MICROSATELLITE LETTERS

Isolation and characterization of nuclear microsatellite primersfor the Barbary thuja, Tetraclinis articulata (Vahl) Mast.(Cupressaceae)

Marıa Teresa Lorenzo • Ramon Casimiro-Soriguer •

Francisco Balao • Juan Luis Garcıa-Castano •

Jose M. Sanchez-Robles • Anass Terrab

Received: 23 September 2013 / Accepted: 26 September 2013 / Published online: 5 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Fifty nuclear microsatellite primers were ini-

tially developed using next-generation sequencing (454)

data from a single individual of Tetraclinis articulata

(Vahl) Mast. Eleven primers were finally applied in 30

individuals from 3 localities from Morocco, Algeria and

Spain. The number of alleles per locus ranged from one to

ten. The average observed and expected heterozygosities

across the populations studied ranged from 0.10 to 0.80 and

from 0.09 to 0.88, respectively. The microsatellite markers

described here are valuable tools for the population genetic

research of T. articulata, and can be used to obtain infor-

mation for creating suitable management strategies to

conserve this endemic and endangered species.

Keywords Cupressaceae � Microsatellites �Population genetics � nSSR � Tetraclinis articulata

Tetraclinis Mast. is a genus of evergreen coniferous trees in

the cypress family (Cupressaceae), containing currently

only one species, the Barbary thuja. Tetraclinis articulata

(Vahl) Mast. is endemic to the mountainous regions of

North Africa (Morocco, Algeria and Tunisia), with isolated

populations occurring in Malta and S and E Spain. The

largest area of distribution is in Morocco, where it is found

in the Rif mountains (E Morocco), E & W Middle Atlas, C

& E plateau, High Atlas and Anti Atlas.

We extracted genomic DNA from leaf tissue from a

single T. articulata using an Invisorb Spin Plant Mini Kit

(Invitek, Berlin, Germany). The technique applied to

develop the microsatellites was Next Generation

Sequencing (NGS). The screening sequence data and the

design of primers were made following the procedures

described in Sanchez-Robles et al. (2012).

From 5,981 reads obtained through the NGS, only in 503

of them primers could be designed. Out of these 503 primers

pairs, we discarded 103 because of their low quality. Con-

sequently, 373 high quality primers pairs were obtained. A

total of 50 primer pairs were tested for their amplification

quality. For this, two quality DNA samples were used. Out of

the 50 loci, eleven originated clear patterns of amplification

although two of them were monomorphic (Tetra16 and

Tetra18). The rest of the selected primer pairs were discarded

as eighteen did not amplified and the other twenty-one pro-

duced unclear, difficult to score patterns.

PCR products were run on a 3,730 DNA Analyzer

sequencer (Applied Biosystem, Foster City, CA, USA) and

sized with LIZ 500 standard (Applied Biosystem, Foster

City, CA, USA). Polymerase chain reactions were per-

formed in 20 ll, the reaction mixture containing approxi-

mately 50 ng of genomic DNA, 19 PCR Buffer, 1 U/ll

i-Start Taq DNA polymerase (iNtRON Biotecnology Inc.,

Sungman, Korea), 0.25 lM primer with the 50-GTTT tail,

0.06 lM primer with the 50-CAG or the M13R tail, and

0.25 lM dye-labeled CAG or M13R universal primer with

FAM, NED or VIC fluorescent label, MgCl2, dNTP and

BSA were added in different amounts for different primer

pairs (Table 1).

M. T. Lorenzo � R. Casimiro-Soriguer � F. Balao �J. L. Garcıa-Castano � J. M. Sanchez-Robles � A. Terrab (&)

Departamento de Biologıa Vegetal y Ecologıa, Universidad de

Sevilla, Ap-1095, 41080 Sevilla, Spain

e-mail: anass@us.es

M. T. Lorenzo � F. Balao

Department of Systematic and Evolutionary Botany, University

of Vienna, Rennweg 14, 1030 Vienna, Austria

R. Casimiro-Soriguer

Departamento de Biologıa, Universidad de Cadiz, Campus Rıo

San Pedro, 11510 Puerto Real, Spain

123

Conservation Genet Resour (2014) 6:233–235

DOI 10.1007/s12686-013-0064-9

Ta

ble

1P

rim

erse

qu

ence

san

dch

arac

teri

stic

so

fel

even

Tet

raci

lin

isa

rtic

ula

tam

icro

sate

llit

em

ark

ers

Lo

cus

Pri

mer

seq

uen

ce(50 –

30 )

aR

epea

tm

oti

fS

ize

(bp

)T

a(�

C)

AM

gC

l 2(m

M)

dN

TP

(mM

)

BS

A

(mg

/ml)

PC

R

pro

file

Gen

Ban

k

acce

ssio

nn

o.

Tet

ra1

F-G

GA

AA

CA

GC

TA

TG

AC

CA

TT

AT

TG

AG

GA

AG

AA

TT

GA

GA

TC

(10

)1

78

65?

45

71

.50

.20

.02

5C

KF

26

77

06

R-G

TT

TG

GA

AT

TC

TA

CC

TC

CC

TA

TC

C

Tet

ra2

F-G

GA

AA

CA

GC

TA

TG

AC

CA

TA

CC

AC

AA

CC

TT

CT

CT

CA

AG

(11

)2

68

55?

45

11

2.0

0.1

0.0

25

BK

F2

67

70

7

R-G

TT

TG

GA

TA

CC

CT

CT

AA

TG

AT

CA

CT

G

Tet

ra4

F-G

TT

TA

AC

CT

TT

GC

TT

TG

AT

AC

CA

CA

AG

(8)

13

16

0?

50

32

.00

.10

.02

5B

KF

26

77

08

R-C

AG

TC

GG

GC

GT

CA

TC

AA

CA

TT

AA

TG

GA

AG

AA

CA

CC

Tet

ra1

5F

-GT

TT

GC

AG

AT

TA

TG

GA

GT

GG

AT

TC

AC

AT

(8)A

T(3

)2

53

60?

50

42

.00

.10

.02

5B

KF

26

77

09

R-C

AG

TC

GG

GC

GT

CA

TC

AT

GT

TG

GA

CA

AG

CA

CA

TA

TT

G

Tet

ra1

6F

-GG

AA

AC

AG

CT

AT

GA

CC

AT

CT

TG

CC

AA

AT

AT

GA

GA

TT

AA

GA

TC

(7)

12

76

0?

50

12

.50

.20

.02

5A

KF

26

77

10

R-G

TT

TG

GA

GG

AT

GA

AG

CT

CA

AG

TA

G

Tet

ra1

8F

-GG

AA

AC

AG

CT

AT

GA

CC

AT

CA

AC

AA

TC

CG

AA

GA

AT

AT

GG

AT

C(7

)2

80

60?

50

12

.50

.20

.02

5A

KF

26

77

11

R-G

TT

TG

AG

TT

GT

TT

GG

GA

GT

CT

TT

G

Tet

ra1

9F

-GT

TT

GC

CT

TC

TC

CA

CT

AG

TT

TG

AA

G(9

)CT

(3)

17

76

0?

50

52

.50

.20

.02

5A

KF

26

77

12

R-G

GA

AA

CA

GC

TA

TG

AC

CA

TT

TG

AG

GC

TA

AA

CA

TT

CA

CC

Tet

ra2

2F

-GG

AA

AC

AG

CT

AT

GA

CC

AT

GA

AA

CA

AC

TA

GG

CA

CC

AG

AA

G(8

)CA

(3)

15

46

0?

50

22

.50

.20

.02

5A

KF

26

77

13

R-G

TT

TC

CG

GT

CT

TC

TT

AG

GA

GT

GA

C

Tet

ra2

9F

-GG

AA

AC

AG

CT

AT

GA

CC

AT

GA

AT

CT

TC

TC

GG

CA

AA

TG

AA

G(1

6)T

C(3

)2

28

60?

50

13

2.5

0.2

0.0

25

AK

F2

67

71

4

R-G

TT

TG

AG

TT

GC

AA

GG

AG

TG

TC

AA

G

Tet

ra4

4F

-CA

GT

CG

GG

CG

TC

AT

CA

CA

TC

TG

TT

GG

AG

CA

AT

TT

GA

C(1

3)

23

95

0?

40

52

.50

.20

.05

0D

KF

26

77

15

R-G

TT

TG

GA

AC

CC

AT

GA

GA

TA

AT

GT

G

Tet

ra4

9F

-GT

TT

CT

CC

AC

CA

TT

GT

CA

CT

TG

TC

AA

G(1

3)

27

06

5?

55

12

2.5

0.2

0.0

50

AK

F2

67

71

6

R-G

GA

AA

CA

GC

TA

TG

AC

CA

TA

AC

CA

AG

AA

GA

TA

GC

CA

AA

G

aS

equ

ence

sin

bo

ldit

alic

sar

eu

sed

toin

tro

du

cesi

tes

for

the

un

iver

sal

pri

mer

.U

nd

erli

ned

bas

esin

dic

ate

shar

ing

of

nu

cleo

tid

esb

etw

een

the

CA

Gta

g,

the

M1

3R

tag

,o

rth

eG

TT

Tta

ilan

dth

e

locu

s-sp

ecifi

cp

rim

erb

ind

ing

site

;T

a=

ann

eali

ng

tem

per

atu

reu

sed

inth

e‘‘

tou

chd

ow

n’’

po

lym

eras

ech

ain

reac

tio

ns

(Do

net

al.

19

91

),A

=n

um

ber

of

alle

les,

PC

Rp

rofi

le=

Sam

ple

sw

ere

incu

bat

edw

ith

the

foll

ow

ing

con

dit

ion

s:in

itia

ld

enat

ura

tio

nat

94

�Cfo

r5

min

;5

cycl

esat

94

�Cfo

r3

0s,

init

ial

Ta

for

30

s,an

d7

2�C

for

30

s;2

1to

uch

do

wn

cycl

esat

94

�Cfo

r3

0s,

init

ial

Ta—

0.5

�Cp

ercy

cle

(pro

file

A,

Ban

dD

)/-

1�C

per

cycl

e(p

rofi

leC

)fo

r3

0s

and

72

�Cfo

r3

0s;

then

30

cycl

es(p

rofi

leA

)/1

5cy

cles

(pro

file

Ban

dC

)/3

5cy

cles

(pro

file

D)

at9

4�C

for

30

s,

the

last

Ta

for

30

san

d7

2�C

for

30

s;an

d7

2�C

for

7m

in

234 Conservation Genet Resour (2014) 6:233–235

123

Fragments were analyzed with the software GENE-

MARKER version 1.8 (SoftGenetics, State College, PA,

USA). We estimated the number of alleles per locus (A),

the observed and expected heterozygosity (Ho and He) and

Hardy–Weinberg equilibrium tests (HWE) for each locus

using GeneAlEx 6.5 (Peakall and Smouse 2012). Tetra44

locus showed deviation from Hardy–Weinberg equilibrium

in the Moroccan population after Bonferroni correction for

multiple comparisons (adjusted P value for 5 % nominal

level = 0.002). We calculated the null allele frequency

(An) with Micro-Checker 2.2.3 (Van Oosterhout et al.

2004) and linkage disequilibrium (LD) between pairs of

loci was tested using GENEPOP 4.0.10 software (Rousset

2008). None of the loci pairs showed significant linkage

disequilibrium (LD) in the 33 comparisons (Table 2).

We have developed eleven new nuclear microsatellite

primer pairs for T. articulata. Nine of the loci showed high

levels of polymorphism, suggesting great potential for

genetic diversity studies. These primers will enable the

development of biogeographic and conservation genetic

studies to investigate the origin of the European T. artic-

ulata populations. Additionally, the microsatellite markers

reported here provide a valuable tool for forest manage-

ment and they could be tested on other Cupressaceae

species.

Acknowledgments The authors thank L. Navarro-Sampedro for

helpful advice in the laboratory. This study was financially supported

by the Spanish Ministerio Educacion y Ciencia to AT (CGL2009-

08713). The authors thank the Herbarium and Biology Research

Services (CITIUS) of the University of Seville for allowing the use of

their facilities.

References

Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)

‘‘Touchdown’’ PCR to circumvent spurious priming during gene

amplification. Nucleic Acids Res 19:4008

Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel.

Population genetic software for teaching and research—an

update. Bioinformatics 28:2537–2539

Rousset F (2008) Genepop’007: a complete re-implementation of the

GENEPOP software for Windows and Linux. Mol Ecol Res

8:103–106

Sanchez-Robles JM, Balao F, Garcıa-Castano JL, Terrab A, Navarro-

Sampedro L, Talavera S (2012) Nuclear microsatellite primers

for the endangered relict fir, Abies pinsapo (Pinaceae) and cross-

amplification in related mediterranean species. Int J Mol Sci

13:14243–14250

Van Oosterhout C, Hutchinson W, Wills D, Shipley P (2004) Micro-

Checker: software for identifying and correcting genotyping

errors in microsatellite data. Mol Ecol Notes 4:535–538

Table 2 Genetic diversity estimates for three Tetraclinis articulata populations

Locus Morocco (N = 10) Spain (N = 10) Algeria (N = 10)

A Ho He HWE An A Ho He HWE An A Ho He HWE An

Tetra1 3 0.300 0.395 0.667 0.068 4 0.700 0.570 0.893 0.000 6 0.600 0.550 0.993 0.000

Tetra2 7 0.700 0.800 0.278 0.056 4 0.200 0.270 0.003 0.055 8 0.700 0.805 0.137 0.058

Tetra4 1 0.000 0.000 – – 2 0.100 0.095 0.868 0.000 2 0.300 0.255 0.577 0.000

Tetra15 2 0.200 0.320 0.236 0.091 2 0.200 0.180 0.725 0.000 4 0.700 0.705 0.710 0.003

Tetra16 1 0.000 0.000 – – 1 0.000 0.000 – – 1 0.000 0.000 – –

Tetra18 1 0.000 0.000 – – 1 0.000 0.000 – – 1 0.000 0.000 – –

Tetra19 4 0.200 0.570 0.113 0.236 3 0.500 0.645 0.321 0.088 5 0.200 0.700 0.004 0.294

Tetra22 2 0.250 0.469 0.351 0.459 2 0.800 0.480 0.136 0.000 2 0.200 0.500 0.180 0.200

Tetra29 7 0.300 0.795 0.053 0.276 5 0.300 0.595 0.012 0.185 10 0.300 0.880 0.005 0.309

Tetra44 4 0.100 0.535 0.001 0.283 3 0.200 0.615 0.028 0.257 4 0.200 0.685 0.005 0.288

Tetra49 9 0.800 0.815 0.508 0.008 4 0.400 0.570 0.640 0.108 7 0.800 0.790 0.141 0.000

N sample size, A number of alleles, Ho observed heterozygosity, He expected heterozygosity, An null allele frequency, HWE Hardy–Weinberg

equilibrium test (P values)

Conservation Genet Resour (2014) 6:233–235 235

123