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Conservation Genetics Resources ISSN 1877-7252 Conservation Genet ResourDOI 10.1007/s12686-012-9846-8

Development of polymorphic microsatellitemarkers of the common toad,Duttaphrynus melanostictus useful forgenetic studies

S. Jegath Janani, Richa Sharma,Karthikeyan Vasudevan, Sushil K. Dutta& Ramesh K. Aggarwal

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TECHNICAL NOTE

Development of polymorphic microsatellite markersof the common toad, Duttaphrynus melanostictus usefulfor genetic studies

S. Jegath Janani • Richa Sharma •

Karthikeyan Vasudevan • Sushil K. Dutta •

Ramesh K. Aggarwal

Received: 2 October 2012 / Accepted: 15 December 2012

� Springer Science+Business Media Dordrecht 2012

Abstract Asian common toad, Duttaphrynus melanos-

tictus, is a widespread habitat generalist species of Indian-

subcontinent and South-East Asia. Here we report, 14 new

microsatellite markers (SSRs) for this toad, developed from

a SSR-enriched genomic library, and validated using 44

unrelated samples of Asian toad and *20 samples belong-

ing to four congeners. Nine SSRs were highly polymor-

phic; amplified 6–22 alleles/marker with Ho and PIC

ranging from 0.477 to 0.955 and 0.518 to 0.918, respec-

tively, while one was found to be monomorphic. Of the

remaining four SSRs, two (DMccmb02, DMccmb17)

amplified only two apparently fixed alleles in all samples,

while the other two (DMccmb22, DMccmb29) amplified

1–4alleles/sample; these four SSRs probably represent

duplicated loci. Interestingly, all the 14 SSRs showed

cross-species transferability to varying extent, with four

SSRs showing 100 % transferability. It is hoped that these

SSRs will greatly facilitate population genetic studies on

the Asian toad species across their geographic distribution.

Keywords Duttaphrynus melanostictus � Asian common

toad � Microsatellite markers � Conservation/population

genetics � Cross-species transferability

The Asian common toad Duttaphrynus melanostictus

belongs to the family ‘‘Bufonidae’’, called true toads, com-

prising of more than 500 species that are wide spread around

the world except Australia, Madagascar and The Antarctic.

The Asian common toad is native to Indian subcontinent and

South-East Asia, and is identified as species complex con-

sisting of more than one species. Traits such as large body

size, use of terrestrial adult niche, presence of parotid glands

make the toad more adaptable to broad range of habitats

(Van Bocxlaer et al. 2010). The species is commonly seen in

human dominated and disturbed areas and breeds through-

out the year using all types of water bodies. The dispersal

and life history traits greatly influence the geographical scale

of the genetic structure of the continuously distributed

population within the species’ distribution range. Though

most of the amphibian populations are considered as meta-

populations, toads like Bufo americanus show complete

absence of isolation by distance within its expanding range

(Leblois et al. 2000). The Asian toad offers to be a model for

understanding the population biology and biogeography of

anurans adapted to diverse landscapes. Moreover, it appears

to be an ideal candidate bio-indicator species for environ-

mental health and ecosystem function (pending validation),

by virtue of being habitat generalist. However, any such

studies will require understanding the genetic structure and

dynamics of its extant populations. To achieve this, genomic

DNA variation based molecular/genetic markers hold great

promise, especially the microsatellite markers that target

hypervariable repetitive simple sequence repeats (SSRs) in

the genome. These are most informative and efficient for the

population level studies, but no such markers have been

developed to-date for the Asian toad. Here, we describe the

first set of 14 SSR markers for the common toad D. mela-

nostictus that would facilitate population genetic studies on

this important bufonid species.

S. J. Janani � R. Sharma � R. K. Aggarwal (&)

Centre for Cellular and Molecular Biology (CSIR-CCMB),

Uppal Road, Tarnaka, Hyderabad 500007,

Andhra Pradesh, India

e-mail: rameshka@ccmb.res.in

K. Vasudevan

Wildlife Institute of India, P.O. Box 18, Dehradun 248 001, India

S. K. Dutta

P.G. Department of Zoology, North Orissa University,

Baripada 757003, India

123

Conservation Genet Resour

DOI 10.1007/s12686-012-9846-8

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In this study, to identify and develop Asian toad specific

microsatellite markers, total genomic DNA was isolated

from the thigh muscle tissue using the standard phenol:

chloroform extraction method (Sambrook et al. 1989),

which was then used to construct a small-insert SSR-enri-

ched genomic library following the method described earlier

by Aggarwal et al. (2004). Briefly, the process involved:

digestion of genomic DNA with frequent 4-base cutters

(HaeIII and RsaI restriction enzymes), elution of agarose-

gel fractionated fragments of 0.3–1.0 kb in size, ligation of

the eluted fragments to Mlu-adaptors (Edwards et al. 1996),

SSR-enrichment of the adaptor-ligated genomic fragments

by Solution hybridization with biotinylated oligonucleotide

probes [(GA)15 (CA)15 (AGA)10 (CAA)10] and solid-phase

separation using strepdavidin coated magnetic beads (Dyn-

abeads, DYNAL), followed by cloning of the selected DNA

fragments into pTZ57R/T vector (Fermentas, USA) and

E. coli DH5a host cells to get the SSR-enriched genomic

library. Plasmids were isolated and cloned inserts were

amplified/sequenced using M13 universal primers on an

automated ABI 3730xl DNA Analyzer following manufac-

turer’s instructions (Applied Biosystems Inc.). Sequencing

of *150 clones, revealed 96 SSR-positive sequences, of

which [30 were found to be part of large tandem mini

satellite repeats and thus not considered for marker devel-

opment. Thirty-six of the remaining SSR-positive sequences

that had a minimum repeat core (simple or compound) of

18 bp were identified manually and used for primer design

using the Primer3 tool (http://biotools.umassmed.edu/bio

apps/primer3_www.cgi). Each forward primer was 50-tag-

ged with FAM fluorophore. The standardization of PCR

amplification conditions (annealing temperature and Mg??

ion concentration) were attempted for all the 36 primer pairs,

and the 14 working primer-pairs (that gave good, clean and

robust amplicons) were further tested for their potential

utility as genetic markers using a panel of 44 unrelated

samples of D. melanostictus collected from south India. All

PCR amplifications were performed in PTC-200 thermal-

cycler (MJ Research Inc.) in 15 ll reaction volumes that

comprised 5 ng template DNA, 2 pM of each primer, 1U

AmpliTaqGold DNA polymerase (Applied Biosystems,

USA), 1 9 PCR buffer, 100 lM of each dNTP and 1.5 mM

MgCl2. PCR conditions comprised: initial denaturation at

94 �C for 10 min, followed by 35 cycles of 3-step amplifi-

cation—(denaturation at 94 �C for 30 s, annealing at primer

specific temperature for 30 s and extension at 72 �C for

45 s), and a final extension at 72 �C for 15 min. The

SSR amplicons were resolved by fragment analysis on

ABI-3730xl DNA Analyzer with LIZ500 as internal size

standard, and were precisely sized using the software

GeneMapperTM ver3.7 (Applied Biosystems, USA). The 14

primer pairs/validated SSRs were also tested for their

cross-species transferability using four of the congeners

Duttaphrynus scaber, Duttaphrynus stomaticus, Duttaphry-

nus himalayanus and a new D. melanostictus cryptic species

(our unpublished data).

Characteristics of the new SSR markers, such as, marker

designation, repeat motifs, primer sequences, allele attri-

butes (number of alleles, size range of alleles, observed and

expected heterozygosity, polymorphism information con-

tent) and GenBank accessions are summarized in Table 1,

while their cross-species transferability status is shown in

Table 2. Nine of the 14 SSRs were found to be highly

polymorphic in the 44 Asian toad individuals, while one

Table 2 Cross-species transferability of the D. melanostictus microsatellites

Locus D. scaber D. stomaticus D. himalayanus D. melanostictus sp2

n/status/Na Allele range N/status/Na Allele range N/status/Na Allele range N/status/Na Allele range

DMccmb04 NA 4/P/6 150–193 NA 12/P/12 158–209

DMccmb08 NA NA NA 10/P/10 191–248

DMccmb09 NA NA NA 10/P/7 152–169

DMccmb12 NA NA 3/P/6 259–295 10/P/7 200–252

DMccmb13 3/P/3 128–158 2/P/2 178, 211 NA 11/P/13 136–171

DMccmb24 4/P/5 120–136 4/P/5 124–134 3/M/1 124, 130 12/P/9 124–175

DMccmb27 4/P/6 252–279 4/P/3 266–274 3/M/1 249 12/P/9 259–289

DMccmb28 4/P/5 121–155 4/P/4 120–132 3/P/3 121–128 11/P/8 120–141

DMccmb32 4/P/6 185–201 NA 3/P/2 187, 191 9/P/7 176–239

DMccmb16 3/P/4 174–183 3/P/3 162–218 3/M/2 171, 250 9/P/8 181–245

DMccmb17 NA 4/M/1 188 NA 10/P/13 150–200

DMccmb02 4/P/3 100–136 4/P/4 130–146 3/M/2 126, 133 9/M/2 126, 133

DMccmb22 NA NA NA 10/P/10 122–151

DMccmb29 4/P/4 180–211 4/P/11 172–233 3/P/3 182–202 12/P/17 152–231

n number of samples, Na number of alleles observed, NA no amplification, p polymorphic

Conservation Genet Resour

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(DMccmb16) was monomorphic in the tested samples. For

the nine polymorphic markers, the PIC values ranged from

0.518 to 0.918 amplifying 6–22 alleles/markers with

observed heterozygosity ranging from 0.477 to 0.955. None

of these markers showed linkage disequilibrium, but two

(DMccmb13, DMccmb28) deviated from Hardy–Weinberg

equilibrium showing heterozygote deficit, which is indicated

to be due to null alleles (Table 1). Of the remaining four

markers, two markers, DMccmb22 and DMccmb29,

amplified 1–4 alleles per individual and were highly poly-

morphic in the test samples, whereas the other two markers

DMccmb02 and DMccmb17 amplified only two alleles that

were almost fixed in the test population. In our opinion,

these markers probably represent duplicated loci as have

been observed in other animal/plant systems (Hendre et al.

2008). Notably, all the new markers also showed cross

species transferability to varying degree (overall 60 % for

three congeners and 100 % to closely related cryptic

species).

Thus, in this study, we have developed and validated 14

new SSR markers of Asian toad, which are expected to be

greatly helpful in undertaking population genetics studies

on this important generalist species, as well as, other

related bufonids.

Acknowledgments The authors thank the Department of Biotech-

nology, India for the financial support under the CCMB-WII-NOU

collaborative project (DBT, Govt of India, BT/PR8354/NDB/51/141/

2006) on anuran DNA barcoding; and the Council for Scientific and

Industrial Research for the lab facilities; Phanindranath and Justin for

the technical support extended for the study.

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