Novel Microsatellite Loci Identified from the Australian Eastern Small-Eyed

Molecular Ecology Notes (2005) 5, 54–56 doi: 10.1111/j.1471-8286.2004.00832.x

PRIMERBlackwell Publishing, Ltd. NOTE Novel microsatellite loci identified from the Australian eastern small-eyed snake (Elapidae: Rhinocephalus nigrescens) and cross species amplification in the related genus Suta

JESSICA STAPLEY,* CHRISTINE M. HAYES,* JONATHAN K. WEBB† and J. SCOTT KEOGH* *School of Botany and Zoology, Australian National University, Canberra ACT 0200, Australia, †School of Biological Sciences A08, University of Sydney, Sydney 2006, Australia

Abstract A total of 15 microsatellite primers pairs were developed for the Australian small-eyed snake Rhinoplocephalus nigrescens. Five primers were used to screen 93 individuals of R. nigrescens and were also tested against eight species of the closely related genus Suta. Allelic diversity in R. nigrescens was high in three loci (12–27) and there was high heterozygosity (0.58–0.82). Observed heterozygosity did not deviate from Hardy–Weinberg expectations for the five loci tested. These primers will be useful in studies of population genetics and mating systems of small-eyed snakes and related species. Keywords: Australia, Elapidae, microsatellite primers, snake Received 18 August 2004; revision accepted 17 September 2004

The venomous Australian small-eyed snakes of the genus (Amersham-Biosciences). Using γ -dATP end labelled Rhinoplocephalus comprise five species (Cogger 2000) that oligonucleotide mixture, clones were screened for micro- are closely related to the nine species in the genus Suta satellites of the following repeats; (ACTC)7 (AACG)7 (ACAG)7 (Keogh et al. 1998, 2000; Keogh 1999). Very little is known (AGAT)7 (GA)15 (GT)15 (GTG)10 (GTC)10 (GTT)10 (GTA)10 about the biology of these secretive nocturnal snakes, (GCG)10 (GAA)10 (GAT)10 (GAG)10 (AGC)10 (ATA)10. A total of but long-term mark-recapture studies on a population of 250 putative recombinants clones were picked, suspended Rhinoplocephalus nigrescens from Morton National Park in in 50 µL of TE and boiled for 5 min. Each sample was used as southeastern Australia have provided information on popu- template for PCR (polymerase chain reaction) amplification lation structure, growth rates, diets, and habitat selection using universal M13 primers (Forward: 5′-TGTAAAACGA- (Webb et al. 2002, 2003, 2004). Currently, two of us (JKW CGGCCAGT-3′, Reverse: 5′-CAGGAAACAGCTATGAC-3′). and JSK) are studying the mating systems and genetic A total of 70 products of appropriate size were dotted on structure of populations of R. nigrescens from southeastern Hybond-NX nylon membrane (Amersham-Bioscience) and Australia. To facilitate future genetic studies on Rhinoploce- secondarily screened with the same – dATP end labelled phalus and Suta, we developed polymorphic microsatellite oligonucleotide mixture as described above. A total of 33 primers for R. nigrescens and tested them against eight species positives were chosen and sequenced using ABI Big Dye of Suta. Genomic DNA (gDNA) was extracted from a liver terminator 3.1 (Applied Biosystems). Products were visualized sample preserved in ethanol using a modified protocol on an ABI3100 Capillary Sequencer. Of the sequenced clones, (Sambrook et al. 1989). A partial genomic library was made 15 contained base pair repeats of greater then seven and following a procedure we have described previously sequence, which are useful for primer design. Primer pairs (Scott et al. 2001). An estimated 25 000 transformants were were identified for the flanking region of sequence using recovered and transferred Hybond-NX nylon membrane Primer 3.0 (Whitehead Institute of Biomedical Research, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_http:// www.cgi) (Table 1). The universal M13 Forward primer Correspondence: Dr Scott Keogh. Fax: 61-2-6125-5573, E-mail: (5′-TGTAAAACGACGGGCCAGTA) was added to each [email protected] forward microsatellite primer. All primers were tested on

PRIMER NOTE 55

Table 1 Characteristics of loci and accession number (Acc.No.) with forward and reverse primers, repeat motif, fragment (Frag.) length of PCR product and allele size range for loci screened for 93 R. nigrescens individuals. ND represents loci that were not screened

Locus Frag Allele acc. no Primer: 5′ to 3′ Repeat length (bp) range (bp)

Rn75 F: TGTAAAACGACGGGCCAGTAGCAGGAAGCAGCATCAAGTA (CA)14 236 381–387 AY714255 R: TTGTCTGCCATCCAAAAACAA

Rn81 F: TGTAAAACGACGGGCCAGTTCCACCCATCAAAATCTCAT (CTT)55 324 238–266 AY714257 R: TTGGCAGCAGCAAAAACACACAT

Rn94 F: TGTAAAACGACGGGCCAGTCAGGAATATGGCAACCTGTGACCTA (GT)28 171 174–214 AY714265 R: TTGGTACATGGGTTGATGAACTG

Rn114 F: TGTAAAACGACGGGCCAGTGACAGCCTCATCCTGTTTCCAAT (GT)11 250 262–278 AY714261 R: TTGCAGAATCTCCTCTGCCATCAT

Rn128 F: TGTAAAACGACGGGCCAGTAATGGCTGAGTTAAGCCAACAAT (CTGT)5(CTAT)19(GT)6 338 344–404 AY714264 R: TTGAGGTGGGGAGAGGGAGTA

Rn32 F: TGTAAAACGACGGGCCAGTAAGCCAGTCATAGAATGCAA (GT)17 151 NA* AY714251 R: TTGCAAAGATGCAGTTGCTGTCA

Rn33 F: TGTAAAACGACGGGCCAGTATGGTTCCAATAGGCATTGA (GT)12 125 NA* AY714252 R: TTGCAGAATCTCCTCTGCCATCAT

Rn50 F: TGTAAAACGACGGGCCAGTTGGGTTGACACCCTCTTCTT (CTAT)56 384 NA* AY714253 R: TTGGATCCAGAACCTCACACC

Rn78 F: TGTAAAACGACGGGCCAGTGGGTCTCAAACCAACAGGAAAT (AGT)8 176 NA* AY714256 R: TTGCCTCATGAGTTCCTGCCATT

Rn84 F: TGTAAAACGACGGGCCAGTGTACCCCTGTGCATCTTGGAAAAT (TAGA)13 254 NA* AY714258 R: TTGCGTTTCAGGAGAGCAAGTCTGTA

Rn93 F: TGTAAAACGACGGGCCAGTGCCACCAGTCATAAATCAAGGACTA (GT)17 162 NA* AY714259 R: TTGCTGCACAGTGACACAAAGAT

Rn111 F: TGTAAAACGACGGGCCAGTCCAGTTCAAGTTGACTATCCTCAC (GT)24 239 NA* AY714260 R: TTGTTAAACTCCCCTTCCCTGGT

Rn117 F: TGTAAAACGACGGGCCAGTCCCCTTCCACTTGTGACTGTA (TTCG)8 318 NA* AY714262 R: TTGATGGAATGGAATGGAATGGAAT

Rn126 F: TGTAAAACGACGGGCCAGTTCAAGCATAGCCCTGGAGAT (TG)19 319 NA* AY714263 R: TTGATGGAGTGGTTTGCCTTCAG

Rn54 F: TGTAAAACGACGGGCCAGTATAGCCCCGGAGATGTTTTTA (GT)18 215 ND* AY714254 R: TTGCCACTTTAAGGCACACACC

*NA refers to loci that did not amplify clean product.

eight R. nigrescens individuals using the following protocol: size standard on AB13100 (Applied Biosystems). From each M13 forward primer was labelled with a different fluore- this initial screening, five primer pairs were chosen on the scent dyes (FAM, HEX or NED, GibcoBRl Life Technologies) basis of ease of scoring and allele diversity. These five loci and was used in amplification reactions of total volume 40 µL. were then used to screen 93 R. nigrescens individuals and Reactions contained 10 ng of gDNA, 10 µµ in each primer, were tested with eight Suta species (Table 2). The loci were µ µ × 3 m MgSO4, 2 m dNTPs, 1 PCR Amplification Buffer scored using genemapper v3.0 (Applied Biosystems) (Invitrogen), 1 × PCR Enhancer (GibcoBRl Life Technologies), and allele frequencies were calculated using genalex 5.4 and 1 unit of PLATINUM® Taq-DNA polymerase (GibcoBRl (Peakall & Smouse 2001). Three of the five loci exhibited Life Technologies). The reactions were amplified using a high allelic diversity (12–27) and high expected and step down PCR protocol; initial denaturation was 94 °C observed heterozygosity. Heterozygosity deviations from 5 min, then 94 °C 30 s, 70 °C 15 s, and 72 °C 1.5 min, which Hardy–Weinberg expectations were tested using genalex was repeated twice. This cycle was subsequently repeated 5.4 and all yielded nonsignificant results (Table 2). There six times, dropping the annealing temperature by 5 °C each was no evidence of Linkage Disequilibrium between cycle. The final stage consisted of 94 °C 30 s, 35 °C 15 s, and loci as estimated by genepop (Raymond & Rousset 1995) 72 °C 1.5 min, which was repeated 30 times, followed by a (Table 2). No null alleles were detected in the five loci final 45 min extension step at 72 °C. A maximum of three screened. Three of the loci (Rn75, Rn81, Rn128) amplified products of different colour and size were combined into a product in the Suta species tested may be useful in future single well and run with GeneScan™ 500 LIZ™ internal studies on this group.

Table 2 The number of alleles (Na) and the observed (HO) and expected (HE) heterozygosity, deviations from Hardy–Weinberg expectations and the probability of Linkage disequilibrium (LD) for 93 individuals of R. nigrescens. Number of alleles recovered and number of snakes genotyped for eight species of Suta. (NA = alleles didn’t amplify, NS = not significant)

R. nigrescens Suta

Na HO HE HW LD dwyeri monachus nigriceps punctata spectabalis suta flagellum gouldii

Rn75 3 0.24 0.46 NS NS 2(1) 2(2) 2(2) 3(1) 2(2) 2(2) 2(2) 3(3) Rn81 16 0.64 0.88 NS NS 2(1) 2(1) NA 3(4) 2(2) 2(2) 2(2) 3(4) Rn94 12 0.58 0.71 NS NS NA NA NA NA NA NA NA NA Rn114 6 0.39 0.57 NS NS NA NA NA NA NA NA NA NA Rn128 27 0.82 0.91 NS NS 2(1) 4(2) 4(2) NA 2(3) 2(3) 2(4) 3(4)

Acknowledgements b and 16S rRNA sequences. Molecular Phylogenetics and Evolution, 10, 67–81. This study was funded by grants from the Australian Research Council Peakall R, Smouse P (2001) GENEALX: Genetic analysis in excel. to JKW and JSK, and a University of Sydney Sesqui Research Fellow- National University, Canberra, Australia. ship to JKW. We thank P. Webb and O. Keogh for helpful comments Raymond M, Rousset F (1995) genepop: population genetics soft- on the manuscript. This work was carried out in accordance with the ware for exact tests and ecumemiscism. Journal of Heredity, 86, University of Sydney Animal Care and Ethics Committee (Approval 248–249. LO4/5- 2003/2/3753 to JKW) and under a scientific license from the Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A NSW National Parks and Wildlife Service (license S10029 to JKW). Laboratory Manual. Cold Spring Harbour Laboratory Press, New York. References Scott IAW, Hayes CM, Keogh JS, Morrison S (2001) Isolation and characterization of novel microsatellite markers from the Cogger HG (2000) Reptiles and Amphibians of Australia, 6th edn. Australian water skink Eulamprus kosciuskoi and cross-species Reed Books, Chatswood, Australia. amplification in other members of the species group. Molecular Keogh JS (1999) Evolutionary implications of hemipenial mor- Ecology Notes, 1, 28–30. phology in the terrestrial Australian elapid snakes. Zoological Webb JK, Brook BW, Shine R (2002) What makes a species Journal of the Linnean Society, 125, 239–278. vulnerable to extinction? Comparative life-history traits of two Keogh JS, Scott IAW, Scanlon JD (2000) Molecular phylogeny sympatric snakes. Ecological Research, 17, 59–67. of viviparous Australian elapid snakes: affinities of ‘Echiopsis’ Webb JK, Brook BW, Shine R (2003) Does foraging mode influence atriceps (Storr, 1980) and ‘Drysdalia’ coronata (Schlegel, 1837), life-history traits? A comparative study of growth, maturation, with description of a new genus. Journal of Zoology, London, 252, and survival of two species of sympatric snakes from southeastern 317–326. Australia. Austral Ecology, 28, 601–610. Keogh JS, Shine R, Donnellan S (1998) Phylogenetic relationships of Webb JK, Pringle RM, Shine RR (2004) How do nocturnal snakes terrestrial Australo-Papuan elapid snakes based on cytochrome select diurnal retreat sites? Copiea, 2004 (in press).