Microsatellite marker development from next-generation sequencing in the New England cottontail (Sylvilagus transitionalis) and cross-amplification in the eastern cottontail (S. floridanus)
King et al. BMC Res Notes
Microsatellite marker development from next-generation sequencing in the New England cottontail (Sylvilagus transitionalis) and cross-amplification in the eastern cottontail (S. floridanus)
Timothy L. King 2
Michael Eackles 2
Aaron Aunins 0
Thomas J. McGreevy Jr. 1
Thomas P. Husband 1
Anthony Tur 4
Adrienne I. Kovach 3
0 Natural Systems Analysts, Leetown Science Center , 11649 Leetown Road, Kearneysville, WV 25430 , USA
1 Department of Natural Resources Science, University of Rhode Island , 1 Greenhouse Road, Kingston, RI 02881 , USA
2 U.S. Geological Survey, Leetown Science Center , 11649 Leetown Road, Kearneysville, WV 25430 , USA
3 Department of Natural Resources and the Environment, University of New Hampshire , 56 College Road, Durham, NH 03824 , USA
4 United States Fish and Wildlife Service , 300 Westgate Center Drive, Hadley, MA 01035 , USA
Objective: The New England cottontail (Sylvilagus transitionalis) is a species of high conservation priority in the Northeastern United States, and was a candidate for federal listing under the Endangered Species Act until a recent decision determined that conservation actions were sufficient to preclude listing. The aim of this study was to develop a suite of microsatellite loci to guide future research efforts such as the analysis of population genetic structure, genetic variation, dispersal, and genetic mark-recapture population estimation. Results: Thirty-five microsatellite markers containing tri- and tetranucleotide sequences were developed from shotgun genomic sequencing of tissue from S. transitionalis, S. obscurus, and S. floridanus. These loci were screened in n = 33 wild S. transitionalis sampled from a population in eastern Massachusetts, USA. Thirty-two of the 35 loci were polymorphic with 2-6 alleles, and observed heterozygosities of 0.06-0.82. All loci conformed to Hardy-Weinberg Equilibrium proportions and there was no evidence of linkage disequilibrium or null alleles. Primers for 33 of the 35 loci amplified DNA extracted from n = 6 eastern cottontail (S. floridanus) samples, of which nine revealed putative species-diagnostic alleles. These loci will provide a useful tool for conservation genetics investigations of S. transitionalis and a potential diagnostic species assay for differentiating sympatric eastern and New England cottontails.
Microsatellites; Cross-amplification; Sylvilagus transitionalis; Sylvilagus floridanus; Sylvilagus obscurus; Nextgeneration sequencing
The New England cottontail (Sylvilagus transitionalis) is
a narrow niche specialist that relies on the dense
understory vegetation of early successional or shrubland
]. These ephemeral habitats have declined steeply
in recent decades in the Northeastern United States
]. As a result, many shrubland species, including the
New England cottontail, face severe population declines
]. Remnant New England cottontail populations are
found today in < 14% of the species’ historical range
and occur in five geographically and genetically distinct
populations located in southern Maine and
southeastern New Hampshire, central New Hampshire, eastern
Massachusetts on Cape Cod, eastern Connecticut and
Rhode Island, and western Connecticut and New York
]. Within these areas, cottontails face the consequences
of population isolation and loss of genetic diversity [
As a result of uncertainty in long-term species’ viability,
the New England cottontail is a high priority for
conservation in the Northeastern United States. To help recover
the species, state and Federal natural resource managers
collaborated with academicians and other
stakeholders to develop and implement a conservation strategy to
improve the outlook for the species [
]. As a result, the
United States Fish and Wildlife Service determined
listing of the species under the Endangered Species Act was
no longer warranted [
]. Despite this decision, much
uncertainty remains about the species’ viability and
population status [
]. To evaluate the effectiveness of the
conservation strategy and inform adaptive modifications
to improve outcomes, extensive population monitoring
and research into population structure and genetic
diversity are needed . To this end, our goal was to develop a
suite of microsatellite markers to aid future conservation
genetics research on the New England cottontail as well
as other Sylvilagus species.
For microsatellite marker development, total genomic
DNA was extracted from tissue samples obtained from
13 New England cottontail, one eastern cottontail (S.
floridanus), and two Appalachian cottontail (S. obscurus)
using the DNEasy Blood and Tissue kit (Qiagen,
Germantown, MD). We chose to sequence multiple species
to maximize the number of microsatellites available for
screening in the target species S. transitionalis.
Sequencing libraries were created from these genomic DNA
samples for sequencing on the Ion Torrent PGM and Ion
Proton (ThermoFisher Scientific, Frederick, MD). For
each sequencing library, the extracted DNA was
quantified with a Nanodrop spectrophotometer (ThermoFisher
Scientific), and 100 ng of DNA from each individual was
used for construction of multiple 200 base pair libraries
following the manufacturer’s protocol. For libraries with
multiple individuals in one run, equimolar proportions of
each library were pooled prior to chip loading.
Sequence reads from each completed run were
imported into Qiagen CLC Genomics Workbench (ver.
7) for processing. Reads were length trimmed to a
minimum size of 30 bp, and the quality trimming threshold
was set to 0.01 corresponding to a Phred score of 20.
Sequence reads from each of the 5 separate runs were
then screened individually for all possible di-, tri-, tetra,
penta-, and hexanucleotide microsatellite repeat motifs
with a minimum repeat length of five with the program
QDD (ver. 3) [
]. Primers were designed for each
putative microsatellite locus within QDD using the integrated
PRIMER 3 code [
]. Seventy-five tri- and tetra
nucleotide loci identified by QDD with predicted amplified
lengths between 100 and 250 bp were selected for
screening within a collection of n = 8 wild-caught New
England cottontails sampled from across the species range.
A universal M13 sequence was added to the 5′ end of
either the forward or reverse primer of each primer pair
enabling an M13 FAM labeled fluorescent dye
complementary to the universal tail to be incorporated into the
polymerase chain reaction (PCR) product [
Polymerase chain reactions were performed in 25 μl volumes,
consisting of 10 ng of DNA, 1 X PCR Buffer (Promega,
Madison, WI), 0.25 μM of labeled forward primer,
0.5 μM of unlabeled reverse primer, 0.1 μM of labeled
M13, 2.0 mM MgCl2, 0.2 mM of each dNTP, 0.25 units/μl
Bovine Serum Albumin (New England Biolabs, Ipswich,
MA), and 0.06 units/μl of Taq polymerase (Promega),
using the following cycling conditions: 94 °C for 15 min,
29 cycles of 94 °C for 1 min, 58 °C for 45 s, and 72 °C for
45 s, 5 cycles of 94 °C for 1 min, 52 °C for 45 s, and 72 °C
for 45 s, all followed by 72 °C for 10 min. PCR products
for each locus were electrophoresed separately on an ABI
3130 Genetic Analyzer (ThermoFisher Scientific)
automated DNA sequencer. Alleles were called using
GeneMapper (ver. 4) (ThermoFisher Scientific) following the
protocols described in [
Loci that amplified consistently and were easily
scoreable on the sample of n = 8 wild New England cottontails
were then tested on DNA extracted from an additional
sample of n = 33 New England cottontails from a wild
population in eastern Massachusetts, on Cape Cod. In
addition, DNA extracted from a sample of n = 6
sympatric eastern cottontails, also collected from eastern
Massachusetts were tested for cross-species amplification.
Genotype data from the Cape Cod New England
cottontail collection (n = 33) were analyzed for null alleles,
large allele dropout, and scoring errors using
MICROCHECKER (ver 2.2.3) [
]. Genetic diversity and
heterozygosity were quantified using GenAlEx 6.5 [
Exact tests in GENEPOP [
] were used to determine
if genotypes at each locus conformed to
Hardy–Weinberg equilibrium (HWE). Multi-locus tests of
conformance to HWE were completed using Fisher’s method in
GENEPOP. Linkage disequilibrium (LD) was tested for
all pairs of loci using contingency tables in GENEPOP.
All tests of HWE and LD tests in GENEPOP used the
default Markov chain parameters. Significance levels for
HWE and LD tests were adjusted using the sequential
Bonferroni correction [
To evaluate the utility of these loci for population
genetic studies, multiple analyses were performed with
the n = 33 New England cottontail collection. All
multilocus genotypes were subjected to analysis via GENECAP
] to identify matching samples, calculate match
probabilities, and estimate the sibling probability of identity
]. To assess the randomness of the collection
(e.g., to ensure the collection did not consist of a small
number of families), we analyzed for the presence of
fullsibling families using the program COLONY v2.0 [
Settings for COLONY analyses included the assumption
of male and female polygamy, no per locus genotyping
error information, no inbreeding, long run length with
full likelihood analysis, high likelihood precision, no
allele frequency updates, and no sibship prior.
Individual New England cottontails were analyzed as offspring
without assignment of individuals as candidate males
(fathers) or females (mothers), as these data were not
available. While the inference of family relationships is
weakened in this situation with no sex, age, relationship
information, and the assumption of polygamy for both
sexes, COLONY is predicted to be more accurate than
pairwise estimates of relationships [
significance of any full-sib relationships was assessed through
the P values reported by COLONY. Within a sample of
individuals taken at random (with respect to kin) from a
population, the frequencies of full and half sib dyads can
be used to estimate the current effective size (Ne) of the
population. Therefore, COLONY was also used to
estimate Ne and associated 95% confidence interval utilizing
the estimates from the sibship assignment full likelihood
method. To estimate whether the Ne has remained
constant (i.e., achieved mutation-drift equilibrium; see [
we conducted analyses with BOTTLENECK [
implementing a two-phased model of mutation (5% IAM; 95%
]). Statistical significance of the
BOTTLENECK analyses was determined with a Wilcoxon
signedrank test performed by the software.
Results and discussion
Within the 44,084,987 million sequence reads analyzed
across five runs of the Ion Torrent PGM and Ion
Proton platforms, 1,420,351 million were identified by QDD
software as containing microsatellites using our chosen
filtering criteria. Of the 75 loci selected from among
these sequences for screening in the sample of n = 8 New
England cottontails, all but three amplified consistently.
An additional 24 loci were monomorphic. Of the
remaining 48 loci, 17 had two alleles, 16 had three alleles, 10
had four alleles, three had five alleles, and two had seven
alleles. From this set of loci, we selected the 35 most
polymorphic, including all with three to seven alleles and
a few with two alleles for additional testing in the wild
collection of n = 33 New England Cottontails and n = 6
eastern cottontails from the same population to test for
cross-specific amplification. The primer sequences of
these 35 loci are provided in Table 1.
Microchecker reported no instances of large allele
dropout or scoring errors. Analyses of family structure in
COLONY indicated the presence of no full sibling dyads,
and therefore all individuals from the n = 33 collection
were retained for subsequent analyses. Three loci were
monomorphic in the Massachusetts wild New England
cottontail population, while the remaining 32 loci
averaged 3.1 alleles per locus (range 2–6; Table 1). Unique
multilocus genotypes were generated for each
individual with the probability of 6.4 × 10−8 that two siblings
would share identical genotypes (PIsibs; [
heterozygosity (He) ranged widely across loci from 6.0%
(StrQ23, StrQ48) to 75.0% (StrQ30) and averaged 47.0%
for the Massachusetts wild New England cottontail
population. Tests for conformance to HWE revealed that locus
StrQ19 was not in HWE (P = 0.016), though this result
did not remain significant after sequential Bonferroni
correction. In addition, no statistically significant linkage
disequilibrium (GENEPOP) was detected after sequential
COLONY estimated the Ne (and 95% confidence
limits) for the Massachusetts collection to be 48 (30–80),
and BOTTLENECK indicated a statistically significant
heterozygote excess for the Massachusetts collection
(P < 0.001; Wilcoxon signed-rank test), suggesting a
recent reduction in the effective population size. These
results are consistent with previous work, showing that
New England cottontails in eastern Massachusetts have
low effective population sizes and have undergone a
recent population decline [
All but two loci amplified in the eastern cottontail
samples, and an additional three loci were
monomorphic (including two that were also monomorphic in New
England cottontails). The remaining 30 loci had two to
six alleles in eastern cottontail. Eight polymorphic loci
(StrQ23, Str25, Str41, Str42, Str43, Str44, Str48, and
Str63) had species-specific amplification patterns with
no overlapping alleles across the screened individuals.
In additional, StrQ72 was monomorphic for a different
allele in each species. These results suggest a subset of
these loci will be useful as a diagnostic screening tool for
samples collected during noninvasive genetic monitoring
]. However, more extensive testing of
samples throughout the range where New England cottontail
and eastern cottontail are sympatric is needed to confirm
In conclusion, the markers developed in this study
could prove useful for future population monitoring and
conservation genetics research of New England
cottontail. The high discriminatory power of these loci
indicate that they will be particularly useful for noninvasive
genetic surveys, where robust individual identification
from a small panel of markers is required. Further, the
cross amplification in eastern cottontails will facilitate
use of these markers in this species as well as possibly
others in the Sylvilagus genus.
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These loci have been thoroughly screened in one of the
geographically distinct populations of New England
cottontails. Given effects of population isolation and genetic
drift, it is likely that these markers may have different
levels of polymorphism or null alleles in other geographic
populations across the species range. Future work should
screen the loci carefully in each geographic area prior
to embarking on a new study. In addition, while we did
not assess the genotyping error rate for these loci, future
studies using them could measure this parameter to
ensure accuracy of the genotype data.
TLK performed bioinformatics analyses, helped conceive the study, and
helped draft the manuscript. He passed away before completion of the
final draft. His contributions to the field of conservation genetics are noted,
and he will be missed. ME carried out the high throughput sequencing and
laboratory screening of the markers and helped draft the manuscript. TJM
helped in the development of the study, conducted quality control screening
of the markers and helped write the manuscript. AA performed bioinformatic
analyses and helped write the manuscript. TPH helped in the development
of the study, secure funding for the project, and provided samples. AT helped
in the development of the study, assisted with sample collection, and edited
the manuscript. AIK helped conceive the study and write the manuscript. All
authors read and approved the final manuscript.
Mary Sullivan (University of Rhode Island) helped process New England
cottontail samples. We would like to thank the following individuals and their
affiliates for providing New England cottontail samples, as the study would
not have been possible without their efforts: Eileen McGourty (USFWS), Annie
Curtis (MA Army National Guard), David Scarpitti (MA Division of Fish and
Wildlife), Howard Kilpatrick (CT Department of Energy and Environmental
Protection), Heidi Holman (NH Fish and Game Department), Wally Jakubas (ME
Department of Inland Fisheries and Wildlife), Michael McBride and Lou Perrotti
(Roger Williams Park Zoo), Jacob McCumber (MA Army National Guard), Tony
Perry (Mashpee Wampanoag Tribe). Use of trade, product, or firm names does
not imply endorsement by the US Government.
The authors declare that they have no competing interests.
Availability of data and materials
The microsatellite sequences generated from this study are available through
NCBI’s GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and are accessible
via the GenBank Accession Numbers KX530819–KX530853.
Consent for publication
Ethics approval and consent to participate
Tissue samples of wild S. transitionalis were collected under the University of
Rhode Island Institutional Animal Care and Use Committee approved protocol
number AN11-12-011. One DNA sample from S. obscurus was kindly provided
by Marian Litvaitis (University of New Hampshire), and the other S. obscurus
tissue sample (specimen 2235-KB) was provided by the Frostburg State
University Mammal Museum from a historical sample collected in 1998. Tissue
samples of S. floridanus were provided by Anthony Tur of the USFWS, whom
obtained the samples from licensed hunters.
Funding for this study was provided by a U.S. Fish and Wildlife Service—U.S.
Geological Survey Science Support Partnership Grant.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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