Conservation Genetics 2: 183–185, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
183
Microsatellite DNA markers for tuatara (Sphenodon spp.) Nicola Aitken1 , Jennifer M. Hay1 , Stephen D. Sarre1∗ , David M. Lambert1 & Charles H. Daugherty2 1 Institute
of Molecular BioSciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand; University of Wellington, PO Box 600, Wellington, New Zealand (∗ Author for correspondence: E-mail:
[email protected]. Now at Applied Ecology Research Group, University of Canberra, ACT 2601, Australia)
2 Victoria
Received 14 November 2000; accepted 5 March 2001
Key words: conservation, microsatellite, polymorphism, reptilia, sphenodontia, tuatara
Tuatara (Sphenodon punctatus and S. guntheri) are the last surviving members of the reptilian order, Sphenodontia (Benton 1993). The distribution of tuatara has declined severely since human arrival approximately 1000 years ago, being once extensive throughout New Zealand, but now restricted to 34 offshore islands in New Zealand, and zoological collections world-wide. Sphenodon guntheri occurs naturally only on North Brother Island with around 300 adults (Thompson et al. 1990). Sphenodon punctatus is present in 11 island groups and is managed as two distinct genetic groups (Cook Strait and northern groups, Daugherty et al. 1990; Cree and Butler 1993). The phylogenetic distinctiveness and limited distribution of the tuatara have made them a target for illegal trade (P. Younger pers. comm.) and the dramatic, recent contraction in their range has made the study of their biology essential for conservation purposes. Ecological studies of the tuatara are difficult owing to a range of life history characteristics. In particular, tuatara mature late (13– 20 years), have a slow reproductive cycle (4 to 5 years, Cree 1994), and a long life span (>100 years, Robb 1977). Therefore, we have isolated and characterised six microsatellite loci in Cook Strait Sphenodon punctatus, and have tested these loci for amplification in populations of S. guntheri and northern and Cook Strait S. punctatus. Microsatellites were isolated using an enrichment procedure modified from Armour et al. (1994). Genomic DNA from three males and three females was digested to completion with Sau 3AI. A 300–600 bp fraction of the combined digests was ligated to SAU linkers. This fraction was enriched for microsatel-
lites by hybridisation with 3 mm2 pieces of nylon membrane saturated with CA/GT and GA/CT repeats. The microsatellite enriched fraction was washed in 1× SSC (0.15 M NaCl, 15 mM sodium citrate) and 0.1% SDS, stripped from the membranes, and PCR amplified using SAU LA as the primer (95 ◦ C, 5 min; (67 ◦ C, 60 sec; 70 ◦ C, 60 s; 95 ◦ C, 60 s) ×35; 70 ◦ C, 4 min). The linkers were removed from the amplification products by digestion with Nde II, the purified enriched fraction ligated into pUC18 Bam HI, and the plasmid transformed into Max Efficiency DH5α competent cells (Gibco BRL). Cells were grown on L-Agar with ampicillin (Amp), BCIG, and IPTG. Approximately 650 recombinant clones were cultured. 154 (24%) of the recombinant clones were positive when hybridised with 32 P labelled (CA)n . Of these, 50 clones were sequenced and 11 primer pairs designed. For six of those loci, reverse primers were fluorescently tagged with either 6-Fam or Tet for genotyping multiple populations. Genomic DNA was extracted from nucleated erythrocytes by standard procedures of cell lysis, proteinase K digestion, phenol/chloroform purification and ethanol precipitation (Millar et al. 1992). The 15 µl PCR reactions contained 200 µM of each dNTP, 1.5–2.5 mM MgCl2 , 0.5 µM each primer, 10 µg bovine serum albumin, 0.5 units AmpliTaq polymerase (Applied Biosystems) and 5 ng genomic DNA. Allele sizes were determined on an ABI Prism 377 using GeneScan version 3.1 with Tamra size standards (Applied Biosystems). PCR conditions were optimized individually for each primer pair as follows
184 Table 1. Tuatara microsatellite loci primers, PCR conditions, size and number of alleles per population, and the expected and observed heterozygosity per locus averaged over populations calculated using GENEPOP 3.1d (Raymond and Rousset 1995, 2000). See text for PCR conditions. [MgCl2 ] is final concentration in mM. Populations: TR is Tawhiti Rahi in the Poor Knights group, LA is Lady Alice and Wh is Whatupuke, both in the Hen and Chickens group, Gr is Green Mercury in the Mercury group, Stp is Stephens Island in western Cook Strait, Bro is North Brother Island in eastern Cook Strait, the only natural population of Sphenodon guntheri, (n) = sample size per population. Locus
Repeat motif in clone
Primer sequences
1/A6
(AC)2 TC(AC)9 TC(AC)6
F: CGC CTG AGG AGT CTA TGC T R: ACT TGA GGT TTC CAT TGT TG
1/B3
(GT)9 GC(GT)13 (GA)6 GG(GA)4
F: CTT GTC GCA GTA CCA TTG AC R: GGG AGG ATA GGG AAG TGA AG
1/C1
GTGC(GT)18 (GA)5
F: AGT CTC ATT GCT TTT CCC AG R: CCT CTT CTC CGC CTT ACA CT
1/C2
(AC)14
F: TCA CTG TCA GCA GGC TCT TC R: GAA TGC GGG GAA TGT GAG G
1/C12
(AC)17
F: ACT GGA ATG ACT GTT GCT GC R: TCC CCT ACT GTT CTC CCA TC
2/A12
(TC)2 (TG)23
PCR condit.
[MgCl2 ] Clone size (bp)
Allele size (bp)
TDAR 57◦ ×15
[2.5]
204
190–212
6
2
3
4
2
2
1
0.52
0.64
2stpTD 56◦ ×25
[2.5]
190
176–216 17
6
6
4
4
8
1
0.78
0.76
TDAR 57◦ ×32
[2.5]
200
184–208
9
1
5
5
2
3
3
0.53
0.51
TA 65◦ ×35
[2.5]
144
144–154
5
2
3
2
2
2
1
0.55
0.52
TDAR 57◦ ×40
[1.5]
164
158–210 17
7
6
6
3
6
3
0.69
0.70
F: GGA GAA GGG AGG AGA ATA ATC TDAR 60◦ ×30 R: ATC ACT GCT CAT TTC AGC C
[2.0]
172
156–198 19
4
8
5
3
7
2
0.75
0.74
(refer to Table 1): TDAR60◦×30; 94 ◦ C 1 min, (94 ◦ C 20 sec, 65 ◦ C 15 s, 72 ◦ C 20 s) −0.5 ◦ C per cycle × 10 cycles; (94 ◦ C 20 s, 60 ◦ C 15 s, 72 ◦ C 20 s) × 30 cycles; 72 ◦ C 7 min. TDAR57◦ ×15–40; 94 ◦ C 1 min, (94 ◦ C 20 sec, 65 ◦ C 15 s, 72 ◦ C 20 s) −0.5 ◦ C per cycle × 16 cycles; (94 ◦ C 20 s, 57 ◦ C 15 s, 72 ◦ C 20 s) × 15–40 cycles; 72 ◦ C 7 min. 2StpTD56◦×25; 94 ◦ C 2 min, (94 ◦ C 20 s, 60 ◦ C 20 s) × 5 cycles; (94 ◦ C 20 s, 57 ◦ C 20 s) × 5 cycles, (94 ◦ C 20 s, 56 ◦ C 20 s) × 25 cycles; 72 ◦ C 4 min. TA65◦×35; 94 ◦ C 1 min, (94 ◦ C 20 s, 65 ◦ C 15 s, 72 ◦ C 20 s) × 35 cycles; 72 ◦ C 7 min. To test the ability of these loci to discriminate between island populations, we genotyped six tuatara populations (n = 6–7 individuals per population) from five island groups that are representative of the latitudinal and longitudinal range of extant populations and species (Table 1). All loci were polymorphic with five to 19 alleles per locus. The mean observed levels of heterozygosity within populations was 0.56 with the highest observed in S. punctatus on Lady Alice and Stephens Islands (0.74) and the lowest in S. guntheri (0.20; Table 1). The high variability observed suggests that microsatellites will be useful for investigations into the mating systems, zoological pedigrees, and the genetic status of populations and species of tuatara. The utility of these primers for studies of the closest relatives of tuatara (squamates, testudines and archosaurs) was tested at the conditions given above, and at the reduced annealing temperature of 50 ◦ C, by amplifying from a gecko, skink, turtle,
Total Number of alleles per population Av. no. of TR LA Wh Gr Stp Bro exp. alleles (6) (6) (6) (6) (7) (6) het.
Av. obs. het.
chicken and rhea. Some DNA bands were amplified under these conditions. The most prominent of these were sequenced but showed no homology to tuatara and contained no microsatellites. This apparent lack of homology between tuatara and the other taxa is consistent with the large time (∼235Mybp; Benton 1993) since tuatara separated from their closest reltives. Among tuatara, these six microsatellite loci enabled a high rate of successful assignment of individuals to their population of origin (Assignment test, Doh analysis package, Brzustowski 2001) with only 4 (10.8%) of the 37 individuals genotyped assigned to an incorrect population. These mis-assignments were between the two islands from the Hen and Chickens group (Lady Alice and Whatupuke) and Stephens Island. When the two Hen and Chickens populations were combined and the assignment test repeated, only one individual (2.7%) was mis-assigned (from the Hen and Chickens group to Stephens Island). This high rate of correct assignment, although obtained using relatively small sample sizes, suggests that the island groups are genetically distinguishable, and that the provenance of smuggled tuatara, animals in zoos for breeding purposes, or museum specimens, should be able to be determined with reasonable probability, at least to the level of island group.
185 Acknowledgements This work was supported in part by Marsden Grant #MAU801, a Foundation for Research Science and Technology Post-doctoral Fellowship (to JMH) and Massey University. We thank Nicola Nelson, Susan Keall, Michael Thompson, Michelle Finch and Graham Ussher for providing samples. This research was conducted with the approval of the Environmental Risk Management Authority, application number GM099/MU/31.
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