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253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus)

August 2008
Conservation Genetics 10(4):963-980

DOI:10.1007/s10592-008-9600-7

Authors:

Latonia Miles

Steward Health Care System

Sally R Isberg

Centre for Crocodile Research, Noonamah, Northern Territory, Australia

Chris Moran

The University of Sydney

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Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425.

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TECHNICAL NOTE

253 Novel polymorphic microsatellites for the saltwater crocodile

(Crocodylus porosus)

Lee G. Miles ÆSally R. Isberg ÆChris Moran Æ

Cris Hagen ÆTravis C. Glenn

Received: 14 April 2008 / Accepted: 18 April 2008 / Published online: 1 May 2008

ÓSpringer Science+Business Media B.V. 2008

Abstract Genomic elucidation and mapping of novel

organisms requires the generation of large genetic resources.

In this study, 253 novel and polymorphic microsatellite loci

were isolated and characterized for the saltwater crocodile

(Crocodylus porosus) by constructing libraries enriched for

microsatellite DNA. All markers were evaluated on animals

obtained from Darwin Crocodile Farm in the Northern

Territory, Australia, and are intended for future use in the

construction of a genetic-linkage map for the saltwater

crocodile. The 253 loci yielded an average of 4.12 alleles per

locus, and those selected for mapping had an average

polymorphic information content (PIC) of 0.425.

Keywords Estuarine crocodile Reptile SSRs 

Primers Tetra-nucleotide repeats Tri-nucleotide repeats 

Di-nucleotide repeats Enrichment

Microsatellites have been isolated from a number of

crocodilians including Alligator mississipiensis (Glenn

et al. 1998), Caiman latirostris (Zucoloto et al. 2002),

Crocodylus moreletii (Dever and Densmore 2001), Croc-

odylus johnsoni and Crocodylus porosus (Fitzsimmons

et al. 2001). These markers have been employed in a range

of species to assess genetic diversity, mating behaviour,

hybridisation, as well as dispersal systems (Glenn et al.

1996,1998; Fitzsimmons et al. 2001; Dever et al. 2002;

Dever and Densmore 2001; Davis et al. 2002; Dessauer

et al. 2002; Verdade et al. 2002; Isberg et al. 2004;

Zucoloto et al. 2006).

The saltwater crocodile (Crocodylus porosus), which is

distributed throughout much of South East Asia, is relatively

uncharacterized genetically. Microsatellite markers have

previously been generated in C. porosus for use in parentage

and species identiﬁcation (Fitzsimmons et al. 2001,2002;

Isberg et al. 2004). Using the 21 microsatellites available for

C. porosus, Isberg et al. (2006) recently demonstrated the

ﬁrst evidence of genetic linkage in the order Crocodylia, as

well as within the class Reptilia. These results have

encouraged further investigation. Consequently, new

resources, including both pedigrees and markers have been

developed to further characterize the genome of C. porosus.

Herein we describe the isolation, primer development, and

polymerase chain reaction (PCR) conditions to amplify a

total of 253 novel and informative microsatellites compris-

ing 41 dinucleotide, 30 trinucleotide, 172 tetranucleotide

and 10 mixed-composite microsatellite DNA loci from

C. porosus.

Small insert libraries enriched for microsatellite repeats

were constructed following the protocol of Glenn and

Schable (2005). Brieﬂy, the genomic C. porosus DNA was

digested with Rsa I, ligated to SuperSNX linkers (Forward

50-GTTTAAGGCCTAGCTAGCAGCAGAATC; Reverse

50-GATTCTGCTAGCTAGGCCTTAAACAA), indepen-

dently hybridized to various mixes of biotinylated

oligonucleotides probes [(AACC)

, (ATCC)

, (AAAC)

L. G. Miles (&)S. R. Isberg C. Moran

Faculty of Veterinary Science, University of Sydney, Room 513,

RMC Gunn Building, Sydney, NSW 2006, Australia

e-mail: l.miles@usyd.edu.au

S. R. Isberg

Porosus Pty Ltd, P.O. Box 86, Palmerston, NT 0831, Australia

C. Hagen T. C. Glenn

Savannah River Ecology Laboratory, University of Georgia,

P.O. Drawer E, Aiken, SC 29802, USA

T. C. Glenn

Department of Environmental Health Science, University

of Georgia, Athens, GA 30602, USA

123

Conserv Genet (2009) 10:963–980

DOI 10.1007/s10592-008-9600-7

Table 1 Microsatellite primer sequence and locus information including sample size (N), observed number of alleles (K), observed heterozygosity (H

obs

), expected heterozygosity (H

exp

), and

polymorphic information content (PIC) for Crocodylus porosus

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpDi04 GTTTTTCTAAACAGTCCAGGATGA (AC)

TD65 148–176 10 7 0.800 0.742 0.650

EU593278 CAGTCGGGCGTCATCATAAATTTCACTAGATGCCATAA

CpDi06 CAGTCGGGCGTCATCATGTTGGGCACTTTGAAC (AC)

TD65 254–307 20 11 0.700 0.768 0.721

EU593279 GTTTAAGAAAAATGGTGGAAAAC

CpDi08 CAGTCGGGCGTCATCACCAGGTTGGAAAATATCT (AC)

TD65 285–301 10 4 0.400 0.337 0.270

EU593281 GTTTTGGTTAACACGTGGTAACTA

CpDi10 GTTTCCCTTTTGCATAGACTAGATA (AC)

(CT)

TD65 261–269 20 4 0.300 0.246 0.211

EU593283 CAGTCGGGCGTCATCACCTAACTTTGGTCCTGAAG

CpDi11 GTTTTGAAATGCCACTAATACAC (AC)

TD65 185–196 20 5 0.700 0.686 0.608

EU593284 CAGTCGGGCGTCATCATAGAGGCAGATAGACAAGAG

CpDi12 CAGTCGGGCGTCATCATCTGAGAAAGAGGGACAC (AG)

TD65 243–253 10 4 0.200 0.189 0.164

EU593285 GTTTCGCACTGGTTCTACACA

CpDi13 GTTTGTGTCAGCCTATACATGTT (AC)

TD65 329–355 20 6 0.600 0.738 0.673

EU593286 CAGTCGGGCGTCATCAGTCTCAGAGTATGCCTAGAA

CpDi15 GTTTAACCCAAGAACTTACAACT (AC)

TD65 279–294 10 7 0.800 0.663 0.587

EU593288 CAGTCGGGCGTCATCAGTAAATCGGGACAGTA

CpDi16 CAGTCGGGCGTCATCACTTCCTAGCCAACACA (AG)

TD65 161–177 10 3 0.300 0.279 0.247

EU593289 GTTTTTATTCAGTGAGTGGGTAGA

CpDi21 CAGTCGGGCGTCATCAAAACAGTTGGCTCTGTG (AC)

TD65 177–192 20 6 0.450 0.518 0.469

EU593290 GTTTATACTTCCTGTGGCATCAT

CpDi23 GTTTTCAAGTGACCCCTTTTC (AC)

TD55 231–256 10 5 0.800 0.716 0.627

EU593292 CAGTCGGGCGTCATCATATTTCTTGTTTGGCACAT

CpDi24 GTTTACACAGCCCTAATACACA (AC)

TD65 156–189 20 6 0.450 0.458 0.416

EU593293 CAGTCGGGCGTCATCATTTTGAGTGGGGATAATAA

CpDi28 CAGTCGGGCGTCATCACTATGCACTCCCTGATTTAAG (AC)

TD65 135–142 20 2 0.350 0.409 0.319

EU593295 GTTTCCCACTCACGAATCTAAAG

CpDi29 GAAACAGCCAAATGTGAG (AC)

TD65 228–254 20 4 0.250 0.549 0.436

EU593296 CAGTCGGGCGTCATCAGGTAGCTCCAAGTAGTTTATT

CpDi31 GTTTGTGCGACTCAACAGTGTGT (AC)

TD65 350–361 10 3 0.500 0.416 0.347

EU593297 CAGTCGGGCGTCATCAGGCGTGTTTGTTAGAA

CpDi33 CAGTCGGGCGTCATCATTCAGATGTCAGCTGGATAG (AG)

TD65 409–417 10 5 0.200 0.353 0.303

EU593299 GTTTTTGAGTGATATGGCAATAAA

CpDi41 CAGTCGGGCGTCATCAGGGCTGTTTATTGTTAGTT (AC)

TD65 207–213 20 3 0.400 0.465 0.398

EU593301 GTTTCCTCCTCCAGTGAAAGAC

964 Conserv Genet (2009) 10:963–980

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpDi42 GTTTTTCAGTTTATTTGCCAAAG (AC)

TD65 114–124 20 4 0.650 0.677 0.586

EU593302 CAGTCGGGCGTCATCAGATTGGGGAGGGAAGT

CpDi43 GTTTACATCCAATTGTGTTGTAAT (AC)

TD65 195–201 10 4 0.300 0.521 0.372

EU593303 CAGTCGGGCGTCATCAATAGCTGTTTTGGCAGTAGT

CpDi48 CAGTCGGGCGTCATCACTGTTTGATATGGCAGAGA (AG)

TD65 180–234 10 4 0.400 0.699 0.611

EU593304 GTTTCCCTCCTTTCAGCAAGTA

CpDi54 CAGTCGGGCGTCATCAAAAACGGTTGCTAGACTA (AG)

TD65 379–395 10 6 0.700 0.647 0.544

EU593305 GTTTGATGCCAAATTTAGGTCATA

CpDi58 CAGTCGGGCGTCATCACTTTGATAACAGGAACTCTG (AG)

TD65 421–437 10 4 0.400 0.542 0.424

EU593306 GTTTTGCATTGCCTATAAATCATA

CpF202 GTTTCCCACTGTGAGTGAGT (AC)

TD55 213–229 10 5 0.700 0.695 0.611

EU593307 CAGTCGGGCGTCATCAGTCTCTGGGATTTGTAGTCTA

CpF401 CAGTCGGGCGTCATCACTAAAAACAAAACCAAACAA (AAAC)

TD65 364–371 10 2 0.500 0.479 0.351

EU593310 ATTCGCACTCACCAGTAA

CpF509 CAGTCGGGCGTCATCAACACAAAGGAGCATACAC (AC)

TD65 328–339 20 5 0.450 0.491 0.448

EU593315 GTTTAGCCAATTCCCATATCT

CpP1001 CAGTCGGGCGTCATCAGCAGAAACGAAAGATGTAGT (AGAT)

TD65 260–277 10 3 0.300 0.416 0.347

EU593396 GTTTGGTTCCGTTGGTTTATT

CpP1002 CAGTCGGGCGTCATCATTTGGGCTCCACAATTC (AAAC)

TD65 276–401 10 8 0.800 0.737 0.651

EU593397 GTTTCATCCCTTGGAGCTCTT

CpP1003 CAGTCGGGCGTCATCATCCAGTCCAGGGCTGAAG (AGAT)

TD65 303–319 10 3 0.440 0.462 0.386

EU593398 GTTTGGCCATGTGTTCAATCAAT

CpP1004 CAGTCGGGCGTCATCACTCAAGTGGGCTGTGT (ACAT)

TD65 193–203 10 3 0.400 0.505 0.365

EU593399 GTTTAGGTCACTATCCCAACTA

CpP1005 CAGTCGGGCGTCATCACTTGGGTCTAATGAGGACTG (AGAT)

TD65 236–249 10 3 0.300 0.574 0.441

EU593400 GTTTATTGAATACCCAAACTCAGA

CpP103 CAGTCGGGCGTCATCACAATCTCTCGTTTTCTTGA (AGAT)

TD65 327–351 10 4 0.500 0.503 0.404

EU593321 GTTTGCTTCCTTTAATAATAGTCAC

CpP104 GTTTAGTTTGCCCACAGTTTATTTG (ATCT)

TD65 277–290 10 2 0.500 0.521 0.372

EU593322 CAGTCGGGCGTCATCATTACACTTGGAAACATTTCAC

CpP106 CAGTCGGGCGTCATCATAGAAAGAAATGGGCTAGTGT (ATAG)

TD65 234–254 20 4 0.350 0.309 0.276

EU593323 GTTTGTATACCCAGGAAAGATTTTG

CpP107 CAGTCGGGCGTCATCATGGCTTGCATTAAGAT (AAAC)

TD55 166–179 10 4 0.600 0.721 0.632

EU593324 GTTTCTCTAAAAACCAAAAACA

CpP1103 GTTTAGAGATCTTTCCCATAACTGTT (AAAC)

TD65 173–185 10 2 0.500 0.395 0.305

EU593405 CAGTCGGGCGTCATCACCATCTTCATGCCTTAAC

Conserv Genet (2009) 10:963–980 965

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP112 CAGTCGGGCGTCATCATATCACATTTACCCAAAGAA (AAAC)

TD55 208–210 10 2 0.400 0.505 0.365

EU593327 GTTTCCAGTTGCCCTTAGATAG

CpP113 CAGTCGGGCGTCATCACAGCTAGATCAGCAGTTCT (ACAT)

TD55 323–326 10 2 0.300 0.521 0.372

EU593328 GTTTGTGGCACATTTCACTCTTAT

CpP114 GTTTGCTACCCAGATTATATCTTA (AGAT)

TD65 185–195 20 3 0.600 0.540 0.431

EU593329 CAGTCGGGCGTCATCACTATTCGGGAACAG

CpP115 GTTTATCCCCTTACTTCACAT (AAAC)

TD55 261–278 10 4 0.600 0.511 0.406

EU593330 CAGTCGGGCGTCATCAGAAGGTTACATAGGGTTAAT

CpP116 CAGTCGGGCGTCATCAATGTGACAGCCAATGTG (AGAT)

TD55 269–274 20 2 0.500 0.508 0.372

EU593331 GTTTACCTCCTATGGATTGACTG

CpP1201 CAGTCGGGCGTCATCAGCAGATGTGGCAAATAGTTC (ACAG)

TD65 123–141 10 3 0.300 0.395 0.305

EU593406 GTTTAAACTCGCCTCACTACAGA

CpP1207 GTTTCTCTTCTGGGTGTGTTAGTAT (AAAC)

TD65 195–199 10 2 0.100 0.100 0.090

EU593408 CAGTCGGGCGTCATCAATTGGGAAGTTCATCA

CpP1208 GTTTAAAACTGGCAAAATGAC (AAAC)

TD65 267–275 10 3 0.500 0.479 0.351

EU593409 CAGTCGGGCGTCATCATCTCACTTGCAGCCTCTA

CpP121 CAGTCGGGCGTCATCAATATTTGTTTCTGGGATCA (AGAT)

TD65 162–187 20 6 0.550 0.621 0.531

EU593333 GTTTAGGAAATGAGCCCTAATAGT

CpP1301 GTTTGATTTCTCTGACCCTAAGT (AAAC)

TD55 190–202 10 3 0.200 0.337 0.269

EU593412 CAGTCGGGCGTCATCACCACTGTATGCTGTCAA

CpP1303 GTTTGACATGGCTTTCTTATACTC (AAAC)

TD55 235–239 20 3 0.450 0.465 0.371

EU593414 CAGTCGGGCGTCATCAAATCCATGCCAGTTTA

CpP1305 GTTTAATAATCCCTGTGCTCATACTA (AAAC)

TD55 289–299 10 3 0.200 0.279 0.247

EU593415 CAGTCGGGCGTCATCACTACAGCAACTACCCAACT

CpP1306 CAGTCGGGCGTCATCATTCTCTCTAGGAGCCACTCAC (ACTC)

TD65 103–250 10 14 0.800 0.863 0.798

EU593416 GTTTAGGGAGACATCTAGGAAGAAC

CpP1308 CAGTCGGGCGTCATCAACCTGAAAATGGATACTG (AAAC)

TD55 153–271 10 8 0.700 0.732 0.665

EU593418 GTTTACGCTTGTTAACTTCACT

CpP1401 GTTTAAAAGTAACCCAAAATACACA (AGAT)

TD55 171–192 20 6 0.500 0.622 0.547

EU593419 CAGTCGGGCGTCATCACTTCGGCATCTGATTC

CpP1404 GTTTCCCAATAACTCCATAACATAG (AGAT)

TD65 290–298 20 4 0.400 0.445 0.359

EU593421 CAGTCGGGCGTCATCAGGGCTTCAGCACACTA

CpP1405 CAGTCGGGCGTCATCATCCTCTCCACAAAACATCTAAT (ACCT)

(AGAT)

TD65 233–242 10 2 0.500 0.395 0.305

EU593422 GTTTACGTGGCCTTAGTCACA

CpP1406 CAGTCGGGCGTCATCACTCCTCTCCACAAAACATCT (AGAT)

TD65 230–238 10 2 0.500 0.395 0.305

EU593423 GTTTCGTGGCCTTAGTCACATA

966 Conserv Genet (2009) 10:963–980

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP1407 CAGTCGGGCGTCATCAGAAATTCCAGCTCAGTTCA (AGAT)

TD65 204–108 10 2 0.100 0.100 0.090

EU593424 GTTTCTCAAATTCCCAGTTCTAAA

CpP1409 GTTTATGCCCTACTGGTTATCTATC (AGAT)

TD65 255–289 20 8 0.550 0.586 0.541

EU593425 CAGTCGGGCGTCATCAGGGAAGGGGATTTAATAAT

CpP1413 GTTTACACACATTGGCTTTTTTAT (AGAT)

TD55 176–186 10 2 0.100 0.100 0.090

EU593427 CAGTCGGGCGTCATCAGGGGTTGGTCATTCAG

CpP1415 GTTTCAGCCCAAATGTCTATGA (ACTC)

TD65 206–222 10 3 0.300 0.279 0.247

EU593428 CAGTCGGGCGTCATCAGTGCTACCCATCAGTTGTA

CpP1416 GTTTCAAATATATCTTGCCATACA (ACAT)

TD65 212–214 20 2 0.300 0.353 0.283

EU593429 CAGTCGGGCGTCATCAGAACAGCGAAAGAACA

CpP1502 CAGTCGGGCGTCATCAAACCAGAAGCAGAAACT (AGAT)

TD65 115–119 10 2 0.700 0.521 0.372

EU593430 GTTTCTATCCCTATATCTCTATGTC

CpP1512 GTTTGCCGAGACAATCAGTCAG (ACAG)

TD65 224–240 10 3 0.200 0.195 0.177

EU593432 CAGTCGGGCGTCATCAAAGATGGGCAAACAGAAC

CpP1603 GTTTGATTCAGATTCGGAGATTT (AGAT)

TD65 312–328 20 4 0.150 0.145 0.136

EU593433 CAGTCGGGCGTCATCAGTGGGCTGTGCAGTTT

CpP1604 CAGTCGGGCGTCATCAAAGGGAGGGCAGACTTGT (AGAT)

TD65 133–141 10 2 0.500 0.479 0.351

EU593434 GTTTGAAACGGCCAGTGAAAT

CpP1607 GTTTAAACAAGATATCTCGATAGA (AGAT)

TD55 255–268 10 4 0.300 0.395 0.340

EU593435 CAGTCGGGCGTCATCATGAATGGGAGATTAAACT

CpP1608 CAGTCGGGCGTCATCAGTAACCAGGGCATCTGTT (ACAT)

TD55 273–287 10 4 0.500 0.479 0.351

EU593436 GTTTATGCATGTATGGGTGTTT

CpP1610 CAGTCGGGCGTCATCATAGAGGGATTTTGACTGT (AGAT)

TD65 296–304 20 3 0.450 0.465 0.371

EU593437 GTTTGATTATTTTGTCTGGGTTCTT

CpP1611 GTTTGCCAGTCACAACACATCTAT (AGAT)

TD65 292–304 10 2 0.100 0.100 0.090

EU593438 CAGTCGGGCGTCATCAAGGATTTCAGGCTGAT

CpP1708 GTTTCCATTATGGCAAATCTTGTA (ACTC)

TD65 213–226 20 4 0.500 0.445 0.390

EU593440 CAGTCGGGCGTCATCAATTGGGATCTTGGATCTG

CpP1714 GTTTCTGAACAGGTTGTCTTAGTTA (AAAC)

TD55 277–289 10 4 0.400 0.328 0.273

EU593441 CAGTCGGGCGTCATCAGTTCAGTATGCCAGTCA

CpP201 CAGTCGGGCGTCATCATTATTCAAACCCATCATAG (AGAT)

TD55 182–200 10 4 0.200 0.337 0.269

EU593334 GTTTAAAAAGCCTACTGTCTACAT

CpP202 GTTTAAATTGTTATGCCAGTTCTG (AGAT)

TD55 272–310 10 5 0.700 0.574 0.441

EU593335 CAGTCGGGCGTCATCATACATGCAGCCGTGTC

CpP203 CAGTCGGGCGTCATCAGTCCATTGCCAGTTGTAA (ACAG)

TD65 205–283 10 10 0.700 0.832 0.763

EU593336 GTTTCTGAGGGTGAACTTTAGAA

Conserv Genet (2009) 10:963–980 967

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP205 GTTTACCCATCATGTATTTTTAGA (AGAT)

TD65 326–338 20 3 0.500 0.513 0.375

EU593338 CAGTCGGGCGTCATCAGAATCACCACCCAAAG

CpP207 CAGTCGGGCGTCATCAGGAAGGCAGGTAGTTTAGAA (AGAT)

TD65 119–123 10 2 0.600 0.505 0.365

EU593340 GTTTGATGTGCCCATGTAAGATA

CpP208 CAGTCGGGCGTCATCACACATGGCTTTTGTTCTGAG (AGAT)

TD65 211–295 10 11 0.900 0.937 0.880

EU593341 GTTTCCTGCAAAATGTTCTCCTA

CpP209 GTTTACTTAGGGTTTGCTAGTCAC (AGAT)

TD55 288–316 10 5 0.700 0.595 0.482

EU593342 CAGTCGGGCGTCATCATACTGCTAATGCGAGACA

CpP213 CAGTCGGGCGTCATCATGTTGGGCACTTTGAACT (AC)

TD65 257–308 10 5 0.700 0.632 0.540

EU593344 GTTTAGAAGAAAAATGGTGGAAAAC

CpP214 CAGTCGGGCGTCATCAGTCTGTCTTTGCAGTCTTG (AAAG)

(AG)

TD65 274–335 10 9 0.600 0.832 0.760

EU593345 GTTTTCCTGGAATAAACTGACTA

CpP218 GTTTGGCATTTGAATTATTAACT (ACCC)

TD55 164–187 20 2 0.300 0.385 0.305

EU593346 CAGTCGGGCGTCATCACTGGCAAATCACTTCTG

CpP219 CAGTCGGGCGTCATCAGTTGTCCTGGAGAACA (AC)

TD55 185–195 10 5 0.700 0.668 0.559

EU593347 GTTTCCCTTTGTAACAATGTAGT

CpP2201 GTTTCAAGTGACCCCTTTTCAG (ACAG)

TD65 230–256 20 6 0.700 0.617 0.525

EU593442 CAGTCGGGCGTCATCATATTTCTTGTTTGGCACATC

CpP2202 CAGTCGGGCGTCATCAGCAACAAAAGACCTTGACA (AAAG)

TD65 197–220 10 7 0.900 0.805 0.728

EU593443 GTTTGGTTGGGTGGAATTATATAC

CpP2203 GTTTAGTTTCACTTGGGATTCTGTA (AAAC)

TD65 209–218 10 3 0.900 0.521 0.372

EU593444 CAGTCGGGCGTCATCATTTACCTGCGTGCTATTT

CpP2206 GTTTAGGCCAGTTCTTATCTACAT (AAAG)

TD65 258–270 20 4 0.750 0.651 0.559

EU593445 CAGTCGGGCGTCATCAAAGTTCTCCCCACTAAAG

CpP2209 GTTTATGACAAAAGGGATGTGATA (AAAC)

TD55 271–274 10 2 0.400 0.442 0.332

EU593446 CAGTCGGGCGTCATCACAGCATTTGGCTTGTATAA

CpP2502 GTTTCCCCACTCACCATATAG (AGAT)

TD65 142–147 10 2 0.100 0.100 0.090

EU593450 CAGTCGGGCGTCATCAGATAATTGGGTTTCCTACA

CpP2504 CAGTCGGGCGTCATCACTCATATTTCCCAACTATCAC (AGAT)

TD65 306–367 20 8 0.750 0.760 0.704

EU593451 GTTTCATTCCCACAATACACATAA

CpP2505 GTTTATGGCCTGTTCATTCTCT (AGAT)

TD65 133–139 10 3 0.100 0.100 0.090

EU593452 CAGTCGGGCGTCATCAGTTACAGGCAAATGGTCTT

CpP2508 GTTTCGATTCGGATTCAGAGAT (AAC)

TD65 181–187 10 2 0.700 0.521 0.372

EU593453 CAGTCGGGCGTCATCAAGTGGCTCATGGTTCAT

CpP2509 CAGTCGGGCGTCATCAACACAATGTAGGTGGTTCT (AGAT)

TD65 324–332 10 2 0.400 0.337 0.269

EU593454 GTTTCAAATACGTAGTTGGGATAA

968 Conserv Genet (2009) 10:963–980

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP2514 GTTTCTTTGCACACCCTATCTATC (AGAT)

TD65 177–194 20 4 0.500 0.601 0.506

EU593456 CAGTCGGGCGTCATCAGAGTTGAAGGGAAGTTTC

CpP2516 GTTTATTCAGGCCAATACAGAAC (AAC)

TD65 290–296 20 3 0.500 0.647 0.534

EU593457 CAGTCGGGCGTCATCAGTGAGCTTTGGGATGTT

CpP2704 GTTTCCCAAGCCTTCAAGTAGAT (AGAT)

TD65 156–172 20 3 0.250 0.449 0.390

EU593458 CAGTCGGGCGTCATCATGGCATGAAATACTATTGAC

CpP2705 CAGTCGGGCGTCATCAGCACAGCAGATCTCTCT (AGAT)

TD55 165–176 10 4 0.400 0.471 0.346

EU593459 GTTTAAAGGGTTTTGTTGTATATG

CpP2706 CAGTCGGGCGTCATCATCTCTTCCAGGAAAATATGA (AAAC)

TD55 334–342 20 3 0.350 0.396 0.247

EU593460 GTTTGATTTTGGGAAGAGCTAGTT

CpP2708 GTTTAAGAAGGGCATATTTACTG (ACAG)

TD55 247–258 10 3 0.300 0.395 0.305

EU593461 CAGTCGGGCGTCATCAGAGTTTCCCTTTCTCAAT

CpP2808 GTTTATGCCATACAAAGAAGAC (ACT)

TD55 273–286 10 5 0.700 0.647 0.562

EU593463 CAGTCGGGCGTCATCAATCTAATCTTGCCTTACTGT

CpP2813 CAGTCGGGCGTCATCAATGGCTGAGGCACTAGTAG (AGAT)

TD65 356–364 53 3 0.000 0.527 0.370

EU593464 GTTTAGGCAGCCAAACTATCAC

CpP2815 GTTTCCCACCTCAGTAAAAACTAT (ATC)

TD65 166–174 20 4 0.450 0.358 0.288

EU593465 CAGTCGGGCGTCATCAGCTATACCCTTCCTGTGTG

CpP2816 CAGTCGGGCGTCATCACTAAAGAATGCTGGATCAA (ATC)

TD65 291–303 65 5 0.300 0.416 0.347

EU593466 GTTTCAGAGGCATAAACAAACAAT

CpP2902 GTTTCTTATGAACAGTGGGTAGTT (ATC)

TD55 383–389 20 3 0.550 0.457 0.346

EU593468 CAGTCGGGCGTCATCATCCTGTTTTGGTGCTACT

CpP2904 CAGTCGGGCGTCATCAGGTTTCCTCCACACAA (AAC)

TD65 223–231 10 3 0.700 0.563 0.436

EU593469 GTTTCTATGCCACTAACATTAACAG

CpP2905 CAGTCGGGCGTCATCAGATAGTTTTCTAGGGTTGTTTT (ATC)

TD55 387–390 10 2 0.300 0.521 0.372

EU593470 GTTTATATGCGGTATCACTGTTATT

CpP2908 CAGTCGGGCGTCATCAGGAATTGGGAAATGAA (AAC)

TD55 273–287 10 3 0.400 0.442 0.332

EU593471 GTTTGAATAGCCCTTCTGTAACA

CpP2909 GTTTCATACCATCTAGCCCTGTT (ACT)

TD55 182–211 10 4 0.300 0.279 0.247

EU593472 CAGTCGGGCGTCATCACCTCATCTACCCCAGACT

CpP3001 CAGTCGGGCGTCATCAGTAGCATGGCATAAGTGT (AATG)

TD65 200–212 10 4 0.400 0.611 0.492

EU593473 GTTTAGAATGCCATAAATCACAT

CpP3004 CAGTCGGGCGTCATCAGGAGTGAATCTATGCCAGC (AGAT)

(ACCT)

TD55 142–156 20 5 0.550 0.591 0.500

EU593474 GTTTAAAATGTTTTCATATGGTCG

CpP3008 CAGTCGGGCGTCATCAACAACTGGCACATCTCA (ACAG)

TD65 370–432 10 9 0.700 0.826 0.765

EU593475 GTTTCCCGTAGCCTCCTACTG

Conserv Genet (2009) 10:963–980 969

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP301 CAGTCGGGCGTCATCATACATGCATTGGCTCATC (AG)

(CT)

TD65 296–303 10 4 0.400 0.551 0.436

EU593349 GTTTCCTAACTTTGGTCCTGAAG

CpP302 GTTTGGAACCCAAGAACTTACAAC (AC)

TD65 188–217 20 9 0.750 0.762 0.707

EU593350 CAGTCGGGCGTCATCATTGGGTTTAGTCAGCACATA

CpP303 GTTTGAAATGCCACTAATACACA (AC)

TD65 233–243 10 5 0.700 0.660 0.579

EU593351 CAGTCGGGCGTCATCATATAACAAGGCATCAATCAT

CpP305 GTTTGTAGCTGGAACCTGATAGTG (AC)

TD65 183–189 20 3 0.350 0.304 0.265

EU593352 CAGTCGGGCGTCATCAGGTTAACACGTGGTAACTACA

CpP307 CAGTCGGGCGTCATCAGAAACCAGAGGCCAATA (ACTC)

TD55 345–377 20 6 0.750 0.705 0.645

EU593353 GTTTCTTGTCTTTGGCAGATT

CpP309 GTTTAATACCTGGCATGTGTTCTTC (AAAC)

TD65 225–306 20 23 0.900 0.928 0.898

EU593354 CAGTCGGGCGTCATCACATCAGGTTGGCATTTCA

CpP311 GTTTGCGCACACACTATATCA (AAAC)

TD65 277–363 10 17 0.900 0.916 0.857

EU593355 CAGTCGGGCGTCATCATAACATGGCAACACATTT

CpP314 GTTTGAAATGCCACTAATACACACA (AGAT)

TD55 260–279 20 7 0.550 0.565 0.505

EU593357 CAGTCGGGCGTCATCACCAATTCTTCAGGTCCTTAT

CpP315 CAGTCGGGCGTCATCATGAGAAAGAGGGACACAGAG (AG)

TD65 249–254 10 3 0.400 0.337 0.269

EU593358 GTTTCGCACTGGTTCTACACACA

CpP316 GTTTGGCATATGAGGGTGTCA (AC)

TD65 116–124 10 5 0.600 0.689 0.592

EU593359 CAGTCGGGCGTCATCACCACCAAATCAGTCTATCA

CpP3202 GTTTAGGTCTGAAGCCGACTT (AAAC)

TD65 384–399 10 3 0.400 0.468 0.420

EU593476 CAGTCGGGCGTCATCACTAAAGGGCTAGGGAATAA

CpP3205 GTTTGTGTTGGCATGAATGAG (AAAC)

TD65 227–235 10 2 0.100 0.294 0.239

EU593477 CAGTCGGGCGTCATCAGGGACTCTCCAAGTAGAGAAAA

CpP3209 CAGTCGGGCGTCATCATACCAGATGCCAAATTGTC (AGAT)

TD65 361–386 10 7 0.600 0.689 0.588

EU593478 GTTTGCCCTCTTGTGTTCTCTC

CpP3211 CAGTCGGGCGTCATCATTAGAGTGCTGGCAACAC (AAAC)

TD65 325–336 20 3 0.300 0.467 0.351

EU593479 GTTTCTTTACTGGCAATTTACTCA

CpP3212 GTTTAGAGAGGGAGAGGAAAAGTC (AGAT)

TD65 250–254 10 2 0.300 0.268 0.222

EU593480 CAGTCGGGCGTCATCACTACACAGGGAAGGACAG

CpP3215 GTTTGCTCCGATTTTCCATAACA (AGAT)

TD55 334–374 20 6 0.500 0.655 0.574

EU593482 CAGTCGGGCGTCATCATGGCATATAGCAAAGCATAG

CpP3216 CAGTCGGGCGTCATCAGATTAATTCATTGGCTCTC (ACAG)

TD55 135–142 20 2 0.150 0.149 0.135

EU593483 GTTTATGCCTTTGCCTTTAG

CpP3217 GTTTCCCAAGCAGTCACTTT (AAAC)

TD55 132–140 20 3 0.400 0.417 0.371

EU593484 CAGTCGGGCGTCATCACGGAGGCATTACTGTTATA

970 Conserv Genet (2009) 10:963–980

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP3220 CAGTCGGGCGTCATCAGGAATTGGAGGAATCAGT (AAAC)

TD55 146–262 20 18 0.700 0.921 0.889

EU593486 GTTTGGCCAATGCTCTTTTA

CpP3301 CAGTCGGGCGTCATCAAGCAGTAGTGGCATTAAG (AAAC)

TD65 241–250 10 3 0.400 0.337 0.269

EU593487 GTTTGTCAGCATATGGCACTGT

CpP3303 CAGTCGGGCGTCATCATGTCTATTTTTGCCTTGTTT (AACC)

TD65 367–389 20 5 0.750 0.736 0.651

EU593488 GTTTGCCTAATTGCCTGAATAA

CpP3304 CAGTCGGGCGTCATCAGATTCAATGCGGACAG (ACAG)

TD65 204–211 10 2 0.200 0.442 0.332

EU593489 GTTTCTTTGCTTATCTCTGCCTATA

CpP3309 CAGTCGGGCGTCATCATTTATAGGCAAACACAGTC (AGAT)

TD55 177–197 20 5 0.900 0.750 0.688

EU593490 GTTTCCCTTTGCATTAATTCTA

CpP3313 CAGTCGGGCGTCATCACTTCTGTTACTTAGGGACTG (AGAT)

TD55 360–368 20 3 0.600 0.559 0.441

EU593491 GTTTAAAAACCCAGGCAAATA

CpP3314 CAGTCGGGCGTCATCAATTTCCTATGGCTGTAAC (AGAT)

TD65 306–326 20 5 0.600 0.538 0.480

EU593492 GTTTGTAGCTGAAGGGAGGATATA

CpP3401 CAGTCGGGCGTCATCATGGCACAATATGAAGAG (AAC)

TD65 185–192 10 3 0.400 0.442 0.332

EU593494 GTTTGGCCAAATTAAATAACAT

CpP3402 CAGTCGGGCGTCATCAGGCGTTGTTGTTGTTGT (AAC)

(ATC)

TD65 205–231 10 6 0.500 0.679 0.587

EU593495 GTTTGGCTAACCCTTCAATACTC

CpP3408 GTTTGGTTTGCCCTTGTCAG (ACT)

TD65 269–279 10 2 0.200 0.189 0.164

EU593496 CAGTCGGGCGTCATCACATGATTAGAGCAGCAGAG

CpP3416 CAGTCGGGCGTCATCAGTAAATGCAGGGTCAGA (AAC)

TD55 361–371 10 3 0.400 0.526 0.375

EU593498 GTTTGTCCCATTTATGTAACAG

CpP3507 CAGTCGGGCGTCATCACTAGCTTTGCCTTGTTC (AAC)

TD65 174–177 10 2 0.400 0.526 0.375

EU593499 GTTTGGGTCTCAGGATCTCTTTA

CpP3508 CAGTCGGGCGTCATCAAGGGGATGTAAACACT (AAC)

TD55 117–128 10 3 0.300 0.268 0.222

EU593500 GTTTATAACTATCATTGCCATTGT

CpP3601 CAGTCGGGCGTCATCAGCAAAGTCCATTGATTTAC (AAC)

TD55 162–180 20 3 0.300 0.268 0.238

EU593503 GTTTCGCATTCTCATTTGTTCT

CpP3603 CAGTCGGGCGTCATCATCTCTAAACACCCAACTA (AGAT)

TD65 373–378 20 2 0.200 0.262 0.222

EU593504 GTTTCACACTGATGGCTCATGT

CpP3604 CAGTCGGGCGTCATCAAAGTAAGGGTTTTTACAAC (AAC)

TD65 94–103 10 2 0.300 0.479 0.351

EU593505 GTTTAATAGTATAGGAGCCAAACA

CpP3610 CAGTCGGGCGTCATCAGGGCTTTAGACAATGACA (AAC)

TD55 233–240 10 2 0.400 0.337 0.269

EU593507 GTTTTGGCTGATGCTTGACTT

CpP4001 GTTTAGCCCATAGCAAATTTCTA (AGAT)

TD65 134–146 10 3 0.500 0.395 0.305

EU593509 CAGTCGGGCGTCATCAAGTGCTGGTGGACTGATAA

Conserv Genet (2009) 10:963–980 971

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP4004 CAGTCGGGCGTCATCACTGAATTGGGTGGAATAG (AGAT)

TD55 408–435 20 7 0.750 0.744 0.677

EU593510 GTTTATCCACATTTTTCCATGAC

CpP4006 CAGTCGGGCGTCATCAAGTGAGATTTGGGTATATTT (AGAT)

TD55 99–120 20 6 0.500 0.445 0.408

EU593511 GTTTCATTTCCTTACCATGATAG

CpP4008 CAGTCGGGCGTCATCATGCTCAGTCCCTTCTTAC (ACAT)

TD65 337–350 10 4 0.800 0.711 0.619

EU593512 GTTTAGGTGGGAGTTCAAAAATA

CpP4010 CAGTCGGGCGTCATCATTGAGGGCAGAGGTAAC (ACAT)

TD65 200–216 20 5 0.450 0.582 0.476

EU593513 GTTTGGATATCTGCCATCACATT

CpP4013 CAGTCGGGCGTCATCATTATAAATAGCCAACAGACAA (AAAG)

(AAAG)

TD55 384–412 20 8 0.650 0.713 0.665

EU593514 GTTTCCGCTTTACAAATGTTAAG

CpP402 CAGTCGGGCGTCATCAAACCAACCAGGGTAAAA (AGAT)

TD65 241–262 10 2 0.200 0.189 0.164

EU593360 GTTTAGTCATGCCCATACTCATAC

CpP405 CAGTCGGGCGTCATCAAGTTTCAGCCAGCTCTAGAA (AAAG)

TD65 195–245 20 8 0.500 0.626 0.569

EU593361 GTTTCCAGAAGCCACCTAAAGTTA

CpP406 GTTTATTGTCATGCCACACTTCT (AAC)

TD65 244–249 10 2 0.100 0.100 0.090

EU593362 CAGTCGGGCGTCATCACCTTTGGGCATAGACAGA

CpP4104 GTTTGGCCACAGTTATTAGTTTC (AGAT)

TD55 170–182 10 3 0.300 0.294 0.239

EU593515 CAGTCGGGCGTCATCACACCTACCCAAAGAAC

CpP4105 GTTTCTCAGATGCTGTCCTACA (AGAT)

TD65 267–271 10 2 0.400 0.505 0.365

EU593516 CAGTCGGGCGTCATCATAGTCCTTTGGCAGTGAT

CpP4111 CAGTCGGGCGTCATCAGCTCTTAAAAGGCAGAAT (ACAT)

TD55 378–385 10 2 0.200 0.337 0.269

EU593517 GTTTCTATCACAATGGGCTCTT

CpP4116 CAGTCGGGCGTCATCATTTCAAATATCCGTGTCAT (AGAT)

TD55 220–236 20 4 0.350 0.458 0.384

EU593518 GTTTACCGCTTGAACCTTGT

CpP4208 CAGTCGGGCGTCATCACTCTTATTGCACCAGGTATA (AGAT)

TD65 192–216 20 7 0.650 0.701 0.635

EU593520 GTTTAGAGAGATGCGTGATGAT

CpP4209 CAGTCGGGCGTCATCAGGGGTCTGTCACAAGA (ACAT)

TD65 176–192 10 5 0.400 0.705 0.610

EU593521 GTTTGAAGTGAACTTTGGGTCAT

CpP4301 CAGTCGGGCGTCATCAATTATAAAAGTCACCAGTCAC (ACT)

TD55 350–362 20 3 0.700 0.578 0.491

EU593522 GTTTGAAATACAAACGCTTAAA

CpP4304 CAGTCGGGCGTCATCAGGAAAAGGTGCTAGAGAA (AAC)

TD65 111–122 20 4 0.400 0.537 0.410

EU593524 GTTTGAACTGATGCGATAAAGAAT

cpP4308 CAGTCGGGCGTCATCACATATGTAAATTTGGAATGA (ATC)

TD65 111–114 20 2 0.400 0.467 0.351

EU593525 GTTTGATTGAGCCATCCTTAAC

CpP4311 CAGTCGGGCGTCATCAGGCTGCTCTGTGTTTG (AGAT)

TD65 211–241 20 8 0.600 0.690 0.631

EU593526 GTTTGGGTTTAGCATCATGT

972 Conserv Genet (2009) 10:963–980

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP4501 CAGTCGGGCGTCATCATATACACACATGCACCTCTT (AGAT)

TD65 101–113 10 4 0.600 0.647 0.554

EU593527 GTTTGCACAGCCCTAATAGA

CpP4506 GTTTGTTGAAGGGCAAAGTGTA (AAAC)

TD65 348–352 10 2 0.600 0.505 0.365

EU593528 CAGTCGGGCGTCATCAAAACTTGGCTTGCTGTC

CpP4508 CAGTCGGGCGTCATCAATGCGTTTGTCCTCTAC (AGAT)

TD65 186–205 10 5 0.500 0.689 0.580

EU593529 GTTTCGTAAACATGCATAAAACTG

CpP501 CAGTCGGGCGTCATCACCTGATAGACTGCCTACAA (ACAG)

TD65 290–375 10 15 0.900 0.958 0.903

EU593363 GTTTGTTAGTTCCCACTGAAGAAG

CpP504 CAGTCGGGCGTCATCATTAGGCAAAGGCAAATAAA (AAAG)

TD55 356–370 10 3 0.400 0.337 0.269

EU593365 GTTTGCGGTAATGATCAGATGTG

CpP602 CAGTCGGGCGTCATCATTGAATGAGGGTTTGTT (AAAC)

TD55 156–160 10 2 0.300 0.368 0.222

EU593367 GTTTGCCTGACTCTTCGTTAATAC

CpP606 CAGTCGGGCGTCATCATTCATTTAGACCTCCCAAGT (AAAC)

TD65 237–249 10 4 0.700 0.569 0.441

EU593368 GTTTTAGCCAGCCTTCAGTT

CpP607 CAGTCGGGCGTCATCATACACTTGCCTGGTTGA (AAAC)

TD65 305–309 10 2 0.300 0.395 0.305

EU593369 GTTTAGAAGGTGCCATAGTTACA

CpP609 CAGTCGGGCGTCATCATTGAATGAGGGTTTGTT (AAAC)

TD65 155–159 10 2 0.300 0.268 0.222

EU593370 GTTTGCCTGACTCTTCGTTAATAC

CpP610 GTTTCTATTGGCACGCTGTCTA (ACAG)

TD55 288–331 20 12 0.700 0.859 0.817

EU593371 CAGTCGGGCGTCATCACGGCACTTTAAATCTGA

CpP612 CAGTCGGGCGTCATCATGGCTTGCATTAAGATA (AAAC)

TD55 225–237 10 4 0.600 0.732 0.640

EU593372 GTTTGGTCCTGAATACCATATAGT

CpP701 GTTTCCCTCCCAACAGACAG (ACAG)

TD65 142–196 10 9 0.600 0.795 0.723

EU593373 CAGTCGGGCGTCATCATGTCCATTGCCAGTTGTA

CpP706 CAGTCGGGCGTCATCAAGTGATGTTGGGTCATAG (ACAG)

TD65 86–150 20 2 0.350 0.286 0.239

EU593375 GTTTCTCGCCTCACTACAGA

CpP714 CAGTCGGGCGTCATCAGCAGACTGAGCCAATA (AAAC)

TD65 217–225 10 3 0.000 0.189 0.164

EU593378 GTTTCGGTGTATAAAGTACAT

CpP715 CAGTCGGGCGTCATCACTTGAGGCAGACTTCTGACT (ACAG)

TD65 215–219 10 2 0.300 0.395 0.305

EU593379 GTTTGCCTGTAATCATCCTTCAC

CpP717 GTTTCCAAAGTAGCAAATTTAAG (AAAC)

TD65 161–165 10 2 0.300 0.395 0.305

EU593380 CAGTCGGGCGTCATCACAGCAGAGCCAAACTAA

CpP722 GTTTGAATTGTTTTAGTGTCTGTC (ACAG)

(AG)

TD55 156–206 20 10 0.450 0.770 0.708

EU593381 CAGTCGGGCGTCATCAAGGGTATGCGAGTTTA

CpP801 CAGTCGGGCGTCATCATTGGCATTAGATTGGTAGAC (AGAT)

TD65 159–195 20 7 0.350 0.411 0.380

EU593382 GTTTCTATGCCAAAGCTACAAC

Conserv Genet (2009) 10:963–980 973

123

Table 1 continued

Locus/accession Primer sequences 5’ to 3’ Repeat motif PCR proﬁle Allele range (bp) NKH

obs

exp

PIC

CpP804 GTTTGGGAATGGTGGCATATAAAT (AGAT)

TD65 185–210 20 6 0.500 0.409 0.341

EU593383 CAGTCGGGCGTCATCAGGAGAATGGGCATCTAAG

CpP805 GTTTCACTTATGGAGCTGCAATAC (AGAT)

TD65 296–304 10 2 0.200 0.189 0.164

EU593384 CAGTCGGGCGTCATCAATCCAAGCGAAACACTG

CpP806 CAGTCGGGCGTCATCACCATCCATCTACCCATCAT (ACAG)

TD65 181–185 10 2 0.400 0.526 0.375

EU593385 GTTTCTGCCACCTAAGAGTCATAG

CpP809 GTTTGGATAATTGTCCCTCTA (AGAT)

TD65 179–183 10 2 0.200 0.189 0.164

EU593387 CAGTCGGGCGTCATCAGGCATTTGCAGTCTCTATT

CpP815 CAGTCGGGCGTCATCAGGTTAAGTGCCACACAAGT (AGAT)

TD65 243–268 20 7 0.550 0.651 0.575

EU593390 GTTTGGCCAATTTCTAATGAA

CpP903 GTTTATTTATTTTTCCAGCACTTC (ACT)

TD65 235–258 20 6 0.600 0.586 0.525

EU593391 CAGTCGGGCGTCATCAGGGCATCTTTGTTGT

CpP906 CAGTCGGGCGTCATCATTATGCATTTGCCTCAGAC (ACAG)

TD65 289–327 20 7 0.500 0.462 0.424

EU593392 GTTTCACATCTTCATGGCAATTAT

CpP910 CAGTCGGGCGTCATCAAGGGCTGCTCTGTGTT (AGAT)

TD65 213–233 10 6 0.600 0.679 0.590

EU593393 GTTTGGGTTTAGCATCATGTAT

CpP911 GTTTGCACTTCTAAAGTGATAGATA (AGAT)

TD65 184–200 10 5 0.300 0.542 0.460

EU593394 CAGTCGGGCGTCATCATGAAAGAAGCAGCTACTA

CpP914 CAGTCGGGCGTCATCAACATGGCAACTTCAGAG (AGAT)

TD65 257–299 20 8 0.800 0.722 0.661

EU593395 GTTTCGAATAAATGCAGCATAA

974 Conserv Genet (2009) 10:963–980

123

Table 2 Microsatellite primer sequence and locus information including sample size (N), observed number of alleles (K), in Crocodylus porosus

Locus/accession Primer sequences 50to 30Repeat motif PCR

proﬁle

Allele range

(bp)

CpDi01 GTTTAGCTCTGCCCTTATGT (AG)

(AC)

TD55 345–356 8 2

EU593277 CAGTCGGGCGTCATCAGGGAAAAGTCAGAATGA

CpDi07 GTTTGTGTATGTATAGGTGGGTTGT (AC)

TD65 310–324 8 2

EU593280 CAGTCGGGCGTCATCATTAGACAGGAGGGTGTG

CpDi09 CAGTCGGGCGTCATCAGCAGCTTGGAAGTTTAG (AC)

TD55 350–356 8 2

EU593282 GTTTCTCAGTAAATCGGGACAGTA

CpDi14 GTTTCACACGCACACTCTCT (AC)

(AC)

TD55 331–373 8 6

EU593287 CAGTCGGGCGTCATCACTTTTTGGTGCCTTAATAG

CpDi22 GTTTAGGCAGGAGGTGATTTAT (AC)

TD55 188–194 8 2

EU593291 CAGTCGGGCGTCATCAAGGAATTGGCTGAGGTAAT

CpDi25 GTTTAATCCTTGCCTCTCACTTA (AC)

TD65 320–322 8 2

EU593294 CAGTCGGGCGTCATCAGTTAGCTTACAGCCAACAGA

CpDi32 CAGTCGGGCGTCATCACACGAAAGGTGCAGTGT (AC)

TD55 367–377 8 2

EU593298 GTTTGAGTATTGTAATGGGTGTATG

CpDi35 CAGTCGGGCGTCATCACTGATGCAGCAAACAATAAT (AC)

TD65 163–172 8 3

EU593300 GTTTAGTAGCTCCGCTGATGA

CpF205 CAGTCGGGCGTCATCAGGAGCATACACCTTAATAC (AC)

TD65 320–322 8 2

EU593308 GTTTGCCAATTCCCATATCTC

CpF206 CAGTCGGGCGTCATCAATATATGCAGCACAACTGA (ATC)

TD65 187–215 8 2

EU593309 GTTTACCACGAGTTTCAAAGAGA

CpF403 GTTTCAGTCGGGCGTCATCATGTTCTCTGCCAGCTTAT (ACAT)

TD55 357–376 8 4

EU593311 GTTTGATGAAAAAGGCTATGAATC

CpF501 CAGTCGGGCGTCATCACAAGGCAAAGGAGAGTC (AGAT)

TD55 265–273 8 3

EU593312 GTTTCACAGATAGATGGCATTTC

CpF507 CAGTCGGGCGTCATCATTTCACCAGGTCTTTTAG (ACAT)

TD55 191–221 8 4

EU593313 GTTTAATAGTCTGTAAGTGCCATTT

CpF508 CAGTCGGGCGTCATCAGGGCTCAGACAGTAAAGAA (AGAT)

TD55 329–335 8 3

EU593314 GTTTATCCCAAGCCTTCAAGTA

CpF703 CAGTCGGGCGTCATCACTTTGTCTGGGAACAT (AAC)

TD55 142–148 8 3

EU593316 GTTTCTTGTTCAATGCAAATA

CpF801 GTTTATGTTCTCTGCCAGCTTAT (ACAT)

TD55 164–211 8 2

EU593317 CAGTCGGGCGTCATCAGGGGTTGCTTTTTCAG

CpF901 GTTTAGTAAGAGACAGTCCCTATAC (AC)

TD55 196–223 8 4

EU593318 CAGTCGGGCGTCATCATTGGGCTTATGTCCTAGTT

Conserv Genet (2009) 10:963–980 975

123

Table 2 continued

Locus/accession Primer sequences 50to 30Repeat motif PCR

proﬁle

Allele range

(bp)

CpF904 CAGTCGGGCGTCATCAGACAGAGCAGCATCATTTA (AG)

TD55 114–132 8 4

EU593319 GTTTAATGGAGCCAATATCAAGA

CpF905 CAGTCGGGCGTCATCAGGCACTGAGCAGAGAG (AC)

TD55 127–135 8 3

EU593320 GTTTGACTGTCCCAGCAGAAG

CpP1006 GTTTACCGCTGCACCTTTATA (AGAT)

TD55 148–180 8 3

EU593401 CAGTCGGGCGTCATCACCAAAGAGCCTTGTCT

CpP1007 GTTTGCCTTTAGCTCTGTTA (AGAT)

TD55 163–198 8 2

EU593402 CAGTCGGGCGTCATCAGACACTCCGTAATTTTTATAAT

CpP109 CAGTCGGGCGTCATCATGAGTCTGGGCAAATG (AAAC)

TD65 342–361 8 4

EU593325 GTTTCTAAACCTTTGCTTTGTTATT

CpP1101 GTTTGGAGGAAACCTGACTGATTC (AATC)

TD65 164–172 8 2

EU593403 CAGTCGGGCGTCATCATGTGTCTGTGTGCGTGTT

CpP1102 CAGTCGGGCGTCATCAGGGAGTGAGGATTGATT (AAAC)

TD65 150–154 8 2

EU593404 GTTTAAAGAAGTAGGGCTAGAA

CpP111 GTTTCCCTCCATATCTATTTTTAAT (ACAT)

TD55 189–192 8 2

EU593326 CAGTCGGGCGTCATCATCACTGTTTCCCAAACT

CpP120 CAGTCGGGCGTCATCAACAAATGGCCTCACTTC (AAAG)

TD55 273–296 8 2

EU593332 GTTTCAGTCAACTTGGCTACTT

CpP1202 GTTTGAAGGTACCAGGCTACAG (AAAC)

TD65 303–319 8 4

EU593407 CAGTCGGGCGTCATCATTTCAGGCACAAGTAAG

CpP1209 GTTTCTTATATCCAGCCTGAAAC (AAAC)

TD65 338–386 8 3

EU593410 CAGTCGGGCGTCATCAAAGAATGGGGAAAAA

CpP1212 GTTTCACTGTTCTAACCCTTTTCTC (ACAG)

TD65 220–258 8 7

EU593411 CAGTCGGGCGTCATCAGCAGCAACTCCCAGTAG

CpP1302 CAGTCGGGCGTCATCAGAGAGAATTCAGCCAAGA (AAAC)

TD55 239–249 8 3

EU593413 GTTTGCTATGGTGCAGTAATAAAT

CpP1307 GTTTACTACCCAACAGTCTCATT (AGAT)

TD65 176–184 8 3

EU593417 CAGTCGGGCGTCATCATGGCGTTATTCTTAAAG

CpP1402 GTTTGGAATCTGGGTTTTCTCTAAT (ACTC)

TD65 270–278 8 2

EU593420 CAGTCGGGCGTCATCATCCTGGTTTTCTGTGATAA

CpP1412 CAGTCGGGCGTCATCAGATTCGATTCGGAGAGATTA (AGAT)

TD55 189–197 8 2

EU593426 GTTTCCACCCACTTAGTGAGAG

CpP1511 GTTTGAATCTTGCCCTGGACT (AAAC)

TD65 275–281 8 3

EU593431 CAGTCGGGCGTCATCAAAATCTTTGCCTCTCACAT

976 Conserv Genet (2009) 10:963–980

123

Table 2 continued

Locus/accession Primer sequences 50to 30Repeat motif PCR

proﬁle

Allele range

(bp)

CpP1707 GTTTGATGTTCTTTGCCATGATTAGAT (ACAG)

TD65 223–227 8 3

EU593439 CAGTCGGGCGTCATCACCATTTACTTCCTCCCTATTT

CpP204 GTTTAACTGGACGTGATATACCTA (AG)

TD65 205–207 8 2

EU593337 CAGTCGGGCGTCATCACAAGGAGGCAAGAATG

CpP206 CAGTCGGGCGTCATCAGTTGATTTGGATCGCTGTC (AGAT)

TD65 204–208 8 2

EU593339 GTTTCCCAATCCCTACAGATGTCA

CpP210 CAGTCGGGCGTCATCATTTGTTTCTGGGATCAT (AGAT)

TD55 180–192 8 3

EU593343 GTTTCCAGGTAGTAGTTCTGATTAG

CpP226 GTTTGTCCATTGCCAGTTGTAA (ACAG)

TD65 202–246 8 4

EU593348 CAGTCGGGCGTCATCATCTGAGGGTGAACTTTAGAA

CpP2402 GTTTATTGACTACTGCCCTCTG (AAAC)

TD55 279–333 8 5

EU593447 CAGTCGGGCGTCATCAGGGGGTTGGACTTGA

CpP2403 CAGTCGGGCGTCATCACTTTAAGGGTTGGGTTTT (AAAC)

TD55 166–170 8 2

EU593448 GTTTACGCCAACAATGAACA

CpP2406 CAGTCGGGCGTCATCAAACCAGGATTTTCTTTAG (AAAC)

TD65 204–208 8 2

EU593449 GTTTCACTTCGCTGGAAATAA

CpP2510 GTTTACGCATGAATTTACACTG (AGAT)

TD55 240–246 8 2

EU593455 CAGTCGGGCGTCATCATCCTGGTTTTCTCTGATT

CpP2710 GTTTGTACTGTGCTCCAAACATC (ACAG)

TD55 190–234 8 9

EU593462 CAGTCGGGCGTCATCACCAAAAGGAGCAACTAAA

CpP2817 GTTTAAATCATTCTGGCACATAC (AAC)

TD65 395–401 8 2

EU593467 CAGTCGGGCGTCATCAACTGCTTTGGCAAGTCT

CpP313 CAGTCGGGCGTCATCAACATTTTTCTTTTAATACAC (AGAT)

TD55 210–218 8 3

EU593356 GTTTGCCAGAATAAAAATAT

CpP3214 GTTTCAAGTAACGGCACAGTG (AAAC)

TD65 200–204 8 2

EU593481 CAGTCGGGCGTCATCACTTAAACCCAAACTCAAAT

CpP3219 CAGTCGGGCGTCATCAGCAAGGGCATATAATAGTTC (AGAT)

TD55 305–313 8 3

EU593485 GTTTAAGTTGCCTTAAAATTGTAAA

CpP3316 CAGTCGGGCGTCATCAGCTGGCATAGTTTACATACAT (AGAT)

TD65 303–336 8 5

EU593493 GTTTAGCCCAAATCTGAAAGTATT

CpP3414 GTTTATTCACGTCGACAGTATTAAA (AGAT)

TD65 344–395 8 4

EU593497 CAGTCGGGCGTCATCAGCAAGCAAGATATCCACTAAC

CpP3513 GTTTGTCAGACCTACCGAAATTA (AAC)

TD65 271–274 8 2

EU593501 CAGTCGGGCGTCATCACTTGGGTTCTGGATTAGTG

Conserv Genet (2009) 10:963–980 977

123

Table 2 continued

Locus/accession Primer sequences 50to 30Repeat motif PCR

proﬁle

Allele range

(bp)

CpP3515 CAGTCGGGCGTCATCATGTTCATCCCCATTCA (ATC)

TD55 356–359 8 2

EU593502 GTTTAACCTTTCTTGCCTCTC

CpP3608 CAGTCGGGCGTCATCAGGGCACCATAATAGAGAG (AAAC)

TD55 145–149 8 2

EU593506 GTTTCTGGTGAGACGCAGTC

CpP3804 GTTTCTGATTGCGCTCACAC (ACTC)

TD65 171–195 8 4

EU593508 CAGTCGGGCGTCATCACAGCATCCCAAATCTGTT

CpP4206 CAGTCGGGCGTCATCAAAGCGTTTCTGAAGTAAG (ACAG)

TD65 294–300 8 2

EU593519 GTTTGCCAACCCAAACTATTATG

CpP4302 CAGTCGGGCGTCATCAGTGACTAGTGTGCGGTAT (ATC)

TD65 349–361 8 3

EU593523 GTTTAAGGCAATCTCTTAAACTTC

CpP502 CAGTCGGGCGTCATCAAGGTAAGATTGGGTTTATG (ACTG)

TD55 363–383 8 6

EU593364 GTTTCTGCGTAATGGTTTCTAGT

CpP508 CAGTCGGGCGTCATCATGCTCAGGTACACACA (ACTC)

TD55 209–226 8 4

EU593366 GTTTAATAATGCTCCTCTTCTG

CpP705 CAGTCGGGCGTCATCAGAGGCAAGACACTACACTC (AAAC)

TD65 167–180 8 4

EU593374 GTTTAGGCAAGAGATCCATAAATA

CpP708 GTTTGAAGAAGGGAAGAAGAGTAC (AAAC)

TD55 194–210 8 2

EU593376 CAGTCGGGCGTCATCATTATAGGGCTGTGTGAAG

CpP713 GTTTAGTAGCTTTGGGACCTGTAA (AATC)

TD55 307–319 8 3

EU593377 CAGTCGGGCGTCATCAGGGCAGCATAAAGATCA

CpP808 CAGTCGGGCGTCATCATCCTTGTTCCCTTATGT (AGAT)

TD65 244–248 8 2

EU593386 GTTTGGTTCAGGCATATTTTATAT

CpP811 GTTTCTCCAATGGCAGGCTGTT (AGAT)

TD55 305–309 8 2

EU593388 CAGTCGGGCGTCATCAGGCGAGCAAGAGAGA

CpP812 GTTTAAATTGTGTGGCCGTAAC (ACAT)

TD55 284–288 8 2

EU593389 CAGTCGGGCGTCATCAGCATGTATGGGTGTTTA

978 Conserv Genet (2009) 10:963–980

123

(AAAG)

, (AATC)

, (AATG)

, (ACCT)

, (ACAG)

(ACTC)

, (ACTG)

, (AAAT)

, (AACT)

, (AAGT)

(ACAT)

, (ACAT)

, (AGAT)

, (AAG)

, (ATC)

, (AAC)

(AAT)

, (ACT)

(AG)

, (TG)

, ], and captured on

magnetic Streptavidin beads (Dynal). Captured DNA was

recovered by PCR using SuperSNX24-f primer.

The TOPO TA Cloning

Kit (Invitrogen) containing

pCR

2.1TOPO

vector and TOP 10 competent E. coli

cells was used to clone the enriched DNA. Transformed

colonies were screened for inserts using blue-white

screening. PCR products were generated from positive

colonies using M13 forward and reverse primers. PCR

products of 500–1,000 base pairs (bp) were prepared for

sequencing using Exonuclease I and Antarctic Phosphatase

(New England Biolabs) and sequenced using Big-Dye

Terminator Cycle Sequencing v3.1 and an AB 3130xl

automated DNA sequence.

Sequences were edited and assembled using SEQUEN-

CHER 4.1.2 (Gene Codes, Ann Arbor, MI) and exported to

Ephemeris 1.0 (available at http://www.uga.edu/srel/DNA_

Lab/dnacomputer_programs.htm) to search for microsat-

ellite repeats. From 4,128 sequences examined by eye,

*80% contained di-, tri, or tetra-nucleotide repeats char-

acteristic of microsatellite loci. However, there was a high

level of sequence redundancy and many sequences were

not favorable for primer design. From the sequences cho-

sen, 397 tetranucleotide, 83 trinucleotide, 69 dinucleotide

and 31 mixed composite microsatellite PCR primers were

designed to amplify 580 loci for future physical and link-

age mapping. Primers were developed using Oligo 6.8

(Molecular Biology Insights), and a custom CAG-tag

(50-CAGTCGGGCGTCATCA-30) was added to the 50end

of one primer from each primer pair to facilitate ﬂuorescent

size detection using three-primer PCR (Shuelke 2000).

GTTT ‘‘pigtails’’ were added to the 50end of each primer

lacking the CAG-tag to facilitate the non-template addition

of adenosine by Taq polymerase (Brownstein et al. 1996).

Primer pairs were optimized and evaluated for poly-

morphic content using DNA samples from between eight to

16 wild-caught adult C. porosus individuals obtained from

Darwin Crocodile Farm, Noonamah, Australia. DNA was

isolated from blood using modiﬁed phenol chloroform

protocol adapted from Sambrook et al. (1989). PCR

ampliﬁcations were performed in 12.5 ll volumes on PTC-

100 (MJ Research) and GeneAmp PCR System 9700

(Applied Biosystems) thermocyclers. Final concentrations

for optimizing reactions were 10 mM Tris pH 8.4, 50 mM

KCl, 0.5 lM unlabeled primer, 0.05 lM tag labeled pri-

mer, 0.45 lM universal dye labeled primer, 2.0 mM

MgCl

, 0.5 mM dNTPs, 0.5U units JumpStart Taq DNA

Polymerase (Sigma), and *20 ng DNA. CAG-primers

were labeled with either VIC, 6-FAM or NED ﬂuorescent

dyes. Reactions were placed on stratiﬁed touchdown

proﬁles (Don et al. 1991), with each proﬁle encompassing

a10°C span of annealing temperatures (ranges: 65–55°C

and 55–45°C).

Cycling parameters are as follows for TD65: initial

denature step of 95°C for 3 min, followed by four cycles of

[95°C for 20 s; 65°C for 20 s; and 72°C for 30 s], followed

by four cycles of [95°C for 20 s; 62°C for 20 s; and 72°C

for 30 s] followed by eight cycles of [95°C for 20 s; 60°C

for 20 s; and 72°C for 30 s], followed by 24 cycles of

[95°C for 20 s; 55°C for 20 s; and 72°C for 30 s], and a

ﬁnal extension step at 72°C for 7 min. Cycling parameters

for TD55 were identical to TD65, except that the annealing

temperature for the respective stages were reduced by

10°C.

PCR products for the 580 loci were brieﬂy checked for

ampliﬁcation on agarose and analyzed on an ABI 3130xl

capillary sequencer. Genotypes were scored using

GeneMapper v4.0 software (Applied Biosystems) and the

optimal touchdown cycling proﬁles were identiﬁed. Of the

580 microsatellite primer pairs designed and evaluated, 421

loci successfully ampliﬁed on one or both proﬁles, and 253

microsatellite loci were identiﬁed as polymorphic using

eight individuals from the Darwin Crocodile Farm map-

ping resource.

A total 189 of the informative loci were ampliﬁed on a

further 75 individuals (ﬁve families) for future linkage

analyses. Following map construction, 72 of these loci

were also ampliﬁed on an additional 400 individuals (10

families). Herein, we focus on results from the parents

only. Conditions and characteristics of all informative loci

are given in Tables 1and 2. Since the number of parents

was limited and the parents are not from a single breeding

population, calculation of Hardy–Weinberg equilibrium

was considered inappropriate. However, CERVUS version

3.0 (Kalinowski et al. 2007) was used to estimate the

number of alleles per locus (k), observed and expected

heterozygosity (H

obs

and H

exp

) and polymorphic informa-

tion content (PIC) for each locus where sufﬁcient

individuals permitted.

Mapping of the C. porosus genome is currently under-

way to generate the ﬁrst framework genetic-linkage map

using these microsatellite DNA loci. We believe that the

primers described herein will provide a valuable resource

for many other studies of crocodilians.

Acknowledgements This research was supported by Rural Indus-

tries Research and Development Corporation grant US-139A to the

University of Sydney, and US Department of Energy award DE-

FC09-07SR22506 to the University of Georgia. All research took

place at the University of Sydney, Australia, and the Savannah River

Ecology Laboratory (SREL), of the University of Georgia, USA.

Tissue samples were provided by Darwin Crocodile Farm, NT,

Australia. Capture, handling and blood sampling of crocodiles was

approved by Australian Animal Ethic Committee, permit No. N00/

8-2005/3/4177.

Conserv Genet (2009) 10:963–980 979

123

Disclaimer This report was prepared as an account of work spon-

sored by an agency of the United States Government. Neither the

United States Government nor any agency thereof, nor any of their

employees, makes any warranty, express or implied, or assumes any

legal liability or responsibility for the accuracy, completeness, or

usefulness of any information, apparatus, product, or process dis-

closed, or represents that its use would not infringe privately owned

rights. Reference herein to any speciﬁc commercial product, process,

or service by trade name, trademark, manufacturer, or otherwise does

not necessarily constitute or imply its endorsement, recommendation,

or favoring by the United States Government or any agency thereof.

The views and opinions of authors expressed herein do not necessarily

state or reﬂect those of the United States Government or any agency

thereof.

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COPEIA

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Microsatellite DNA markers have been used to assess genetic diversity and to study ecological behavioral character-istics in animals. Although these markers are powerful tools, their development is labor intensive and costly. Thus, before new markers are developed it is important to prospect the use of markers from related species. In the present study we investigated the possibility of using microsatellite markers developed for Alligator mississipiensis and Cai-man latirostris in South American crocodilians. Our results demonstrate the use of microsatellite markers for Paleosuchus palpebrosus, Caiman crocodilus and Caiman yacare.

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Sep 2001

Genetic Diversity, Population Subdivision, and Gene Flow in Morelet's Crocodile (Crocodylus moreletii) from Belize, Central America

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Dec 2002

The lack of information surrounding natural history and ecology of the endan- gered Morelet's crocodile (Crocodylus moreletii) has prompted a baseline study of the population genetics for this species. Nine microsatellite loci have been used to estimate genetic structure within and gene flow patterns among crocodiles (using a recently described maximum likelihood approach) from seven localities in north- central Belize. Individuals from the seven localities grouped into four apparent pop- ulations. Within localities, a high degree of genetic heterogeneity was observed. Among all localities, some subdivision was present (F ST 5 0.062; RST 5 0.100). Fur- thermore, among the apparent populations, we found a significant correlation be- tween geographic distance and genetic subdivision. Our findings suggest a relatively high level of migration among populations (Nm 5 5.15) and are consistent with an isolation-by-distance model of gene flow. Two contiguous subpopulations in partic- ular, New River and New River Lagoon, may form an important source for genetic variation for smaller populations throughout the region. These data will allow us to test hypotheses of relatedness among C. moreletii for other drainages in Belize and will be useful in optimizing future management programs for C. moreletii.

Microsatellite markers for Crocodylus: new genetic tools for population genetics, mating system studies and forensics

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Jan 2011

ABSTRACT Questions of population identification and gene flow have become more important in the conservation and management of crocodilians. Additionally there has long been an interest in understanding crocodilian mating systems. To address such questions we designed nuclear microsatellite markers from the DNA of C. acutus, C. porosus and C. johnstoni. We report on the development and testing of 26 new microsatellite loci for Crocodylus spp, which represent the first microsatellite loci found in Crocodylus.

Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment

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