Puerto Rican boa must include information regarding the distribution of genetic diversity in the species. A preliminary genetic analysis (Puente-Rolo´n et al. 2013) suggested that the species exhibited a lack of phylogeographic structure across the island. However, Puente-Rolo´n et al. (2013) included only three nonspecific microsatellite markers, and there is a great need for better resolution in population genetic parameters for this endangered species. Here we describe the isolation and characterization of novel microsatellite markers developed specifically for the Puerto Rican boa. We extracted whole genomic DNA from tail clips obtained from two individual Puerto Rican boas from Arecibo Municipality, Puerto Rico using the Promega Wizard SV DNA purification system according to the manufacturer’s protocol. These extracts were quantitated using the NanoDrop system, then sent to the Savannah River Ecological Laboratories for microsatellite marker development and primer design. 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 TrueSeq DNA library kit. Illumina sequencing was conducted on a HiSeq machine with 100 bp paired-end reads. Five to 10 million of the resulting reads were analyzed with the Perl script PAL_FINDER_v0.02.04 (Castoe et al. 2012) to identify potentially amplifiable loci (PALs) containing di-, tri-, tetra-, penta-, and hexanucleotide microsatellites. These reads were then batched into the program Primer3 (version 2.3.0) 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 197,407 reads were selected. From these, 48 primer pairs of the resulting 5,973 loci were chosen, representing a variety of repeat motif
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Table 1 Characteristics of 23 polymorphic microsatellite loci developed for C. inornatus Locus
Primer sequence 50 ? 30
Repeat motif
Size (bp)
N
Na
Ci2
F: aCATGCTGCAGAACTTCACCG
AAGGAG
410–446
22
AAGGAG
224–254
ATACAG
Ho
He
7
0.82
0.79
22
6
0.82
0.75
267–303
23
7
0.57
0.73
ATTTT
212-262
22
9
0.86
0.83
ATATT
220–260
23
9
0.70
0.85
R: CAATGCAAGTGTCCAGAAGAGG Ci3
F: aGGCACTTGTCCTGAATCTAATGG R: TGGATCTGTTTGCATCTGGC
Ci5
F: aATTGAAATCAATGAAACTGGAAAGG R: CTTGCTGTGTGTGTATGGATGG
Ci7
F: aTCAACAACATTTAAACAAATGCCC R: TGTGCTCCTGAAGGAAAGGC
Ci8
F: aTTTAAAGTATCAGATGCCAGTTCC R: GACTAGTGCAAGCCTCATATATTTACC
Ci10
F: aCATAAGTGGAGGGCCAAGC R: GCCAAATGTATGTATTACCTCAAAGG
AAATT
247–272
20
6
0.55
0.77
Ci11
F: aAATCAAGATGTGTATGGCCACC
ATAGG
296–326
23
7
0.83
0.77
ATCT
248–276
20
6
0.90
0.78
ATCT
257–273
24
5
0.63
0.68
ATCT
272–300
18
8
0.83
0.83
ATCT
302–318
23
6
0.74
0.70
TTCC
260–276
23
5
0.56
0.72
ATCT
369–397
23
8
0.87
0.79
R: TACCGAGGTGCTGGGATAGC Ci18
F: aAGTGAGCTTCCAGGAGAGAGG R: GGACCACCCATTCAAACC
Ci20
F: aAGTCATAACCAATGTCACATACAGC GGCACCAAACTTCTTTGTGG
Ci24
F: aCTCTCTGGAAAGATGCCAGG R: TTTGCCATTTATTGTCTACTTGG
Ci25
F: aTTCGTACTCCAAGAAGTGCTGC R: TCCTAGCAAGACAAAGCTGCC
Ci27
F: aGCAAGTGGCTGGACATGG R: AAAGTTGATGCTGTGTTTATATCATGG
Ci30
F: aAAGACTCATCAATATTCCTAGTCAGCC R: TCAGAAGCTTTGCTACAGCCC
Ci32
F: aTGCACTAATGCCTAGTTGAAAGG R: CAGGGATGGATTAGGTTCTGC
AAAG
270–304
23
9
0.87
0.84
Ci33
F: aAGTGGAATTGAATGTATTTAACAAGG
TTCC
269–301
23
9
0.87
0.84
ATCT
263–291
23
8
0.65
0.80
AAAG
340–368
24
8
0.67
0.80
AAAG
221–289
24
15
0.79
0.88
ATCT
254–278
24
7
0.75
0.74
AAAG
437–465
23
7
0.78
0.81
R: AAAGGGAAAGAAGTGTTGAGTGG Ci34
F: aTCATGGATATGAGATGGGCG R: CATGAAGGCACTCATGCAGC
Ci35
F: aTCAGGTTATCATTGCCTCATGC R: CATTCTCCAGTCACTTTGGGC
Ci36
F: aCTTAGTGACTGAAGATGCATTGCC R: AGCTGCAAGACGCACACC
Ci37
F: aGATAGACCTCGTGTATCTATTGGTGC R: TCCTCAGTTGCTGCATTTGG
Ci41
F: aCGCTTTATAGGCATGTTGTCTATCC R: GCCCATATCCAGCTATATTTCCC
Ci43
F: aCAAACTCTTCTCAGTTAACCACGC
ATCT
320–344
23
7
0.87
0.82
Ci45
R: ACGGCACCTCCAGAATGG F: aGCACTAAAGCCAACATACAGTAATCC
TTCC
202–230
21
8
0.14
0.86
ATCT
183–215
24
9
0.63
0.79
R: CCACCTCACACACCTTGGG Ci47
F: aTTGGTTGTTCTGGTTCTGGC R: GGCAAACTGCTTGAGTTCCC
Number of individuals genotyped is N; Na is the number of alleles observed; Ho and He are observed and expected heterozygosity, respectively a
The M13 sequence tag (50 -CACGACGTTGTAAAACGAC-30 )
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lengths. The forward primer from each primer pair was modified on the 50 end with a 19 bp sequence tag (50 -CAC GACGTTGTAAAACGAC-30 ) to allow for the use of a third fluorescently-labeled PCR primer. The 48 primer pairs were tested for amplification and polymorphism using DNA obtained from four individual Puerto Rican boas selected from the larger dataset. ‘‘Touchdown’’ PCR reactions were done in an Eppendorf Mastercycler Pro with the following conditions: denaturation at 95 °C for 5 min; 10 cycles at 95 °C for 20 s, 60–50 °C for 60 s, and 72 °C for 40 s. stepping down 1 °C each cycle from 60 to 50 °C; 20 cycles at 95 °C for 20 s, 48 °C for 20 s, and 72 °C for 40 s; and a final extension at 72 °C for 10 min. A third forward primer labeled on the 50 end with one of four dyes (6-FAM, PET, VIC, or NED) was included in each PCR. We visualized PCR products by gel electrophoresis and resolved genotypes on an automated sequencer (ABI 3730XL) at Massachusetts General Hospital DNA Core Facility, Cambridge, MA using GeneScanTM 500 LIZ size standard and PEAK SCANNER 1.0 software (ABI) with manual verification of peak calling. Out of the 48 primer pairs, we found 23 polymorphic loci which we selected for further characterization. We selected a total of 24 individual boas to genotype at these loci (subset from Puente-Rolo´n et al. 2013). We used MICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004) to investigate whether our genotype profiles showed evidence of allele-dropout or null alleles. Basic descriptive statistics were calculated using GENALEX 6.4 (Peakall and Smouse 2006). We tested for departures from Hardy–Weinberg equilibrium (HWE) using GENEPOP 4.0 (Raymond and Rousset 1995). All 23 loci were highly polymorphic, with a mean of 7.6 alleles per locus (range 5–15 alleles/locus) found among the 24 individuals (Table 1). We found limited evidence for null alleles in loci Ci10 and Ci34 at estimated frequencies of 0.24 and 0.18, though neither was statistically significant [Van Oosterhout et al. 2004]. We found
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evidence for significant deviation from Hardy–Weinberg equilibrium after Bonferroni correction in locus Ci45 due to heterozygote deficiency. We anticipate that these loci will be extremely beneficial to ongoing conservation work for the endangered Puerto Rican boa. Acknowledgments We thank K. Winchell and speleological volunteers for assistance in the field. We are grateful to R. Beasley, S. Lance, and the Molecular Ecology Laboratory at the Savannah River Ecological Lab for assistance with sequencing and primer design. We are also grateful to the Puerto Rico Departamento de Recursos Naturales y Ambientales (DRNA) for permits and assistance. All samples were collected under DRNA permits 2012-EPE-001 (to RGR) and 00-EPE-16 (to ARPR), U.S. Fish and Wildlife Native Endangered Species Recovery Permit # TE63270A-0 (to RGR), and U.S. Department of Agriculture Special Use Permit # CNF-2118 (to RGR). We are grateful for funding from the United States Fish and Wildlife Service Southeast Region Endangered Species Recovery Implementation. This work was approved by the University of Massachusetts Boston Institutional Animal Care and Use Committee (IACUC) Protocol no. 2011006.
References Castoe TA, Poole AW, Jason de Koning AP, Jones KL, Tomback DF, Oyler-McCance SJ, Fike JA, Lance SL, Streicher JW, Smith EN, Pollock DD (2012) Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake. PLoS ONE 7:e30953 Peakall R, Smouse PE (2006) Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295 Puente-Rolo´n AR, Reynolds RG, Revell LJ (2013) Preliminary genetic analysis supports cave populations as targets for conservation in the endemic, endangered Puerto Rican boa (Boidae: Epicrates inornatus). PLoS ONE 8(5):e63899 Raymond M, Rousset F (1995) GenePop v1.2: population genetics software for exact test and ecumenicism. J Hered 86:248–249 Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538
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