Molecular Ecology Notes (2003)

3

, 431–434 doi: 10.1046/j.1471-8286.2003.00477.x

© 2003 Blackwell Publishing Ltd

Blackwell Publishing Ltd.

PRIMER NOTE

New polymorphic microsatellite loci for different camel species

D . EVDOTCHENKO, Y . HAN, H. BARTENSCHLAGER, S . PREUSS and H. GELDERMANN

Department of Animal Breeding and Biotechnology, University of Hohenheim, D-70593 Stuttgart, Germany

Abstract

New microsatellite loci were screened and sequenced from the genomic DNA of male

Camelus bactrianus

. Among 32 loci, 23 were amplified in bactrian and dromedary species,19 in llama and 20 in alpaca. The different species had similar fragment lengths per locus,with more striking similarities between bactrian and dromedary and between llama andalpaca, respectively. Seven loci had more than 10 alleles each, nine were monomorphicin all species, and one was monomorphic in Old World and polymorphic in New Worldcamels. The results show that the informative microsatellite loci can be widely applied toseveral species.

Keywords

: camel, cross-species amplification, DNA-marker, microsatellites, polymorphism

Received 6 December 2002; revision received 28 April 2003; accepted 6 May 2003

Until recently, at least 54 microsatellite loci have beenanalysed for South American camels (Lang

et al

. 1996: 15loci; Obreque

et al

. 1998, 1999: 23 loci; Penedo

et al

. 1999a,b:14 loci; McPartlan

et al

. 1998: 2 loci). In this study, 32 newmicrosatellite loci were screened and sequenced on thebasis of the genomic DNA (gDNA) of a male

Camelusbactrianus

(bactrain) and were tested for polymorphismand cross-species amplification in four camel species.Genomic DNA from blood samples of different camelspecies (20

Camelus bactrianus

from the Russian Altairegion, 20

Camelus dromedarius

(dromedary) of differentbreeds from Kenya, seven

Camelus bactrianus

, six

Lamaglama

(llama)

,

four

Lama pacos

(alpaca) from German zoos)was isolated by Phenol/Chloroform extraction.

The DNA from a male

Camelus bactrianus

was restrictedwith

Sau

3AI, and the restriction fragments were enrichedusing magnetic beads (Dynabeads M-280 streptavidin,DYNAL) coated with the biotinylated oligonucleotides(CA)

10

/(GT)

10

and (ATCC)

7

according to the provider’sprotocol. Following the separation of the enriched frag-ments by electrophoresis, the 200–400 bp fragments wereligated in the vector pT7 Blue (Novagen) and cloned in

Escherichia coli

NovaBlue. Positive clones were sequenced.DNA sequences flanking 23 microsatellite loci were

submitted to GenBank (Accession nos AF329137–AF329151,AF329153–AF329155, AF329157–AF329160, AF380345).

The primer sequences for polymorphic loci are given inTable 1; forward primers were fluorescence labelled (Cy-5or fluorescein). Polymerase chain rection (PCR) was per-formed using a 25

µ

L reaction mix containing a buffer ofInvitek, 1.5 m

m

MgCl

2

, 0.1 m

m

of each dNTP, 5–10 pmol ofeach primer, 1.25 U of

Taq

DNA Polymerase (Invitek) and100 ng gDNA. The thermocycler profile was 94

°

C for5 min, 30 cycles of 94

°

C for 45 s, 48–60

°

C (according to theannealing temperature of primers) for 45 s, and 72

°

C for30 s, followed by a final extension at 72

°

C for 5 min. Foroptimization, the annealing temperature and concentra-tion of MgCl

2

were adjusted for each primer set on thebasis of

Camelus bactrianus

gDNA (Table 1). PCR amplifi-cates were separated together with internal and externalstandard fragments (Fermentas) by electrophoresis on 5%Hydrolink gels using a DNA sequencer (ALF, AmershamPharmacia Biotech). Fragment sizes were analysed withthe software

allelelinks

(Amersham Pharmacia Biotech).Analysis of locus specific allelic fragment sizes estimatedby the ALF analysis and basic statistical tests (e.g. Hardy–Weinberg equilibrium) were conducted using the softwarepackage

biosys

-1.7 (Swofford & Selander 1989). Polymor-phism information content (Botstein

et al

. 1980) and exclu-sion probabilities for parentage controls ( Jamieson 1994)were calculated by assuming genetic equilibria per animalgroup.

Correspondence: Hermann Geldermann. Garbenstr. 17, 70599Stuttgart, Germany. Fax: + 49 711 4593101; E-mail: tzunihoh@unihohenheim.de

432

P R I M E R N O T E

© 2003 Blackwell Publishing Ltd,

Molecular Ecology

Notes

, 3, 431–434

Specific PCR products were generated for the 23 loci inbactrian as well as in dromedary. Amongst them, 19 locicould be amplified in llama and 20 in alpaca (data notshown). Different repeat motifs were found for the loci,which were not always complementary to the oligonucle-otides (CA)

10

(TG)

10

and (ATCC)

7

used for screening. Frag-ment length intervals of the polymorphic loci given inTable 1 point to similar allelic fragment lengths per locusand species. Fragment lengths were more similar betweenllama and alpaca and between dromedary and bactrianthan between New and Old World

Camelides

. For theloci CMS16, CMS32 and CMS36, no common fragmentlength interval was found between Old and New World

Camelides

.The loci were largely different with regard to their poly-

morphism (Table 2). No significant (

P

< 0.05) deviationfrom Hardy–Weinberg equilibrium was observed for theanimal groups of bactrian and dromedary, and the groups

of llama or alpaca were not tested because of small num-bers of animals. Among the 32 loci investigated, five weremonomorphic and had identical fragment sizes in allinvestigated species. One locus was monomorphic in bac-trian and dromedary and could not be amplified in theother species. Three loci were monomorphic in each of thespecies, but had different fragment sizes in individual spe-cies. More than one allele within a species were observedin 13 loci for bactrian, 12 loci for dromedary, 11 loci forllama and 12 loci for alpaca. Seven (50%) of the polymor-phic loci showed more then 10 alleles, and in most loci theallelic fragment lengths were similar in different species.Since microsatellite alleles of the same length can be differ-ent from one species to another, sequence analysis of allelicmicrosatellite regions in the different species is recom-mended. Nevertheless, the new informative microsatelliteloci can be efficiently used in several species, e.g. for par-entage control, gene mapping or phylogenetic analysis.

Table 1 Details of microsatellite loci identified in four camel species

Locus (Accession no.) Primer sequence (5′−3′)

Ta (°C) (MgCl2)

Consensus repeat motif*

Fragment length (bp)/NA (NI)†

B D L A

CMS3 F: GCTCTCTGTGCCTCAATATG 55 (TTATAA)9 258 258 251–258 251–258(AF329138) R: ACCATAGTTCTGGGTAGCA (1.5 mm) 1 (24) 1 (18) 2 (6) 2 (4)CMS9 F: TGCTTTAGACGACTTTTACTTTAC 55 (GT)24 233–256 231–243 227–247 229–237(AF329160) R: ATTTCACTTTCTTCATACTTGTGAT (1.5 mm) 7 (26) 4 (20) 5 (6) 2 (4)CMS13 F: TAGCCTGACTCTATCCATTTCTC 55 (AC)27 248–265 238–254 242–261 246–265(AF329158) R: ATTATTTGGAATTCAACTGTAAGG (1.5 mm) 6 (26) 6 (20) 6 (6) 3 (4)CMS15 F: AAATACTTAAAGGTTCCCAGA 55 (TG)23 140–159 121–144 140–146 138–146(AF329151) R: TTGTAAACTAAAGCCAGAAAG (1.5 mm) 9 (26) 6 (19) 4 (6) 3 (4)CMS16 F: ATTTTGCAATTTGTTCGTTCTTTC 60 (TG)34 183–193 179–207 — —(AF329157) R: GGAGTTTATTTGCTTCCAACACTT (1.5 mm) 3 (26) 7 (20) — —CMS17 F: TATAAAGGATCACTGCCTTC 55 (AT)38 144–149 149–167 135–147 140–161(AF329147) R: AAAATGAACCTCCATAAAGTTAG (1.5 mm) 3 (26) 2 (20) 2 (2) 2 (4)CMS18 F: GAACGACCCTTGAAGACGAA 60 (GT)14 157–186 157–163 165–188 165–182(AF329148) R: AGCAGCTGGTTTTAGGTCCA (1.5 mm) 5 (26) 2 (20) 5 (6) 3 (4)CMS25 F: GATCCTCCTGCGTTCTTATT 58 (CT)33 118–128 93–102 93–95 93–118(AF380345) R: CTAGCCTTTGATTGGAGCAT (1.5 mm) 6 (26) 4 (19) 2 (6) 4 (4)CMS32 F: ACGGACAAGAACTGCTCATA 55 (CA)30 198–204 198–209 167–169 167–169(AF329146) R: ACAACCAATAAATCCCCATT (1.5 mm) 2 (25) 4 (20) 2 (6) 2 (4)CMS36 F: AAATTACTGAATTCCCCCCCTA 55 (AC)9 139–143 141–143 — —(AF329144) R: GTTACAACGCTCTCATTCATC (1.5 mm) 3 (27) 2 (20) — —CMS50 F: TTTATAGTCAGAGAGAGTGCTG 55 (GT)27 154–183 170–190 129–140 129–135(AF329149) R: TGTAGGGTTCATTGTAACA (1.5 mm) 5 (26) 8 (20) 4 (6) 2 (4)CMS58 F: AATATACATCCTCCCAACTGGT 55 (AC)18 107–115 98–116 — 137–142(AF329142) R: TTATTTCTCTTAACCCCTCTCTAA (1.5 mm) 4 (26) 5 (20) — 2 (3)CMS104 F: TTAGGTCCCTGGGCTTTATG 52 (AC)23 105–124 93 94–102 100–108(AF329154) R: AAATTGTGCATTCTCTTGCATC (1.6 mm) 2 (25) 1 (20) 2 (6) 3 (4)CMS121 F: CAAGAGAACTGGTGAGGATTTTC 60 (TG)24 151–159 147–166 128–151 128–157(AF329159) R: AGTTGATAAAAATACAGCTGGAAAG (1.5 mm) 5 (26) 7 (20) 5 (6) 4 (4)

Accession no., GenBank accession number; Ta, annealing temperature; B, bactrian; D, dromedary; L, llama; A, alpaca.*DNA sequence of the locus was screened for tandem repeats with etandem from the emboss package (supplied by the UK HGMP Resource Centre); for loci with several repeat motifs only the one with the highest score is given.†For each species-locus combination, the fragment lengths, the number of alleles (NA) and the number of individuals (NI) in which amplification was observed are given.

P R I M E R N O T E 433

© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 431–434

Tab

le 2

Esti

mat

es o

f pol

ymor

phis

m o

f new

mic

rosa

telli

te lo

ci in

four

dif

fere

nt c

amel

spe

cies

Locu

s

Bact

rian

*D

rom

edar

y*Ll

ama†

Alp

aca†

NA

/EN

AH

O/H

EEP

/PIC

NA

/EN

AH

O/H

EEP

/PIC

NA

/EN

AH

O/H

EE

P/PI

CN

A/E

NA

HO

/HE

EP/

PIC

CM

S31/

1.00

0.00

/0.0

00.

00/0

.00

1/1.

000.

00/0

.00

0.00

/0.0

02/

1.60

0.50

/0.4

10.

15/0

.30

2/1.

880.

75/0

.54

0.18

/0.3

6C

MS9

7/3.

840.

73/0

.75

0.52

/0.7

04/

2.36

0.60

/0.5

90.

34/0

.53

5/3.

600.

67/0

.79

0.49

/0.6

82/

1.60

0.00

/0.4

30.

15/0

.30

CM

S13

6/5.

320.

89/0

.83

0.62

/0.7

96/

2.94

0.75

/0.6

80.

44/0

.62

6/2.

670.

83/0

.68

0.43

/0.6

03/

2.91

1.00

/0.7

50.

36/0

.58

CM

S15

9/4.

150.

65/0

.77

0.55

/0.7

36/

3.88

0.63

/0.7

60.

52/0

.70

4/2.

770.

67/0

.70

0.37

/0.5

73/

2.13

0.25

/0.6

10.

28/0

.47

CM

S16

3/1.

310.

27/0

.24

0.11

/0.2

27/

2.09

0.40

/0.5

40.

34/0

.50

——

——

——

CM

S17

3/2.

960.

50/0

.68

0.37

/0.5

92/

1.05

0.05

/0.0

50.

02/0

.05

2/1.

600.

50/0

.50

0.15

/0.3

02/

2.00

1.00

/0.5

70.

19/0

.38

CM

S18

5/2.

520.

58/0

.62

0.32

/0.5

22/

1.72

0.40

/0.4

30.

17/0

.33

5/3.

130.

33/0

.74

0.46

/0.6

43/

2.13

0.75

/0.6

10.

28/0

.47

CM

S25

6/1.

650.

39/0

.40

0.23

/0.3

84/

2.52

0.74

/0.6

20.

32/0

.53

2/1.

180.

17/0

.17

0.07

/0.1

44/

2.91

0.75

/0.7

50.

41/0

.61

CM

S32

2/1.

040.

04/0

.04

0.02

/0.0

44/

2.89

0.60

/0.6

70.

40/0

.60

2/1.

180.

17/0

.17

0.07

/0.1

42/

1.88

0.75

/0.5

40.

18/0

.36

CM

S36

3/2.

220.

63/0

.56

0.25

/0.4

52/

1.28

0.25

/0.2

20.

10/0

.19

——

——

——

CM

S50

5/2.

970.

65/0

.68

0.39

/0.6

08/

4.97

0.70

/0.8

20.

61/0

.77

4/2.

060.

50/0

.56

0.30

/0.4

82/

1.28

0.25

/0.2

50.

10/0

.19

CM

S58

4/3.

110.

69/0

.69

0.39

/0.6

15/

2.46

0.75

/0.6

10.

35/0

.54

——

—2/

1.80

0.67

/0.5

20.

17/0

.35

CM

S104

2/1.

570.

32/0

.37

0.15

/0.3

01/

1.00

0.00

/0.0

00.

00/0

.00

2/1.

380.

33/0

.30

0.12

/0.2

43/

1.68

0.50

/0.4

60.

21/0

.37

CM

S121

5/3.

350.

62/0

.72

0.47

/0.6

67/

2.73

0.65

/0.6

50.

41/0

.59

5/3.

601.

00/0

.79

0.49

/0.6

84/

3.20

1.00

/0.7

90.

43/0

.63

NA

, num

ber

of a

llele

s; E

NA

, eff

ecti

ve n

umbe

r of

alle

les;

HO

, obs

erve

d he

tero

zygo

sity

; HE, u

nbia

sed

expe

cted

het

eroz

ygos

ity;

EP,

exc

lusi

on p

roba

bilit

y; P

IC, p

olym

orph

ism

info

rmat

ion

cont

ent.

*No

sign

ific

ant d

evia

tion

(P <

0.0

5) fr

om H

ardy

–Wei

nber

g eq

uilib

rium

.†N

ot te

sted

for

equi

libri

um b

ecau

se o

f sm

all n

umbe

r of

ani

mal

s (6

llam

a, 4

alp

aca)

.

434 P R I M E R N O T E

© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 431–434

Acknowledgements

We thank Dr B. Kaufmann, Tierhaltung und Tierzüchtung in denTropen und Subtropen, University Hohenheim, for the collectionof blood samples from Kenyan dromedaries. This research wassupported by the Ministry of Science, Research & Art, Baden-Württemberg, Germany.

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