Microsatellite loci isolated from 00Graphicraft Limited...

Molecular Ecology (2000)

9

, 629–644

© 2000 Blackwell Science Ltd

Blackwell Science, Ltd

PRIMER NOTES

0000Graphicraft Limited, Hong Kong

Microsatellite loci isolated from

Odontesthes argentinensis

and the

O. perugiae

species group and their use in other South American silverside fish

L . B . BEHEREGARAY and P. SUNNUCKS

Department of Biological Sciences, Macquarie University, NSW 2109, Australia

Keywords

: microsatellites,

Odontesthes

, population genetics, radiation, silverside fish

Received 4 September 1999; revision received 18 October 1999; accepted 23 October 1999

Correspondence: L. B. Beheregaray. Fax: (612) 9850 8245. E-mail: [email protected]

Odontesthes

(Atheriniformes: Atherinopsidae) is a diverse andwidespread genus of silverside fish, containing between 15 and22 recognized species distributed across marine, estuarineand freshwater environments of temperate South America(Dyer 1993). The marine-estuarine

O.

argentinensis

ranges fromthe south-east coast of Brazil (25

°

S) to Chubut, in Argentina(43

°

S) (Dyer 1993). It is an economically significant resourcefor local fisheries in Brazil, Uruguay and Argentina (de Buen1953) and a suitable organism for bioassay studies (Phonlor& Cousin 1997). The freshwater

O.

perugiae

species group iscomposed of morphotypes mostly distributed along thecomplex system of lagoons of the coastal plain of southernBrazil (Dyer 1993).

We are using molecular markers to understand historicaland recent biogeographic scenarios that have shaped theevolution of

Odontesthes.

Results based on sequences of threemitochondrial DNA genes revealed that

O. argentinensis

and

O. perugiae

are closely related and have recently radiated inthe coastal areas of southern Brazil (L. Beheregaray, unpub-lished). We developed microsatellites for these taxa to iden-tify population structures, clarify taxonomic problems andreconstruct their phylogeographic history. Evidence for theapplication of these primers in a wide range of Neotropicalsilverside species is presented. To our knowledge, these arethe first microsatellite loci isolated from fish native to SouthAmerica, a continental area with one of the most diverseichthyofaunas of the planet.

Genomic DNA of

O.

argentinensis

and the

O.

perugiae

speciesgroup was extracted by salting-out (Sunnucks & Hales 1996),pooled together, and digested with

Hae

III,

Eco

RV and

Alu

I.Fragments ranging between 430 and 550 bp were cloned into

Sma

I cut pUC18 and a resulting library of approximately25 000 colonies was screened with radiolabelled CA/GT andGA/CT co-polymers according to Taylor

et al

. (1994).Putative positive clones were rescreened, and 31 clones

encompassing different hybridization intensities were selected

for sequencing. This strategy increased our chances of choosingboth long and short repeats as they are expected to inform atdifferent levels of divergence (see below). Thirteen of the clonessequenced contained microsatellites with appropriate flank-ing regions for primer design.

Variation at each of the 13 loci was assessed by the poly-merase chain reaction (PCR) using a 10

µ

L radiolabelledreaction containing 50–100 ng of template DNA, 12 pmol of eachprimer, 0.5 units of

Taq

DNA polymerase (Promega), 200

µ

m

of dCTP, dGTP, and dTTP, 20

µ

m

of dATP, 2 m

m

MgCl

2

, 10 m

m

Tris-HCl (pH 8.3), 50 m

m

KCl, 0.1% Triton X-100 and 0.05

µ

L[

α

-

33

P] dATP at 1000 Ci/mmol overlaid with mineral oil. PCRamplifications were performed in an MJ Research PTC100thermocycler starting with 94

°

C for 3 min, followed bya ‘touchdown’ (32 cycles at 94

°

C/20 s, annealing/45 s and72

°

C/60 s), and a final step of 72

°

C for 4 min. Theannealing temperature of the ‘touchdown’ PCRs decreasedtwo degrees per cycle until reaching the fifth cycle (Table 1).PCR products were separated by 6% polyacrylamide gelelectrophoresis and visualized by autoradiography followingTaylor

et al.

(1994).Twelve loci were successfully amplified and showed high

levels of polymorphism and heterozygosity (Table 1). Numberof alleles per locus ranged from 6 to 33 and expected hetero-zygosities from 0.42 to 0.94. An ongoing screening showsthat loci with short and less variable repeats (e.g. Odont16)better differentiate between marine and freshwater lineages,while loci with long repeats and higher polymorphism (e.g.Odont07) are useful to reveal fine-scale population struc-tures (L. Beheregaray, unpublished). Marine populations haveshown a significant larger number of alleles at all loci thanfreshwater populations (202 and 120 alleles, respectively,

P

after a Wilcoxon rank test = 0.002). This difference in geneticvariability was expected due to the relatively reducedsubpopulation sizes of freshwater fish (Ward

et al

. 1994).All primer sets were also tested without extra optimization

in several

Odontesthes

species (including the commerciallyimportant

O. bonariensis

) and in the diverged

Basilichthyssemotilus,

a silverside endemic to freshwater environmentswest of the Andes Mountains. Amplification and poly-morphism was extensive (Table 2), indicating that thesemarkers are potentially useful for a wide range of popula-tion studies of wild and captive South American silversidespecies.

Acknowledgements

This research was partially supported by CAPES (BrazilianMinistry of Education), which provided funds for the field trip toBrazil and a PhD Scholarship to L.B.B. Additional fundingcame from a Macquarie University Postgraduate Research Awardto L.B.B. This is Key Centre for Biodiversity & Bioresourcespublication 302.

MEC882.fm Page 629 Friday, March 31, 2000 8:44 AM

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© 2000 Blackwell Science Ltd,

Molecular Ecology

, 9, 629–644

References

De Buen F (1953) Los pejerreyes (família Atherinidae) en lafauna uruguaya, con descripción de nuevas espécies.

Boletim doInstituto Oceanográfico da Universidade de Sã

ο

Paulo

,

4

, 3–80.Dyer BS (1993)

A phylogenetic study of atheriniform fishes with asystematic revision of the South American silversides (Atherinomorpha,Atherinopsinae, Sorgentinini)

. PhD Thesis, University of Michigan.

Phonlor G, Cousin JC (1997) Early life history of silverside fishes.In:

Subtropical Convergence Environments: the Coast and Sea inSouthwestern Atlantic

(eds Seeliger U, Odebrecht C, Castello JP),pp. 136–141. Springer-Verlag, Berlin.

Sunnucks P, Hales D (1996) Numerous transposed sequences ofmitochondrial cytochrome oxidase I–II in aphids of the genus

Sitobion

(Hemiptera: Aphididae).

Molecular Biology and Evolution

,

13

, 510–524.

Table 1 Primer sequences and characteristics of 12 Odontesthes argentinensis and O. perugiae microsatellite loci*. Repeat structure is basedon sequence of cloned allele. Ta is the annealing temperature used in the ‘touchdown′ polymerase chain reactions (PCRs) (see text fordetails). (n) is number of O. argentinensis and O. perugiae individuals screened in populations from southern Brazil and HE is expectedheterozygosity

Locus Primer sequences (5′–3′)Repeat structure Ta (°C)

No. of alleles and (n)

Size range (bp) HE

GenBank accession no.

Odont02 F: CGGTCACCTGATGGTTGGTC (GA)22 61 → 53 30 (204) 129–191 0.94 AF177431R: AACCCTCAACCCGGCGCATC

Odont07 F: GTCTGAAAGCATCGAGTGAC (CA)27 55 → 47 26 (402) 153–209 0.94 AF177432R: TCTTTTTCTACATCCCAAGC

Odont08 F: TTTCTTTATCCCGCTATGTC (CA)17 55 → 47 25 (402) 126–178 0.93 AF177433R: TGCTCTTGCCCTTGTGTATG

Odont09 F: CCCCACAGAGATAGGCACAC (CA)17 59 → 51 18 (402) 149–193 0.82 AF177434R: GGAGGGAAAAGGTCCAACAG

Odont11 F: ACACTGGTCCTCCTTACCTG (CA)14 59 → 51 18 (402) 128–164 0.75 AF177435R: CAGAATGTGATTGGTGGAGC

Odont16 F: CTGTGCAGACTTTCACTCTC (GA)11 55 → 47 7 (402) 120–138 0.42 AF177436R: CCTGTGGTAAGACCTTTCTG

Odont23 F: TAAGATGGAGGTGCTG (GA)23 59 → 51 33 (204) 108–180 0.92 AF177437R: TTCTCCCCTTCGTCCATAAC

Odont25 F: AAACGGCTCCCATAGACATC † 61 → 53 12 (10) 102–180 0.80 AF177438R: TAACATCACTGGGGTCACAC

Odont27 F: CCTAATGAAGGTGAAATGC (CA)10 59 → 51 6 (15) 185–199 0.87 AF177439R: GTAAAGGGAGAGGATGGAG

Odont29 F: GAGAAAGCGACCAAAACAGC (CA)9 61 → 53 6 (15) 123–135 0.67 AF177440R: ACAGGAGCTGGGATGGGATG

Odont38 F: ACTAAACGCCCTGATTGTCC ‡ 61 → 53 11 (12) 104–150 0.85 AF177441R: TCAGTCCATCCATCCAAGAG

Odont39 F: CTCCCACTAATCCCATTTG § 59 → 51 18 (402) 163–199 0.85 AF177442R: TACGTGAGCATGTGTGTTG

*OdontO9 and 38 were isolated from O. perugiae and the others from O. argentinensis. †(CACG)4(CA)7,(CA)7,(CA)13; ‡ (CA)11,(AGAC)6; §(CT)5,(CA)9.

Table 2 Number of alleles from cross-species amplification in seven South American silversides. n is the number of individuals tested

Locus (Odont)

Species n 02 07 08 09 11 16 23 25* 27* 29 38* 39

Odontesthes incisa 10 9 10 8 8 9 1 5 8 5 7 8 6O. bonariensis 15 7 6 5 5 6 2 11 3 7 4 5 3O. retropinnis 3 4 4 5 3 2 2 1 2 3 2 4 2O. humensis 3 5 5 5 2 2 2 2 2 4 4 4 4O. mirinensis 40 15 10 13 12 9 2 12 9 6 5 9 4O. hatcheri 3 3 5 3 2 1 2 2 2 3 4 3 2Basilichthys semotilus 1 1 1 2 2 1 1 1 1 1 2 1 2

*extra optimization is advisable to facilitate scoring these loci.


P R I M E R N O T E S 631

© 2000 Blackwell Science Ltd, Molecular Ecology, 9, 629–644

Taylor AC, Sherwin WB, Wayne RK (1994) Genetic variationof microsatellite loci in a bottlenecked species: the NorthernHairy-nosed Wombat, Lasiorhinus krefftii. Molecular Ecology, 3,277–290.

Ward RD, Woodwark M, Skibinski DOF (1994) A comparison ofgenetic diversity in marine, freshwater, and anadromus fishes.Journal of Fish Biology, 44, 213–232.

9no issue no.2000904primer notesprimer notesprimer notes1000Graphicraft Limited, Hong Kong

Microsatellite characterization in the rainbow wrasse Coris julis (Pisces: Labridae)

T. GUILLEMAUD,*† R . STREIFF , *‡ R . SERRÃO SANTOS,§ P. AFONSO,§ T. MORATO§ and M. L . CANCELA*

*Universidade do Algarve, CCMar/UCTRA, P-8000 Faro, Portugal, §Universidade dos Açores, Departamento de Oceanografia e Pescas, P-9900, Horta (Açores), Portugal

Keywords: Dinucleotide repeats, Labridae, tetranucleotide repeats

Received 23 October 1999; revision accepted 12 November 1999

Correspondence: Thomas Guillemaud. Present address: †USVE, INRA, BP 2078, 06606 Antibes cedex, France. Fax: (351) 289818353; E-mail: [email protected] address: ‡INRA, Laboratoire de Modélization et de Biologie évolutive, 488 rue de la Croix-Lavit, 34000 Montpellier, France

The rainbow wrasse, Coris julis (Linnaeus 1758), is one of themost common fish in shallow coastal waters within its geo-graphical distribution (e.g. Sanchez Delgado 1981; Lejeune1987) that includes the Mediterranean, southern Black Seaand the North-Eastern Atlantic (Porteiro et al. 1996). Dispersionis apparently limited in the rainbow wrasse, giving that itshows sedentary habits and that migration movements havenot been described to date in this species (Lejeune 1987).Thus, it constitutes a good model for investigating genetic driftand differentiation in marine fishes. In this study, we havedeveloped microsatellite markers to analyse the genetic popula-tion structure of Coris julis throughout its distribution area.

A (CT)n-microsatellite enriched partial genomic librarywas constructed following a modified protocol of Kijas et al.(1994). Fragments ranging from 300 to 600 bp of Sau3A-digested DNA were ligated into annealed Sau3A adaptors(AdapF: 5′-CTCTTGCTTACGCGTGGACTC-3′ and AdapR:5′-GATCGAGTCCACGCGTAAGCAAGAGCACA-3′). Afterdenaturation, single stranded DNA was enriched for micro-satellites through the hybridization with 5′-biotinylated, 3′-aminated (CT)15 oligonucleotides bound to streptavidin-coatedmagnetic beads (Magnesphere, Promega, Madison, WI). Theenriched DNA was eluted twice in 10 µL water for 20 minand then used as a template for a polymerase chain reaction(PCR) using AdapF as a primer to recover double strandedDNA fragments. The PCR product was directly ligated over-night at 4 °C into pGEM-T easy vector (Promega, Madison,WI) using a 1:1 insert:vector molar ratio. The ligation wasperformed in 10 µL using 50 ng vector, 8 ng purified PCRproduct, and three units of T4 ligase (Promega). One tenth of

the ligation was transformed into 50 µL supercompetant cells(X11-Blue MRF′, Stratagene, La Jolla, CA). Eighty recombinantclones were screened for the presence of (CT)n microsatellitesand 16 for (GT)n microsatellites using the PCR method describedby Waldbieser (1995). Approximately 40% of the clones werepositively scored for both the CT and GT motifs.

From 35 clones sequenced, using the dideoxy chain termina-tion method (Sanger et al. 1977), 28 contained at least onemicrosatellite and three sequences were identical. Nineteenclones contained a CT-motif, four a GT-motif, three both CT- andGT-motifs, one an AAAC-microsatellite, and one an ACG-microsatellite. The two last clones were screened positivelyfor the GT motif. These results seem to indicate that theenrichment procedure and the PCR screening are not highlyspecific to the motif used. Due to the positions of the micro-satellites within the inserts, we could define primer pairs for21 different loci. Seventeen loci were successfully amplifiedusing the following conditions: a total volume of 20 µL con-tained 10 ng DNA, 20 mm Tris-HCl (pH 8.4), 50 mm KCl,between 0.75 and 2.5 mm MgCl2, 100 µm dATP, 60 µm each ofthe other dNTPs, 2.4 µm each primer, one unit of Taq DNApolymerase (Gibco BRL, Life Technologies Inc., Gaithersburg,MD), and 0.16 µL [α-35S]dATP (12.5 mCi/mL, 1250 Ci/mmol).PCR was performed in a Stratagene Robocycler (StratageneCloning Systems, La Jolla, CA) and consisted of a first denatura-tion step at 94 °C for 4 min, followed by 30 cycles of 45 s at94 °C, 45 s at 47 or 50 °C depending on the locus, and 45 sat 72 °C, with a final extension of 10 min at 72 °C. Ten lociwere polymorphic and showed a number of alleles rangingfrom 2 to 23. Eight of them were easily scorable with theconditions showed in Table 1.

These eight microsatellites were scored for 20 individualsfrom Farol Island, Portugal. None of the loci showed a sig-nificant departure from Hardy–Weinberg equilibrium (genepop3.1b; Raymond & Rousset 1995), and no linkage disequilibriumwas detected between each pair of locus. Therefore, theseeight loci seem highly informative for populations studies.

Acknowledgements

We thank Pedro Neves for helping in sample collection fromPortugal, and Jacques Lagnel for technical advice. This work wasfunded in part by project PRAXIS EMG 1957/95 (CLIPE).T.G. was the recipient of postdoctoral fellowship (PRAXISXXI/BPD/4470/96).

References

Kijas JMH, Fowler JCS, Garbett CA, Thomas MR (1994) Enrich-ment of microsatellites from citrus genome using biotinylatedoligonucleotide sequences bound to streptavidin-coated magneticparticules. Biotechniques, 16, 657–662.

Lejeune P (1987) The effect of local stock density on social behaviourand sex change in the Mediterranean labrid Coris julis.Environmental Biology of Fishes, 18, 135–141.

Porteiro FM, Barreiros JP, Santos RS (1996) Wrasses (Teleostei:Labridae) of the Azores. Arquipélago, 14, 23–40.

Raymond M, Rousset F (1995) genepop (Version 1.2): populationgenetics software for exact tests and ecumenicism. Journal ofHeredity, 86, 248–249.


632 P R I M E R N O T E S


Sanchez Delgado F (1981) Contribution al conocimiento delos labridos (familia Labridae) de las costas ibericas. ParteI: Descripcion de las especies. Boletin del Instuto EspañolOceanografico, 6, 19–57.

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing withchain-terminating inhibitors. Proceedings of the National Academyof Sciences of the USA, 74, 5463–5467.

Waldbieser GC (1995) PCR-based identification of AT-rich tri- andtetranucleotide repeat loci in an enriched plasmid library.Biotechniques, 19, 742–744.

9no issue no.2000902primer notesprimer notesprimer note1000Graphicraft Limited, Hong Kong

Characterization of microsatellite loci developed for the wattled curassow, Crax globulosaCOLIN R. HUGHES* and ERIK D. LARSON

*Department of Biology, University of Miami, Box 249118, Coral Gables, FL 33124–0421, USA

Keywords: Aves, Cracidae, curassow, microsatellite, polymorphic, primers

Received 13 November 1999; revision accepted 18 November 1999

Correspondence: C. R. Hughes. Fax: +1 305 284 3039; E-mail: [email protected]

The family Cracidae comprises 50 species of curassows,guans, and chachalacas (Monroe & Sibley 1993). All are largebodied Neotropical birds, and most are forest dwellingfrugivores (Delacour & Amadon 1973). These characteristics

mean that species face double jeopardy; they are extensivelyhunted in addition to suffering from habitat destruction.As a consequence, 13 species are considered threatened spe-cies, including the wattled curassow (Collar et al. 1992). Wedeveloped and characterized six microsatellite loci in thewattled curassow as part of an effort to breed the birds incaptivity. Additionally, we examined the polymorphism ofthese loci in six other members of the Cracidae.

DNA was extracted from blood or tissue using a proteinaseK, phenol–chloroform extraction procedure modified fromMüllenbach (1989). Size selected genomic DNA digested withDpnII was cloned into Lambda Zap Express, Stratagene,La Jolla, CA (Hughes et al. 1998). We screened ≈ 125 000 cloneswith the oligo (AAT)10 (Hughes & Queller 1993), sequenced31 positives (Sequenase ver 2.0 or Thermosequenase, USB),and developed primers for 10 clones containing ≥ 6 uninter-rupted repeats of the sequence AAT.

Polymerase chain reactions (PCRs) (5 µL) contained ≈ 5 ngof DNA, 50 mm KCl, 10 mm Tris/Cl pH 8.3, 1.5% mM MgCl2,0.1% NP40, 100 mm each dNTP, 0.25 U Taq DNA poly-merase (Perkin Elmer), 2.5 pmol each primer, and 0.05 µL35S dATP. Reactions were cycled using the ‘tube-control’function of a Hybaid thermal cycler: 90 s at 92 °C, then 5 sat a suitable annealing temperature for each primer pair(Table 1), 5 s at 72 °C, 5 s at 92 °C, 30 times, and finally 90 s at72 °C. Amplified fragments were resolved using 6% denatur-ing polyacrylamide gels. Allele length was determined bycomparison to the sequencing products of M13.

Six loci tested were found to be polymorphic in thewattled curassow (Table 1). Original clones of these had

Table 1 Microsatellite loci from Coris julis. Number of repeats, repeat motif and size of the amplification product from the clones areindicated. Annealing temperatures and MgCl2 concentrations, number of alleles obtained and expected (HE) heterozygosities are alsoindicated. Number of alleles and heterozygosities are based on a sample of 20 individuals from Farol Island. GeneBank accessionnumbers for the cloned sequences are AF190802–AF190809

Locus Repeats Primer sequence (5′–3′) Size (bp)Anneal.temp. (°C) MgCl2 (mm)

No. ofalleles HE

D-11 (CAAA)2TAAA(CAAA)7 F: GGACACTTCGACCACGAACC 176 50 1.25 6 0.74R: GTCACTCTCTGAGCTAACTGTGC

D-3–3 (CT)6 F: GAAGCACCTTTCCAGGGGAT 85 47 0.75 3 0.55R: CAGAGGATTCTGTGGTAAAC

6–1 (TG)3TAA(GT)3(GA)2AAG(AC)6CCTC(AC)4 F: GGTCACCGTTGTTGTTTGTC 159 47 1 2 0.18R: GATCCCGTATGCCAAACAC

A-1 (CT)8 F: GTGCTGCTGAGACACTGCGA 230 50 0.75 4 0.65R: TGTCTTCAGCTCATGCCTCC

F-7 (TG)3C(GT)9 F: GCTCAGTGAAGTAAACGGAGAGG 243 50 0.75 6 0.67R: TATCAGGAAGCGGCCAGTGTTG

G-2–2 (CT)7(TC)2CC(TC)5 F: AACATCCTTGTGAACACACG 122 50 0.5 5 0.32R: AGAACCTGCTGCCTCTCTGC

F-2–4 (GA)19…(TC)13…?…(TC)5…(TC)5…(TC)12* F: AGGAGAACCAGAACATTACG ~ 250** 47 2 4 0.64R: CTGAACTCAAAATAAAGTCG

E-4 (CT)17 F: ACTTCCTTGCACTCACACTC 232 50 0.75 15 0.81R: GGCTCAGACTTGAGGCTTAG

*The question mark corresponds to an unknown sequence of about 25 bp. **Approximate size as revealed from an agarose gel electrophoresis.




contained over 10 AAT repeats. Four loci with 6, 6, 7, and 8,repeats of AAT were monomorphic.

The six polymorphic loci were used in PCR reactions con-taining DNA from six other species of Cracidae. PCR condi-tions were those described above. Our primers had lessutility in these species (Table 2), although some will be use-ful in studies of other curassows and chachalacas. Theseresults are consistent with previous studies of primer pairutility across taxa (Primmer et al. 1996; Hughes et al. 1998).DNA-DNA hybridization shows curassows and chachalacasto be closely related, and these genera to be less related to theguans (Sibley & Ahlquist 1990). The hybridization data how-ever, show the Cracidae to be distantly related to other birds;so distantly, that these primers are unlikely to be of use out-side this family.

Acknowledgements

This work was supported in part by National Science Founda-tion grant # DEB 9424625, University of Miami, and a grantfrom Louisiana State University Museum of Natural Science. Wethank Daniel Brooks and Chelle Plassé for supplying the bloodsamples.

References

Collar NJ, Gonzaga LP, Krabbe N et al. (1992) Threatened Birds ofthe Americas, 3rd edn, Vol. 2. Smithsonian Institution, Washington.

Delacour J, Amadon D (1973) Curassows and Related Birds. AmericanMuseum of Natural History, New York.

Hughes CR, Kavlie R, Johnson K (1998) Characterization ofpolymorphic trinucleotide microsatellite loci in the great-tailed grackle, Quiscalus mexicanus. Molecular Ecology, 7, 783–784.

Hughes CR, Queller DC (1993) Detection of highly polymorphicmicrosatellite loci in a species with little allozyme poly-morphism. Molecular Ecology, 2, 131–137.

Monroe BL, Sibley CG (1993) A World Checklist of Birds. Yale Uni-versity Press, New Haven.

Mullenbach R, Lagoda PJ, Welter C (1989) An efficient salt-chloroform extraction of DNA from blood and tissues. Trendsin Genetics, 5, 391.

Primmer CRM, Møller AP, Ellegren H (1996) A wide-rangesurvey of cross-species microsatellite amplification in birds.Molecular Ecology, 5, 365–378.

Sibley CG, Ahlquist JE (1990) Phylogeny and Classification of Birds.Yale University Press, New Haven.


Table 1 Polymorphic AAT-repeat microsatellite loci developed for the wattled curassow. HO, is proportion of observed heterozygotes ina sample of 23 individuals, some known to be related. Expected heterozygotes (HE) = 1 – p2

i. GenBank accession numbers: AF189271–AF189276

Locus names Repeat sequence Primer sequences, 5′–3′ HO HE

Productsize range

No. ofalleles Suitable Ta °C

CgAAT11 AAT13 F: GGCCATTTGTTGCACAGTAG 0.52 0.69 228–237 4 60–65R: GATCTGGAGCTGCTTTTATTA

CgAAT32 AAT14 F: GTGCCCCAGCAGTAATAATA 0.76 0.66 146–175 6 55R: CCATTGTACCAAAGTCACAGTA

CgAAT62 AAT6 F: CTGCTGGCCACGATTCTTC 0.78 0.75 112–127 6 55GATAAT7 R: TCACGTACTTTTGTCCGTATTGTA

CgAAT82 ATT16 F: GGTCCCTTCCAAGTTGAATCAT 0.67 0.78 97–118 6 60–65R: AAGCCAAGCATGGAAAGAAAATA

CgAAT85 AAT16 F: CCATAGGTGGGTTGTATTA 0.59 0.71 190–232 6 60–65R: AGCAGAGCCAATATGAAGTAA

CgAAT190 AAT13 F: TCACCACCATTTCCCAACAG 0.60 0.71 192–222 6 55–60R: ATGAGATTTACCTTCAGTTCT

Ta, annealing temperature.

Species Sample size

Locus

11 32 62 82 85 190

Crax daubentoni, yellow-knobbed curassow 5 M P P — P PCrax fasciolata, bare-faced curassow 2 M P P P P PCrax rubra, great curassow 5 P P M M P PMitu tomentosa, crestless curassow 3 M P P — M POrtalis vetula, plain chachalaca 2 M P P — P PAburria pipile, Trinidad piping guan 2 — M M — M P

Table 2 Cross species amplification usingthe primers developed for wattled curassows.Successful amplification of appropriate sizedproduct indicated as P, polymorphic or M,monomorphic. Unsuccessful amplification, —




Microsatellites in rockfish Sebastes thompsoni (Scorpaenidae)

M. SEKINO,* N. TAKAGI , * M. HARA† and H. TAKAHASHI‡

*National Research Institute of Fisheries Engineering, Hasaki, Kashima, Ibaraki, 314–0421, Japan, †National Research Institute of Aquaculture, Nansei, Watarai, Mie, 516–0193, Japan, ‡National Institute of Agro-biological Resources, Kannondai, Tsukuba, Ibaraki, 305–8602, Japan

Keywords: DNA, microsatellites, rockfish, Scorpaenidae, Sebastes thompsoni


Correspondence: M. Sekino. Fax: +81–479–1875; E-mail: [email protected]

The rockfish, Sebastes thompsoni, is an important commercialand recreational fish. Around Japan, the rockfish is mainlydistributed in the northern coast of the Sea of Japan (Amaoka1984; Ikehara 1989). Nagasawa & Kobayashi (1995) reportedthat larval rockfish is carried by the Tsushima warm currentand widely dispersed in the Sea of Japan, however, theirpopulation structure and recruiting dynamics into regionalpopulations around Japan are still unknown. For evaluationand effective use of natural stocks, it is necessary to knowgenetic population structure and gene flow, and an efficienttool is required. Microsatellite markers are highly polymorphicand quite sensitive genetic markers to study fish populations(McConnell et al. 1995). We, therefore, developed microsatel-lite markers in the rockfish and examined their application toother Sebastes species.

A (CA)n-enriched library was constructed using the methodof Takahashi et al. (1996) with modifications. Genomic DNAwas extracted from fin tissues according to Asahida et al.(1996). To obtain nonbiased DNA fragments, genomic DNAwas fragmented by sonication. Sonicated fragments wereblunted by mung bean nuclease (Takara, Shiga, Japan) underthe conditions recommended by the supplier. The fragmentswere electrophored on agarose gel and fragments ranging from300 to 500 bp were recovered. The fragments were repairedby T4 DNA polymerase (Toyobo, Osaka, Japan) according to thesupplier’s manual, and then ligated into Srf I site of pCR-ScriptAmp SK(+) vector (Stratagene, La Jolla, CA, USA). Recombinantplasmid vector was transformed into XL2-Blue MRF′ ultra-competent cells (Stratagene), and single-stranded DNA wasprepared according to Takahashi et al. (1996). Selective second-strand DNA synthesis was employed using (CA)12 oligonucle-otide and cloned pfu DNA polymerase (Stratagene) accordingto Takahashi et al. (1996). Single-stranded DNA remaining inthe reaction mixture was digested with mung bean nuclease.The resultant double-strand DNA was transformed again intoXL2-Blue MRF′ cells and these transformants were referredto a (CA)n-enriched library. From the library, 35 clones wererandomly chosen, and plasmid DNAs were purified. The DNAsequences were determined using Thermo Sequenase™ cyclesequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden)in combination with KS and T3 primers and subjected to aDNA sequencer (ALFexpress, Amersham Pharmacia Biotech).

Of the 35 clones, 21 CA-repeat microsatellites were detected.The length of CA-repeat units varied between 4 and 30 units

with a mean value of 12.1. We developed 16 PCR primer pairsusing a software (Primer Premier, Premier Biosoft Inter-national, Palo Alto, CA, USA). Five clones were discardedbecause they had insufficient sequence information to developprimers and/or the CA-repeat arrays were too short. To detectmicrosatellite polymorphisms, amplification was performed ina 20 µL reaction volume, which included 20 pmols of eachprimer (one of a pair primer was 5′ end-labelled with Cy5),100 µm each dNTP, 10 mm Tris-HCl (pH 8.3), 50 mm KCl,1.5 mm MgCl2, 0.001% gelatin, 0.5 units of Ampli Taq GoldTM

(Perkin Elmer, Foster City, CA, USA), and approximately 200 ngof template DNA using PC-960G Gradient Thermal Cycler(Corbett Research, Mortlake, N.S.W., Australia). Polymerase chainreaction (PCR) was performed: 10 min at 94 °C, 25–30 cycles of30 s at 94 °C, 45 s at a primer-specific temperature, and 45 s at72 °C, and final elongation for 5 min at 72 °C. Analyses of PCRproducts were performed using ALFexpress sequencer and asoftware (Allelelinks, Amersham Pharmacia Biotech). Of the16 primer pairs, 15 were available for PCR amplification, outof which we finally chose eight primer pairs (the remaining sevenhaving been rejected because their polymorphisms were lowand/or they produced unexpected PCR products) and exam-ined further microsatellite polymorphisms in natural rock-fish from the northern coast of the Sea of Japan (see Table 1).

As shown in Table 1, the number of alleles per locus rangedfrom 3 to 22, and the observed and expected hetrozygosityranged from 0.30 to 0.92, and from 0.30 to 0.88, respectively.Genotype frequencies observed that all loci were in agree-ment with Hardy–Weinberg’s expectations in the Markov-chain method (parameters used; Markov-chain steps: 100 000;dememorization steps: 10 000), using the Arlequin ver.1.1software (Schneider et al. 1997).

We attempted cross-species amplification in three otherSebastes species; S. inermis, S. joyneri, and S. vulpes, known to becommercially important fishes. All primer pairs could be usedfor all species under the same PCR condition, and each locusshowed polymorphisms (Table 2). These data suggest that micro-satellite markers developed from a Sebastes species can beapplied to other Sebastes species. The microsatellite markersin this study will contribute to phylogentic studies in theSebastes species besides intraspecies population studies.

Acknowledgements

We express our gratitude to Dr M. Higuchi, Niigata PrefecturalFisheries and Marine Research Institute, for donating fish DNAsamples. We also acknowledge the contributions of N. Takagi andY. Nakamura, National Research Institute of Fisheries Engineering,and M. Minezawa, National Institute of Agro-biological Resources.

References

Amaoka K (1984) Sebastes thompsoni. In: The Fishes of the JapaneseArchipelago (English Text) (eds Masuda H, Amaoka K, Araga C,Ueno T, Yoshino T), p. 311. Tokai University of Press, Tokyo.

Asahida T, Kobayashi T, Saitoh K, Nakayama I (1996) Tissuepreservation and total DNA extraction from fish stored atambient temperature using buffers containing high concentra-tion of urea. Fisheries Science, 62, 723–726.

Ikehara K (1989) Mode of life for Sebastes thompsoni in Tsushima

MEC882.fm 34 Friday, March 31, 2000 8:44 AM

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Table 1 Core repeat and primer sequences, PCR amplification conditions, and results of variability of eight microsatellite loci in a Sebastes thompsoni population

Locus Core repeat sequence (5′–3′) Primer sequence (5′–3′)Anneal °C

No. of samples

No. ofalleles

Size range(bp) HO HE (p*)

GenBank accession no.**

Sth 3A (CA)10 F-ATGGTGACAAGCTAGCAGTGCATTC 56 50 5 113–121 0.60 0.67 (0.29) AB033424R-GACAATGTCCATCTAGGCATGACTG

Sth 3B (CA)13 F-GTCATGCCTAGATGGACATTGTCTAC 55 50 14 147–178 0.92 0.85 (0.77) AB033425R-GAGATAAGAGGAGTTTGAAGGCAGAG

Sth 24 (CA)8 F-AGGACAGGATGTGCCCTTTTACCA 56 50 4 133–139 0.46 0.53 (0.41) AB033426R-GCCTCAGAGGCCGATTTCCTTATT

Sth 37 (CA)16 F-TACAGGAAACAAGACCACGGGTACAG57 49 10 221–243 0.88 0.88 (0.28) AB033427R-GCAACATCCCTTTAAGTCACCTGCAG

Sth 45 (CT)13X5(CT)2X3(CA)5(GA)2A(CA)4 F-CTGGGACCTAGCCTGATTACAGCA 57 49 10 192–212 0.71 0.76 (0.50) AB033428R-AAACTCAGCGACAGCAGACCACA

Sth 56 (TA)2(CA)24 F-CAGCAGCTCCAGTTCAGTGTATGT 55 50 22 164–250 0.74 0.75 (0.20) AB033429R-GGATTGATCCTCATGTGGTGCTCT

Sth 86 (CA)6CG(CA)5C2(CA)3 F-ACCATCACCCACTGTAAACTGCA 54 50 10 189–211 0.82 0.79 (0.40) AB033430R-TACCAGGAAACGTCGTGTCTCAA

Sth 91 (CA)6(TA)3G2A(CA)4 F-TTTCGATATGCTTCGCTAGGGTGTT 57 50 3 216–220 0.30 0.30 (0.19) AB033431R-CCATCAAACTGCACCAACAAAGACA

mean 9.8 0.68 0.69

*p is the exact P-value estimated by a test anologous to Fisher’s exact test described by Schneider et al. (1997). Signifficant differences between HO and HE were not observed at all loci.**The nucleotide sequence data will appear in the DDJB/EMBL/GenBank nucleotide databases with the accession numbers.

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water current areas (in Japanese). Contributions to the FisheriesResearches in Japan Sea Block 15, 71–79.

McConnell SK, O’Reilly P, Hamilton L, Wright JM, Bentzen P(1995) Polymorphic microsatellite loci from Atlantic salmon(Salmo salar): genetic differentiation of North American andEuropean populations. Canadian Journal of Fisheries and AquaticScience, 52, 1863–1872.

Nagasawa T, Kobayashi T (1995) The early life history of the rockfish, Sebastes thompsoni (Scorpaenidae) in the Sea of Japan. Japa-nese Journal of Ichthyology, 41, 385–396.

Schneider S, Kueffer JM, Roessli D, Excoffier L (1997) Arlequin,Version 1.1: A software for population genetic data analysis. Gen-etics and Biometry Laboratory, University of Geneva, Switzerland.

Takahashi H, Nirasawa K, Furukawa T (1996) An efficientmethod to clone chicken microsatellite repeat sequences.Japanese Poultry Science, 33, 292–299.


Characterization of microsatellite loci in the endangered freshwater crayfish Austropotamobius pallipes (Astacidae) and their potential use in other decapods

N. GOUIN, F. GRANDJEAN and C. SOUTY-GROSSET

Laboratoire de Génétique et Biologie des Populations de Crustacés, UMR CNRS 6556, Université de Poitiers, 40 Avenue du recteur Pineau, 86022 Poitiers cedex, France

Keywords: Austropotamobius pallipes, conservation genetics, Crustacea, Decapoda, microsatellite


Correspondence: N. Gouin. Fax: + 33 (0)5 49 45 40 15; E-mail: [email protected]

Conservation genetics is an applied science whose goal is todescribe explicitly the composite genomes of small endangeredpopulations to help in their management. In the white-clawedcrayfish Austropotamobius pallipes, endemic to western Europeanfresh waters and now confined to headwater river systems,the resolution of available molecular markers such as allozymesand mitochondrial DNA (mtDNA) is of limited usefulness

for conservation because of their inability to reveal geneticvariability among populations over its range (Grandjeanet al. 1997; Santucci et al. 1997; Lörtscher et al. 1998). The useof genetic markers with higher resolution, therefore, appearsto be essential in order to understand the genetic structure ofA. pallipes and then to define concrete management plans. Herewe report the isolation and characterization of six microsatel-lite loci from A. pallipes. We examined the polymorphism withinthis species and two closely related taxa, A. italicus italicus andA. i. carsicus, described by Brodsky (1983). Because of the deficitof genetic markers available in decapods, we also examined theconservation of these loci in six species of freshwater crayfishencountered in Europe and in four marine decapod species.

We developed microsatellite markers following the methodsdescribed by Estoup & Martin (1996). DNA for cloning wasisolated from abdominal muscles of one A. pallipes specimenusing phenol–chloroform (Kocher et al. 1989), and 10 µg weredigested with Sau3AI. Fragments, 400–900 bp, were purifiedfrom 1.7% agarose using a DEAE NA45 cellulose membrane(Schleicher and Schuell) and ligated into the pUC18/BamHIplasmid (Pharmacia). XL1-Blue competent cells (Stratagene)were transformed with ligation products. Transformed cells weregrown on LB agar, transferred onto Hybond-N + membranes(Amersham) and screened using digoxigenin-end-labelled(GT)10 (TC)10 and (CAC)5(CA) probes. Among the 942 clonesscreened, 58 were identified as positive and isolated. DNAfrom 40 clones was sequenced using the BigDye TerminatorCycle Sequencing Ready Reaction Kit (Perkin-Elmer AppliedBiosystems) and analysed with an ABI PRISM 310 automatedsequencer. Eleven primer sets were developed using the pro-gam primer 3 by Rozen and Skaletsky (code available at http://www.genome.wi.mit.edu/genome_software/other/primer.html)but five failed to amplify a polymerase chain reaction (PCR)product. Characteristics of the remaining six primer pairs aredescribed in Table 1.

PCR reactions were performed in a final volume of 12.5 µL(1× Taq DNA polymerase buffer (Promega: 50 mm KCl, 10 mmTris-HCl (pH = 9.0), 0.1% Triton X-100), 1.2 mm MgCl2,60 µm each dNTP, 5 pmol each primer, 0.25 U Taq DNA poly-merase (Promega) and 15 ng of DNA template) using a Trio-Thermoblock (Biometra). For each reaction, one primer of eachpair was end-labelled with one fluorescent phosphoramidite(TET, HEX or 6-FAM). Amplification conditions were 95 °Cfor 5 min followed by 30 cycles of 95 °C for 30 s, specificannealing temperature (Table 1) for 30 s, 72 °C for 45 s anda final extension of 72 °C for 10 min. PCR products were runwith the internal size standard GeneScan-500 [TAMRA] (PEApplied Biosystems) on an ABI PRISM 310 automated sequencer.Their size was determined using GeneScan Analysis 2.1.

All these microsatellites appeared polymorphic in the white-clawed crayfish (Table1): Ap2, Ap3, Ap5, Ap6 for A. pallipes,Ap1, Ap2, Ap3, Ap6 for A. i. italicus, and Ap1, Ap3, Ap4, Ap6for A. i. carsicus. However, allelic diversity appeared lower inA. pallipes than in A. italicus. Locus Ap5 failed to be amplified inA. italicus. Amplification products from loci Ap4 for the italicussubspecies and Ap2 for the carcicus appeared longer than thelimit of 500 bp required by the size standard to be analysed.

Concerning cross-species amplifications, annealing tem-peratures were decreased until 48 °C depending on loci and

Table 2 The results of cross-species amplifcation of the eightmicrosatellites in the three Sebastes species

Locus

Species

S. inermis S. joyneri S. vulpes

Sth 3A 4 (15) 2 (2) 4 (6)Sth 3B 6 (15) 1 (2) 5 (6)Sth 24 1 (9) 4 (2) 1 (6)Sth 37 9 (15) 4 (2) 2 (6)Sth 45 2 (16) 1 (2) 3 (6)Sth 56 17 (12) 4 (2) 9 (6)Sth 86 2 (16) 1 (2) 2 (6)Sth 91 3 (15) 1 (2) 2 (6)

The number of alleles detected are given. The number of samples examined for each species is given in parenthesis.




species. Table 2 reveals that conservation of these loci appearedrelatively low between studied decapod species (38%), withno information about the extent of polymorphism.

Acknowledgements

We thank D. Guyonnet and S. Patri for their technical assistanceand the members of the Délégations régionales of the ConseilSupérieur de la Pêche (C.S.P.), D. Mazzoni, F. Quaglio, N. Budhinaand M. Robinson for kindly providing samples. This researchwas funded by the C.S.P., the Ministère de l’Aménagement duTerritoire et de l’Environnement and a doctoral fellowship fromthe Conseil Régional du Poitou-Charentes.

References

Brodsky SY (1983) On the systematics of palearctic crayfishes(Crustacea, Decapoda). Freshwater Crayfish, 5, 464–470.

Estoup A, Martin O (1996) Marqueurs microsatellites: isolementà l’aide de sondes non radioactives, caractérisation et mise au

point. Personal communication, protocols available in the webat http://www.inapg.inra.fr/dsa/microsat/microsat.htm

Grandjean F, Souty-Grosset C, Holdich DM (1997) Geographicalvariation of the mitochondrial DNA between Europeanpopulations of the white-clawed crayfish Austropotamobiuspallipes. Freshwater Biology, 37, 493–501.

Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S,Villablanca FX, Wilson AC (1989) Dynamics of mitochondrialDNA evolution in animals: amplification and sequencing withconserved primers. Proceedings of the National Academy ofScience of USA, 86, 6196–6200.

Lörtscher M, Clalüna M, Scholl A (1998) Genetic populationstructure of Austropotamobius pallipes (Lereboullet 1858)(Decapoda: astacidae) in Switzerland, based on allozyme data.Aquatic Sciences, 60, 118–129.

Santucci F, Iaconelli M, Andreani P, Cianchi R, Nascetti G,Bullini L (1997) Allozyme diversity of European freshwatercrayfish of the genus Austropotamobius. Bulletin Français de laPêche et de la Pisciculture, 347, 664–676.


Table 1 Characteristics of white-clawed crayfish (Austropotamobius pallipes) microsatellites (Tot., total number of alleles per locus;Ta, annealing temperature of primers in C; HO, observed heterozygosity; HE, expected heterozygosity; *, 5′ end-labelled primers;–, nonamplification; ?, not assessed). GenBank accession numbers for cloned sequences are AF204815–AF204820

Locus Primer sequences (5′–3′) Repeat motifSize range (bp) Tot.

No. of alleles (Ta) [HO/HE]

A. pallipes n = 30

A. i. italicus n = 15

A. i. carsicusn = 10

Ap1 F: TCTTGGGGATTGGCTAGTTG (CA)13(CG)2(CA)6 123–141 3 1 2 2R: CCTGAACTAAAAGGTGCTTTGG* (60) (60) (60)

[0/0] [0.08/0.08] [0.22/0.21]Ap2 F: TTCGATATAACCGTTTGACCTG* (CA)31 151–191 7 3 4 ?

R: TCAGACTTTGGCCATTGAAG (60) (58) (58)[0.35/0.50] [0.45/0.61] ?

Ap3 F: CGCCTATCTAACCTTGGTTGTC (CA)25 128–216 12 3 4 6R: GGACTTGGGAAGCCTTGTG* (60) (52) (52)

[0.43/0.49] [0.51/0.72] [0.40/0.72]Ap4 F: GCGGTTCTTATGTTGGTC* (CA)14CC(CA)9AT(CA)37 301–323 4 1 ? 4

R: GAGTGTAAGACAATTTTTGG AA(CA)18 (52) (48) (52)[0/0] ? [0.58/0.79]

Ap5 F: CGGCTGAAAGGTCAAAATG (CG)3(TA)4(CA)50CC(CA)3 389–395 3 3 — —R: TGTCTTCCAGGGACGTGAG* (CT)2(CA)52CG(CA)18 (58)

[0.33/0.55]Ap6 F: GCTGTGTGGGATGGAGGT* (TG)7GGGT(TG)8GG(TG)40 346–370 9 2 4 5

R: CACTAGCGTATTCAAGCAACT TT(TG)9TT(TG)7CA(TG)3 (56) (54) (54)[0.00/0.13] [0.00/0.35] [0.50/0.56]

Species Infra-order Ap1 Ap2 Ap3 Ap4 Ap5 Ap6

Austropotamobius torrentium Astacidea + + – – – +Astacus astacus Astacidea + + – – – *Astacus leptodactylus Astacidea + + + + – *Pacifastacus leniusculus Astacidea + * + + – *Procambarus clarkii Astacidea – + + – – *Orconectes limosus Astacidea + + – + – *Nephrops norvegicus Astacidea – – – – – –Homarus gammarus Astacidea * * – – + *Cancer pagurus Brachyura + – – – + *Galathea strigosa Anomura + + – – – –

Table 2 Cross-species amplification ofsix white-clawed crayfish microsatellitesin decapods (+ , amplification; –, non-amplification; *, multibanding pattern).For each species, only one specimen wasanalysed




Microsatellite loci from common and thick-billed murres, Uria aalge and U. lomvia

G. IBARGUCHI, T. P. B IRT, K . I . WARHEIT, * P. T. BOAG and V. L . FRIESEN

Department of Biology, Queen’s University, Kingston, Ontario, Canada, K7L 3N6 and *Department of Fish and Wildlife, 100 Capitol Way N., Olympia, WA, 98501–1091, USA

Keywords: common murre, microsatellite, primers, thick-billed murre

Received 21 August 1999; revision accepted 2 December 1999

Correspondence: V. L. Friesen. Fax: + 01 613 5336156; E-mail: [email protected]

Murres (guillemots) are colonial seabirds and importantcomponents of arctic and subarctic marine ecosystems. Thick-billed murres (Uria lomvia) generally occupy arctic latitudeswhile common murres (U. aalge) generally are found at borealand subarctic latitudes (Gaston & Jones 1998). To investigatethe hypothesis that natal philopatry has lead to genetic structur-ing both within and among breeding colonies (Birt-Friesenet al. 1992; Friesen et al. 1996), we isolated and characterizedmicrosatellite loci from these species.

Birds from both species were sampled throughout theirrespective ranges and DNA was prepared from either bloodor solid tissues using standard Proteinase-K digestion followedby phenol–chloroform extraction. Microsatellites were isolatedusing protocols similar to those reported by Gibbs et al. (1998).Genomic libraries were made by digesting 10 µg of DNA(2 µg from each of five birds for each species) with AluI,HaeIII and RsaI. Following electrophoresis through 2% agarosegels, DNA fragments ranging in size from approximately250–450 bp were purified and ligated into the SmaI site ofpUC18. Colony lifts were then probed with dinucleotiderepeat tracts (AC, GT, AG and CT) labelled with [α32P]-dCTP.Positive clones were sequenced and polymerase chain reaction(PCR) primers were designed from sequences flanking repeats.

Optimal PCR conditions varied slightly among the five locisubsequently found to be polymorphic. In general, amplifica-tions were done in 10–15 µL reaction cocktails containing 10 mmTris-HCl pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.2 µm dNTPs(each), 4 pmol primers (each) and 0.3 U Taq DNA polymerase(Boehringer Mannheim). One primer in each pair was end-labelled with [γ33P]-dATP using polynucleotide kinase.Resolution of alleles for locus ulo12a12 in common murreswas improved by increasing MgCl2 concentration to 2.0 mm.Optimal annealing temperatures used also varied among loci(Table 1). Amplified products were resolved in 6% denatur-ing polyacrylaminde gels (7 m urea) and visualized usingBIOMAX X-ray film (Kodak).

All five microsatellites developed from U. lomvia (threemarkers) and U. aalge (two markers) amplify and arepolymorphic in both murre species (Table 1). Numbers ofalleles detected vary from 4 to 32, although sample sizesalso vary considerably among loci and species. Heterozygo-sity estimates are similar to those reported for other avianmicrosatellites (e.g. Primmer et al. 1995; Dawson et al. 1997).Generally, observed heterozygosities are similar to expectedvalues except for locus ulo12a12 in common murres, in which Ta

ble

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5826

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6229

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917

0.90

358

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412

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AG

586

139–

151

430.

777

0.74

454

413

7–14

312

80.

441

0.39

1R

: TA

TA

GG

CT

TA

TG

CC

AG

AG

AG

AC

ulo1

4b29

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4(T

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7(T

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2F:

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TT

CC

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AA

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721

120–

172

296

0.80

40.

801

5820

127–

161

128

0.73

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727

R: T

AC

CC

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11F:

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GA

AA

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732

146–

198

296

0.91

20.

892

5820

155–

197

128

0.77

30.

779

R: T

TT

AG

CT

GG

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AA

GT

TA

GT

CA

Gua

a5–8

(AC)

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CA

GT

TT

CT

TT

AA

GT

CG

TG

CC

AG5

78

108–

126

430.

677

0.72

153

910

6–12

212

80.

716

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expected heterozygosity exceeds observed heterozygosity.The most likely explanation is the presence of one or morenull alleles at this locus (Pemberton et al. 1995), but thisexplanation cannot be verified from the present data set.

Primers developed to amplify ulo14b29 in murresgenerate PCR products in marbled murrelets (Brachyramphusmarmoratus), although only two alleles were detected among121 birds (Congdon et al., in preparation). Otherwise theutility of these loci with other species has not been explored.

Acknowledgements

We are grateful to D. Michaud, C. Crossman and H. Jones for experttechnical assistance and to A. J. Gaston for assistance with manyaspects of our seabird studies. V. Bird and J. Piatt provided murresamples from the North Pacific. Financial support was providedby the Natural Sciences and Engineering Council (Collaborativeand Operating Grants to P.T.B. and V.L.F.), the Tenyo MaruTrustee Committee, the Washington Department of Fish andWildlife and Queen’s University (Graduate Research Award to G.I.).

References

Dawson JG, Gibbs HL, Hobson KA, Yzerinac SM (1997) Isolationof microsatellite DNA markers from a passerine bird, Dendroicapetechia (the yellow warbler), and their use in population studies.Heredity, 79, 506–514.

Birt-Friesen VL, Montevecchi WA, Gaston AJ, Davidson WS (1992)Genetic structure of thick-billed murre (Uria lomvia) popula-tions examined using direct sequence analysis of amplified DNA.Evolution, 46, 267–272.

Friesen VL, Montevecchi WA, Baker AJ, Barrett RT, Davidson WS(1996) Population differentiation and evolution in the commonguillemot Uria aalge. Molecular Ecology, 5, 793–805.

Gaston AJ, Jones IL (1998) The Auks. Oxford University Press,Oxford.

Gibbs HL, De Sousa L, Marchetti K, Nakamura H (1998) Iso-lation and characterization of microsatellite DNA loci for anobligate brood parasitic bird, the common cuckoo (Cuculuscanorus). Molecular Ecology, 7, 1437–1439.

Pemberton JM, Slate J, Bancroft DR, Barrett JA (1995) Nonampli-fying alleles at microsatellite loci: a caution for parentage andpopulation studies. Molecular Ecology, 4, 249–252.

Primmer CR, Møller AP, Ellegren H (1995) Resolving geneticrelationships with microsatellite markers: a parentage testing sys-tem for the swallow Hirundo rustica. Molecular Ecology, 4, 493–498.

9no issue no.2000914primer notesprimer notesPRIMER NOTEs1000Graphicraft Limited, Hong Kong

Development of polymorphic microsatellite markers in a tropical tree species, Melaleuca cajuputi (Myrtaceae)M. MIWA, R . TANAKA, M. SHINONE, K . KOJIMA and T. HOGETSU

Asian Natural Environmental Science Center, The University of Tokyo, Midori-cho 1–1–8, Tanashi, Tokyo 188–0002, Japan

Keywords: genet, Melaleuca cajuputi, microsatellite markers, polymorphism, ramet, reproduction

Received 23 October 1999; revision received 2 December 1999; accepted 7 December 1999

Correspondence: M. Miwa. Fax: +81–424–65–5601; E-mail: [email protected]

The tropical tree species, Melaleuca cajuputi, belongs to theMyrtaceae, and is widely distributed from northern Australiathrough Malay Peninsula to Indochina. M. cajuputi trees fre-quently form populations in peat swamps and sandy places inNarathiwat, southern Thailand, where other tree species rarelygrow. Therefore, M. cajuputi is expected to be a suitable treespecies for reforestation in tropical deteriorated lands. The treesenlarge their population both by sexual and vegetative repro-duction. Although knowledge about the reproduction of treespecies which may be suitable for reforestation is of generalimportance, the reproductive pattern of M. cajuputi in the fieldis not well understood. In the present study, polymorphic micro-satellite markers were developed to investigate the balancebetween sexual and vegetative reproduction, and betweenautogamy and allogamy in natural populations of M. cajuputi.

We isolated microsatellite regions from total DNA ofM. cajuputi using a modification of the enrichment techniquedescribed by Edwards et al. (1996). DNA was extracted fromM. cajuputi leaves dried with silica gels by a modified CTABmethod (Zhou et al. 1999), and digested with a restriction enzyme(HindIII, EcoRI, XhoI or SalI). The 5′ projecting ends of theDNA fragments were blunted by a DNA Blunting Kit (TakaraShuzo Co., Tokyo), and ligated with an EcoRI/NotI/BamHIadaptor (Takara Shuzo Co.) by a DNA Ligation Kit version1 (Takara Shuzo Co.). The adaptor-ligated fragments wereamplified by polymerase chain reaction (PCR) using a primerdesigned on the basis of the sequence at their ends. Severalkinds of oligonucleotides among (GCC)10, (CAA)10, (CTG)10,(GTG)8, (GC)15, (GA)15, (GT)15 and (AT)15 were attached tothe membrane filter for enrichment. Microsatellite-containingfragments were cloned into pT7Blue vectors using a pT7BluePerfectly Blunt Cloning Kit (Novagen Co., WI, USA), andpropagated in Escherichia coli, XL1-Blue MRF′ strain. Sequen-cing reactions were performed using a Texas Red T7 primer(Hitachi Instruments Service Co., Tokyo) and a ThermoSequenase Premixed Cycle Sequencing Kit (Hitachi Instru-ments Service Co.) according to the manufacturer’s instructions.Extension products were separated by a sequencer (SQ-5500,Hitachi Co., Tokyo). Primer pairs for amplification of themicrosatellite regions were designed on the basis of thesequences flanking the obtained microsatellites.

To characterize each microsatellite loci, the regions in theDNA extracted by the above method from dried leaves of31 M. cajuputi ramets growing in a sandy place in Narathiwat,southern Thailand, were amplified by PCR using the designedprimer pairs. The amplification was performed in 10 µL ofa reaction mixture containing 10 ng DNA, 0.4 mm of eachdNTP, 0.2 µm of each designed primer pair, 1 × GC buffer I(Takara Shuzo Co.; ingredients are unavailable) in which 2.5 mmof Mg2+ were included, and 0.5 U of LA Taq DNA polymerase(Takara Shuzo Co.), where the reverse primer was labelledwith Texas Red, by a PCR thermal cycler (TP3000, TakaraShuzo Co.). The reaction schedule was as follows: (i) 94 °C for1 min, annealing temperature (Ta) shown in Table 1 for 1 min,




72 °C for 1 min, 29 cycles; (ii) 94 °C for 1 min, Ta for 1 min and72 °C for 5 min, 1 cycle. The PCR products were denaturedby heating and separated by the sequencer on a sequencinggel [6% Long Ranger (FMC BioProducts Co., ME, USA),6.1 m urea and 1.2 × TBE (0.1 m Tris (hydroxymethyl) amino-methane, 3.0 mm EDTA and 0.1 m boric acid) ] in 0.6 × TBE.The band sizes were estimated by computer software fraglysversion 2 (Hitachi Electronics Engineering Co., Tokyo). Also,observed and expected heterozygosities in each microsatellitelocus were calculated by Nei’s equation (Nei 1987).

The sequences of 17 microsatellite loci in DNA of M. cajuputiwere determined. When the DNA was digested with EcoRI,HindIII, SalI and XhoI at the first step of microsatellite isola-tion, the sequences of 3, 7, 3 and 4 microsatellite loci were deter-mined, respectively. Eleven out of 17 loci showed single-locus,polymorphic banding patterns (Table 1). These polymorphicloci had 2–10 alleles per locus and expected heterozygositiesranging from 0.12 to 0.86. Observed heterozygosities in mostloci showed relative consistency with the expected ones. Twoloci, however, were not polymorphic, being amplified intoonly one allele, and in the other four loci, no clear bandswere amplified by the designed primer pairs (data not shown).

We confirmed that the 11 polymorphic markers coulddistinguish genetic differences between several groups of

ramets connected to each other by roots. This suggests thatthe 11 markers are available to identify M. cajuputi genets inthe field. Because the 11 markers are single-locus and poly-morphic, they would also be useful for analyses of gene flowin M. cajuputi populations.

Acknowledgements

This work was supported in part by Grants-in-Aid for ScientificResearch (A) (No. 07556035), Creative Basic Research (No. 09NP0901)and JSPS Fellows (No. 8312) from the Ministry of Education,Science, Sports and Culture of Japan, and a Grant-in-Aid fromPROBRAIN.

References

Edwards KJ, Barker JHA, Daly A, Jones C, Karp A (1996) Micro-satellite libraries enriched for several microsatellite sequencesin plants. Biotechniques, 20, 758–760.

Nei M (1987) Molecular Evolutionary Genetics. Columbia Univer-sity Press, New York.

Zhou Z, Miwa M, Hogetsu T (1999) Analysis of genetic structureof a Suillus grevillei population in a Larix keampferi stand bypolymorphism of inter-simple sequence repeat (ISSR). NewPhytologist, 144, 55–63.

9no issue no.2000917prime notesprime notesPrime notes100Graphicraft Limited, Hong Kong

Table 1 Primer pairs for amplification of 11 polymorphic microsatellite regions in Melaleuca cajuputi and some characteristics of themicrosatellites

Microsatellite locus* Primer pair sequence (5′–3′) Repeat type† Size range (bp) Ta (°C)‡ Allele number HO§ HE¶

Eco-1 R:CCATGGGTAATGTCGGAAGTG (GGC)7(GAT)(GGC) 115–124 57 3 0.00 0.56(AB034970)** F:GCCATACATGCGACTGAGTACHin-2 R:ACCGTCAACCACCACTGTTTG (GCC)5(GCT)(GCC)4 116–130 57 5 0.45 0.66(AB034971) F:GCCAGCAGTGATTAGAGCATCHin-4 R:TTTGGCGTGCTCAGAGCTCT (GA)10AA(GA)4 91–110 57 5 0.13 0.67(AB034971) F:CACCCCAAATATTCCATCTCTCHin-5 R:GTTTGCCAAATCCATTACGGTC(AAG)3(AAT)(AAG)6 146, 134 57 2 0.13 0.12(AB034972) F:CAATGATATTCACGTAGTCGGTGHin-6 R:TCATCGACTTCTCTTTCTGAGC (AG)31 108–234 57 9 1.00 0.86(AB034973) F:CATCAACACACGCAACCACTCHin-7 R:TCACTACCATGTAGGTGCTCC (CT)14…(CA)23 168–226 57 7 1.00 0.79(AB034974) F:TTACAAACATACCTCTGGCCAGSal-1 R:AGTCCCAGTCGTCAACAGAG (CGG)6 91–97 57 3 0.90 0.60(AB034975) F:CCATCAAAGACAAAAGAGCGTCSal-2 R:GAGGATTCTAAGCTGCAGC (GGC)6(GGT)2(GAT) 129–139 57 5 0.71 0.71(AB034976) F:GAAAGGCCATACGGAATCTCC (GGT)(GAT)7Sal-3 R:GCATCATCATCGAGCTGCATG (GT)20(GC)3GT(GA)27 139–192 57 10 1.00 0.86(AB034977) F:ACCAGTGACTAATCGGGTGTGXho-1 R:AGGTGGTGATGGACGAGCTG (GGC)6(GGT)(GGC) 103–117 60 7 0.77 0.70(AB034978) F:GTCGCATTGACATCCGAAGCGXho-4 R:AATCCGCGACTGTGCAGAGG (GT)41(GA)23 155–283 60 7 0.61 0.71(AB034979) F:CTCAAGCCGATGTTCCTCGC

*Microsaellite loci designated as Eco-, Hin-, Sal- and Xho- mean those isolated from the amplified DNA fragments which had been prepared after digestion of DNA with EcoRI, HindIII, SalI and XhoI, respectively.†Sequenced microsatellite repeat type.‡Annealing temperature for polymerase chain reaction amplification.§Observed heterozygosity (n = 31 ramets).¶Expected heterozygosity (n = 31 ramets).**DDBJ/EMBL/GenBank accession number for the cloned sequences. The loci of Hin-2 and Hin-4 were found from the same clone.




Microsatellite markers for investigating population structure in Octopus vulgaris (Mollusca: Cephalopoda)E. C. GREATOREX,* C. S . JONES, J . MURPHY, L . N. KEY, A. M. EMERY and P. R . BOYLE

Department of Zoology, University of Aberdeen, Tillydrone Avenue, AB24 2TZ, UK

Keywords: microsatellites, Octopus vulgaris, population structure

Received 30 October 1999; revision accepted 8 December 1999

Correspondence: E. C. Greatorex. *Present address: Horticulture Research International, Wellesbourne, Warwick, CV35 9EF. Fax: 01789 470552; E-mail: [email protected]

Octopus vulgaris, Cuvier 1787, is probably the most widelyfound cephalopod species, having a worldwide distributionin temperate waters on the continental shelves of Europe,Africa, Asia and the Atlantic coasts of North and SouthAmerica. Whether it is a truly cosmopolitan species or a species-complex has yet to be investigated by modern molecularmethods, but it is nevertheless an important fisheries cat-egory with global catches in excess of 100 000 tonnes. Despiteits fishery importance, there are currently no methods for theinvestigation of population structuring or the identificationof ‘stocks’ for fishery management of this important species.Here we report on the isolation and characterization of thefirst polymorphic microsatellite markers for the study ofpopulation structure in O. vulgaris.

Genomic DNA was extracted from ethanol preserved armtips from six individual octopus specimens from Crete, andsix from Vigo; these locations represent the extremes of therange of sites from where O. vulgaris samples were obtainedduring this project. A phenol–chloroform extraction proce-dure modified for use in molluscs following Vernon et al.(1995) was used.

Two size selected partial genomic libraries (Rassmann et al.1991), the first 300–600 base pairs (bp) and the second 300–900 bp were constructed by ligating Sau3AI digested DNAinto dephosphorylated pUC18 vector digested with BamHI(Pharmacia). The first library was probed with a [α-32P] dATPlabelled dinucleotide polymers (AG)8 and (AC)8 (2034recombinant colonies gave 78 positives), whilst the secondwas probed with [γ-32P] dATP end-labelled tri- and tetra-oligonucleotide repeats, e.g. (TAA)4 (GATA)4 and (GACA)4(3500 colonies gave 25 positives) at high stringency. A selec-tion of di-, tri- and tetranucleotide positives were sequencedusing a Big Dye dye-terminator kit (following manufacturer’sprotocols), and an ABI 377 automated sequencer (PE AppliedBiosystems) was used to detect sequences. Primers weredesigned from unique sequences flanking the microsatelliterepeats, using oligoTM Macintosh version 4.1 (National Bio-sciences Inc., USA).

Amplification efficacy of primer pairs was tested usingindividuals that were used to construct the partial genomiclibrary, and a few randomly chosen individuals. Amplifiedsamples were run on 1% 1 × TBE agarose gels at 80 V for15 min and gels stained with ethidium bromide. Polymerasechain reaction (PCR) mixes contained 20 ng template DNA,2.5 mm MgCl2, 0.2 mm of each dNTP, 5 pmol of each primer,0.04 units of Taq DNA polymerase (Bioline), and the manu-facturers supplied buffer (16 mm (NH4)2SO4, 67 mm Tris HCl,pH 8.8, 0.01% Tween-20), in a final reaction volume of 10 µL.Reactions were performed on a Hybaid ‘Omn E’ PCR thermalcycler using a ‘touchdown’ protocol (Don et al. 1991) whichused an initial set of 10 cycles dropping from 5 °C above thesupposed annealing temperature by 0.5 °C increments eachcycle. Touchdown programmes with annealing temperaturesdropping from 60 °C–50 °C gave best results.

PCR conditions were optimized for six loci (reported inTable 1); bright resolvable, polymorphic products were

Table 1 Microsatellite loci in Octopus vulgaris

Locus* Repeat unit Tm (°C)† Mg2+ (Mm) Product size‡ n§ No. of alleles¶ HE HO Primer sequences (5′–3′)

µOct3 (AT)16(GT)15 55 2.5 147 12 12 91.6 75.0 F: CTCCCTAGTTTTGAATCACGR: GCCACTAATACACTTTTCAAGG

µOct8 (TG)36 56 2.5 160 10 11 92.6 70.0 F: AGGGAGAGAAAATAGAAAAACR: TAAACTGAATAATACATACATACG

µOv04 (TTA)22 44 2.5 126 49 14 72.0 98.0 F: ATACCAGGCCTTGTGCCTTTAGR: CAGCACCGTAATACATCTTCAG

µOv06 (ATT)24 45 2.5 146 49 17 69.5 98.0 F: GGGCCTTATTCCTTAAGCAGR: CCATTTGCATTTGAATATTTTTAAAG

µOv10 (GA)14 52 2.5 122 62 12 68.7 98.4 F: GCAATAAAGGAGAAAACAAAAACAR: GCTATTGTCACAATAAGGCTCTCC

µOv12 (GATA)20 53 2.5 176 61 18 64.7 83.6 F: GCATAATGTGCCGCTAAATGGAACR: GCCTCGTCGGTATTTCCTCTTTCA

*µOct and µOv, locus nomenclature. µOct loci isolated from first library, and µOv from second. †Tm(°C) predicted by oligo™. ‡Predicted product size in base pairs assuming allele size identical to sequenced allele. §Individuals genotyped from two populations of Octopus vulgaris, from Crete, and Vigo ¶Number of alleles at each locus, expected (HE) and observed (HO) heterozygosity are given for these n individuals. GenBank accession numbers (order listed in table): AF197130–AF197135.




produced in all samples of the predicted size. Forward primersof these primer sets were labelled using fluorescent dyescompatible with the PE/Applied Biosystems Genescan sys-tem, and alleles sized from 4% acrylamide denaturing gelsrun for 1 h at 750 V. Primers were tested on putativelyunrelated individuals from two populations, one from Creteand one from Vigo.

All six loci were polymorphic. Some problems with shadowbands were encountered with loci from the first library (µOctloci). This is a common problem with dinucleotide repearts(Litt et al. 1993). The second library was probed with tri- andtetranucleotide oligonucleotides, to avoid problems encoun-tered with the first set of dinucleotides.

This suite of microsatellite primers will be a useful tool inunderstanding population structure in O. vulgaris.

Acknowledgements

Funded by the Commission of the European communities researchprogramme in the fisheries sector (contract FAIR CT 1520). Weacknowledge support from Biotechnology and Biological SciencesResearch Council (Advanced Fellowship to C.S.J, no. 1/AF09056).Thanks to Eileen Dillane, and Jamie Coughlan at the Aquacul-ture Development Centre, University College Cork, Ireland, forradioactively isolating microsatellites.

References

Don RH, Cox PT, Wainright BT, Baker K, Mattick JS (1991)‘Touchdown’ PCR to circumvent spurious priming duringgene amplification. Nucleic Acids Research, 19, 4008.

Litt M, Huage X, Sharma V (1993) Shadow bands seen whentyping polymorphic dinucleotide repeats: Some causes andcures. Biotechniques, 15 (2), 280–284.

Rassmann K, Schlotterer C, Tautz D (1991) Isolation of simplesequence loci for use in polymerase chain reaction-based DNAfingerprinting. Electrophoresis, 12, 113–118.

Vernon JG, Jones CS, Noble LR (1995) Random amplified poly-morphic DNA (RAPD) markers reveal cross-fertilisation inBiomphalaria glabrata from wild populations. Journal of MolluscanStudies, 61, 455–465.


Characterization of microsatellite loci in bannertailed and giant kangaroo rats, Dipodomys spectabilis and Dipodomys ingens

C. DAVIS ,* B . KEANE,† B . SWANSON,‡ S . LOEW,§ P. M. WASER,‡ C . STROBECK* and R . C . FLEISCHER§

*Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada, †Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221, USA, ‡Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA, §Department of Zoological Research, National Zoological Park, Smithsonian Institution, Washington, DC 20008, USA

Keywords: Dipodomys, Heteromyidae, kangaroo rats, microsatellites, pocket mice

Received 6 October 1999; revision accepted 6 January 2000

Correspondence: B. Keane. Fax: +1 513 556 5299; E-mail: [email protected]

Microsatellite genotyping is the current method of choice foranalysing genetic structure and pedigrees in animal popu-lations. Here we describe the isolation and characterizationof six microsatellite loci in the bannertailed kangaroo rat,Dipodomys spectabilis, and three microsatellite loci in the giantkangaroo rat, D. ingens. These are the first microsatellite locito be characterized for any member of the rodent familyHeteromyidae. The primers were developed as part of long-termstudies using genetic data to address questions regardingbetween-population dispersal and within-population inbreed-ing in metapopulations of D. spectabilis and D. ingens (Keaneet al. 1991; Waser & Elliott 1991; Good et al. 1997).

D. spectabilis microsatellites were isolated from small insertgenomic libraries. For each library approximately 50 µg ofgenomic DNA was isolated from muscle tissue by standardmethods (Sambrook et al. 1989) and digested to completionwith Sau3A. The resulting fragments were electrophoresed ona 1% agarose gel and fragments of 150–500 bp were excisedand isolated from the gel using QIAquickTM spin columns(QIAGEN). Size selected fragments were ligated into BamHIdigested dephosphorylated M13mp18RF (Gibco BRL) followedby transfection into Escherichia coli DH5α F′IQ by electro-poration (E. coli PulserTM, Bio-Rad). Approximately 10 000recombinant clones were screened with a biotinylated (GT)11oligonucleotide probe, and clones containing putative micro-satellites were detected using a nonradioactive detection kit(Gibco BRL). A total of 134 positive clones were isolated. Fiftyof these clones were rescreened, and genomic inserts from 39clones were amplified and sequenced as in Zheng et al.(1995). GT dinucleotide repeats in D. spectabilis were commonlyvery long, containing as many as 75 repeat units. Primerswere designed using oligo (National Biosciences Inc., Version4.0) with one primer of each set fluorescently labelled withone of 6-FAM, TET or HEX (Applied Biosystems) for putativemicrosatellites in 30 clones.

In order to assess variation at the loci derived from D.spectabilis, six animals from Portal, Arizona were genotypedusing all 30 primer sets. Total genomic DNA was isolatedfrom 10 to 20 mg ear snips using QIAquickTM spin columnsand approximately 100 ng was used as template in 15 µLpolymerase chain reactions (PCRs) also containing 0.16 µm(2.4 pmol) of each primer, 120 µm dNTP, 2 mm MgCl2, 0.3 Uof Taq DNA polymerase and 1X PCR buffer (10 mm Tris buffer,pH 8.8, 0.1% Triton X-100, 50 mm KCl and 0.16 mg/mL BSA).Cycling was performed in a Perkin Elmer 9600 thermal cyclerunder the conditions: 1 min at 94 °C, three cycles of 30 s at94 °C, 20 s at 54 °C, and 5 s at 72 °C, followed by 33 cycles of15 s at 94 °C, 20 s at 54 °C, and 1 s at 72 °C. Final extension at72 °C for 30 min preceded ramping to 4 °C. PCR products wereresolved on a model 377 DNA Sequencer and analysed usinggenescan and genotyper software (Applied Biosystems).Many primer sets amplified products which appeared asstreaks on the gel image due to excessive slippage of the




polymerase during amplification over long repeat units andcould not be scored. Six loci, with moderate repeat lengths inthe sequenced allele, exhibited clean and variable bandingpatterns and were run on 29 additional individuals fromPortal, AZ (Table 1). These six loci had 7–20 alleles; one locus(Ds19) was sex-linked, and the low HO in another (Ds3)suggested possible null alleles. All six loci were also poly-morphic in a sample of seven D. ingens individuals.

D. ingens trinucleotide microsatellites were developedaccording to the enrichment protocol in Fleischer & Loew(1996). (CAC)n repeats were highly enriched and all of theloci reported here are composed of (CAC)n or some combina-tion of (CAC)n and (GT)n. From the D. ingens sequences wedesigned 11 primer sets, three of which resulted in variableproducts (Table 1). PCR conditions were similar to thosedescribed above, but included 1.5 mm MgCl2, 0.5 U of Taq

Gold DNA polymerase, 200 µm of each dNTP, and 0.5 µm(5 pmol) of each primer in a total reaction volume of 10 µL.The PCR involved a 10-min start at 94 °C followed by 30cycles of 94 °C for 50 s, 56 °C for 80 s and 72 °C for 30 s. In D.spectabilis, two primer sets revealed variable patterns in 10individuals, but the low HO in Di5A suggests there may benull alleles present.

We also tested the effectiveness of the D. spectabilis derivedprimer pairs in six other species within the family Hetero-myidae (Table 2). These results demonstrate that some of theprimer pairs designed from the DNA sequences of D. spectabilismay also be useful in other heteromyid species, particularlywithin the genus Dipodomys. However, the higher percentageof heterospecific primer pairs within the genus Dipodomysindicates that the conservation of microsatellite flankingregions decreases with increasing phylogenetic distance

Table 1 Repeat motif in allele cloned, number of alleles (NA), size of polymerase chain reaction products in bp, observed (HO) andexpected (HE) heterozygosity in Dipodomys spectabilis (Ds) and D. ingens (Di), and primer sequences (5′–3′) for six microsatellite locideveloped in D. spectabilis and three loci in D. ingens. The number and size of alleles, as well as heterozygosities, at each locus are basedon the analysis of 29 D. spectabilis and seven D. ingens individuals for D. spectabilis primers (Dsxx), and on 10 D. spectabilis and 50D. ingens for D. ingens primers (Dixx)

Locus Repeat motif NA Size range HO HE Primer sequences Accession no.

Ds1 (GT)17(AG)19 Ds:8 202–218 0.72 0.82 F: GATCAACCACCCAGCTCTAT AF186399Di:9 170–214 0.86 0.87 R: GCAAAGCCCTGAGTTCAAAAG

Ds3 (GT)16 Ds:7 170–182 0.52 0.73 F: TCAAGCTCCAGGACAGCACAAG AF186400Di:4 174–184 0.43 0.64 R: GTTTCCATTGATGCCCAGCAGATTT

Ds19* (GT)36 Ds:11 94–128 0.48 0.86 F: ATCCTCAGAACCTTCATTCA AF186401Di:8 102–122 0.57 0.87 R: AATCTATGAGTGAGCCAACAG

Ds28 (GT)15 Ds:7 183–209 0.76 0.79 F: GATCTTTATTTATTTGTGTGTTT AF186402Di:6 188–210 0.57 0.79 R: ATTAGAGTCAGAAGCCAGAGCAG

Ds30 (GT)15 Ds:20 246–318 0.97 0.95 F: ATCCTTCCTCCCAATGTTGTAG AF186403Di:9 219–267 0.71 0.86 R: GTTTCCCAGGCAGAAAAGTTTATGATA

Ds46 (GT)14GCG(GT)11 Ds:7 174–188 0.69 0.70 F: CTAATCACCGAGCCAAA AF186404Di:5 147–157 0.71 0.76 R: GTTTATCAACTATAAAAATCATAGAAAA

Di5 (CAC)11CAA(CAC)4 Ds:4 175–199 0.30 0.65 F: TGGTTCCATGTAGTAGCCCTGAGC AF187992Di:2 179–182 0.22 0.31 R: TCCCAGAGTTCCAAGTACCTCC

Di12E (CA)16CCC(CAC)18 Ds:4 210–234 0.60 0.67 F: GTAGCTGGGCTTACAGACATGAG AF187993Di:7 195–225 0.52 0.68 R: GGCAGGTCTCCTTCTGAGATGGC

Di12F (AGC)14(CAC)11 Ds:1 — — — F: CCTTGAGTGATAAGGCTCAG AF187994Di:2 163–214 0.18 0.16 R: CAGTCTCCTGAGTAGCTAGG

*Locus Ds19 is X-linked.

Table 2 Results of cross-species amplification of DNA from six species in the family Heteromyidae using six microsatellite primer pairsdesigned from the bannertailed kangaroo rat, Dipodomys spectabilis. Entries are the size range in number of bp of the polymerase chainreaction (PCR) products detected (number of alleles in parentheses). —, indicates that either no product or a multiband pattern wasobserved with the PCR conditions used

LocusDipodomys merriami (n = 10)

Dipodomys ordii (n = 3)

Perognathus flavus (n = 6)

Chaetodipus baileyi (n = 2)

Chaetodipus hispidus (n = 4)

Chaetodipus penicillatus (n = 4)

Ds1 168–214 (12) 188–217 (3) 243–261 (5) 176 (1) — 176 (1)Ds3 174–204 (9) — — — — —Ds19 96–122 (8) — 243–262 (4) — — —Ds28 — 173–198 (6) — — — —Ds30 242–273 (12) 235–252 (4) — — 262–264 (2) —Ds46 — 159–171 (4) — — — —




within the family Heteromyidae, as has been seen for otherorganisms (e.g. Primmer et al. 1996).

Acknowledgements

The D. spectabilis work was supported by the National ScienceFoundation. B.K., B.S., and P.W. thank K. Yutzey and J. Wintersfor laborabory and field help. R.C.F. and S.L. thank C. Tarr,T. Steeves, S. Good, A. Neumann and especially K. Pilgrim forassistance in the laboratory, and K. Ralls and D. Williams forsamples and logistic aid. The Dipodomys. ingens work was funded bythe California Department of Fish and Game, California Water Board,Sacramento, CA, the Smithsonian Institution, Washington DC, andthe Friends of the National Zoo, Washington DC.

References

Fleischer RC, Loew S (1996) Construction of microsatellite-enrichedgenomic libraries. In: Molecular Zoology: Advances, Strategies and

Protocols (eds Ferraris JD Palumbi SR), pp. 461–468. Wiley-Liss,New York.

Good SV, Williams DF, Ralls K, Fleischer RC (1997) Populationstructure of Dipidomys ingens (Heteromyidae): The role ofspatial heterogeneity in maintaining genetic diversity. Evolution,51, 1296–1310.

Keane B, Waser PM, Danzl-Tauer L, Minchella DM (1991) DNAfingerprinting: estimating background band-sharing in banner-tailed kangaroo rats. Animal Behaviour, 42, 141–143.

Primmer CR, Moller AP, Ellegren H (1996) A wide-range surveyof cross-species microsatellite amplification in birds. MolecularEcology, 5, 365–378.

Sambrook J, Fritch EF, Maniatus T (1989) Molecular Cloning: aLaboratory Manual. 2nd edn. Cold Spring Harbor LaboratoryPress, New York.

Waser PM, Elliott LF (1991) Dispersal and genetic structure inkangaroo rats. Evolution, 45, 935–943.

Zheng W, Stacey NE, Coffin J, Strobeck C (1995) Isolation andcharacterization of microsatellite loci in the goldfish Carassiusauratus. Molecular Ecology, 4, 791–792.


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