Isolation and characterization of microsatellites from Scaptotrigonaxanthotricha (Apidae, Meliponini): a stingless bee in the BrazilianAtlantic rainforest

Olívia Maria Pereira DUARTE1, Fernanda Amato GAIOTTO

1, Anete Pereira SOUZA2, Gustavo Maruyama MORI2,

Marco Antônio COSTA1

1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Rodavia Ilhéus-Itabuna, Km 16, Ilhéus, BahiaCEP 45662-900, Brazil

2Departamento de Biologia Vegetal, Universidade Estadual de Campinas-UNICAMP, Instituto de Biologia, CP 6109,Campinas, São Paulo CEP 13083-970, Brazil

Received 16 June 2011 – Revised 5 October 2011 – Accepted 21 October 2011

Scaptotrigona / population genetics / polymorphism / Hymenoptera

Scaptotrigona bees are efficient pollinators as theyuse odors in marking trails to distant food sources.They build their nests in cavities of large living treesmaking them susceptible to anthropic actions, such asdeforestation. Due to the reduction in population sizeas a result of fragmentation and destruction of naturalhabitat, many species are threatened with extinction(Freitas et al. 2009). Scaptotrigona comprises 30Neotropical species, out of which 18 have beenrecorded in Brazil (Moure et al. 2007). This genusstill harbors many undescribed species and crypticspecies complexes (Silveira et al. 2002).

Scaptotrigona xanthotricha shows a wide distribu-tion in the Atlantic rainforest ranging from southern tothe northeastern Brazil (Silveira et al. 2002). Duarte etal. (2009) reported a remarkable geographical variationin the number and location of the Nucleolus OrganizerRegions in this species. This finding lent support to theexistence of interpopulational divergence or evenadditional taxa, thus reinforcing the need for moredetailed assessment of this group using molecularmarkers.

Studies on the genetic diversity and populationstructure of stingless bees have become frequentdue to the development of microsatellite markersfor several species such as Melipona mondury

(Lopes et al. 2010), Melipona bicolor (Peters et al.1998), and Scaptotrigona postica (Paxton et al.1999), among others. However, these studies arestill scarce when compared to the high diversitywithin this group. The present study aimed at theidentification and characterization of microsatelliteloci for S. xanthotricha.

An enriched genomic library was constructed from apool of DNA from 30 individuals of the same colonyfollowing Billotte et al. (1999). DNA extractionfollowed Han and McPheron (1997). Five microgramsof DNA (300 ng/μl) was digested with RsaI and linkedto RsaI adapters. The library was enriched for (CT)8and (GT)8 repeats using biotinylated probes attached tostreptavidin-labeled magnetic beads as described bythe manufacturer (MagneSphere Paramagnetic Par-ticles, Promega). Selected fragments amplified bypolymerase chain reaction (PCR) using primers withsequences complementary to the adapters and insertedinto the plasmid pGEM-T (Promega) were transformedinto competent XL-1 Blue Escherichia coli cells. Oncemost of the colonies were transformed, 96 wereselected for long-term storage at −80°C into a micro-plate. Forty-eight positive clones were sequenced onan automated sequencer ABI PRISM 377 (AppliedBiosystems) using SP6 and T7 primers and the BigDye terminator kit v3.1 (Applied Biosystems). A totalof 32 sequences contained microsatellite regions.

Corresponding author: M.A. Costa,costama@pq.cnpq.brManuscript editor: James Nieh

Apidologie (2012) 43:432–435 Scientific note* INRA, DIB and Springer-Verlag, France, 2011DOI: 10.1007/s13592-011-0109-6

Tab

leI.Microsatellite

loci

developedforS.

xantho

tricha

.

Locus

Sequence

Repeatmotif

T a(°C)

Range

NA

PIC

HE

HO

FIS

PNExc

I

Sxant

01F5′-G

TTGCGTA

TAGCCGGTCGTG-3′

CT(11)

56.7

322

––

––

––

–R5′-CGCTCCGTGAATGACAGTCC-3′

Sxant

05F5′-G

GAGAACCGACGACGAAGTG-3′

CT(22)

6222

0–24

27

0.74

50.78

70.80

6−0

.023

0.60

30.08

2R5′-TAGTGCCGGCTGGATCATTA

-3′

Sxant06

F5′-G

GACGAACGAGAGAGAGA-3′

AG

(16)

61.8

151–17

310

0.83

40.86

00.94

4−0

.098

0.45

90.03

8R5′-G

GCAAGGATCGTCAGTA

T-3′

Sxant10

F5′-G

ATCGATGCACGAAGAGTTG-3′

AG(20)

61.8

250–30

09

0.81

40.85

00.63

60.25

20.49

70.04

8R5′-TTGCGTCGAGGTTA

CCTTG-3′

Sxant13

F5′-ATCATGGCGACCGAGAAGAA-3′

GT(15)

61.8

172–18

69

0.74

80.78

90.80

6−0

.021

0.59

30.07

9R5′-A

CGAGCATCAGGATGGTTGG-3′

Sxant14

F5′-CGTA

ATA

GGACGATGTCGTG-3′

GA

(12)

6516

2–18

614

0.86

20.88

60.97

0−0

.095

0.39

80.02

7R5′-TCATCTGGACGCGGACACA-3′

Sxant15

F5′-G

CGCACGACATCGTCCTA

TT-3′

GT(12)

5444

0–48

02

0.37

50.51

00.33

30.34

60.87

50.37

5R5′-CGTCAGCCAATCTTGAACTC-3′

Sxant18

F5′-CCAAGATCTGTCATGCGTG-3′

GA

(23)

5814

2–18

619

0.92

70.94

60.91

20.03

60.24

40.00

9R5′-CAGACGTTA

CCTGGACGAA-3′

Sxant

19F5′-TGTCGGTA

ACGCTA

TCAGTC-3′

CT(25)

5151

0–55

611

0.85

80.88

70.63

60.28

30.40

80.02

9R5′-TCGATTCTGTCGGTCTGTC-3′

Sxant20

F5′-A

AGAGAGCAAGAGCGAGAG-3′

CT(26)

56.7

328–37

610

0.85

40.88

40.63

90.27

70.42

30.03

2R5′-A

CGTGTGTTA

CAGGCTA

CAG-3′

Sxant21

F5′-A

GCGGTGGTGATCCTTA

CGA-3′

AC(14)

64.6

243–26

06

0.57

50.64

60.77

8−0

.205

0.77

40.19

5R5′-CTGCCACCGTGTTA

GCCTCT-3′

Sxant23

F5′-G

AACATA

CGTGTGCATA

TAC-3′

CT(25)

48.2

370–42

68

0.82

00.85

70.54

30.36

60.49

20.04

6R5′-G

TACTTGAGCCTA

ACTGATT-3′

Sxant24

F5′-TGAAGACGAGAACAATCATC-3′

AG

(22)

50.5

150–17

810

0.80

00.83

60.70

60.15

60.52

00.05

4R5′-TTA

CAGCAGCCTTA

CCAATA

-3′

Mean

9.58

0.76

80.81

0.72

6–

––

T aannealingtemperature,N

Aallelenumber,PIC

polymorphism

inform

ationcontent,HEexpected

heterozygosity,H

Oobserved

heterozygosity,F

ISfixatio

nindex,

PNExc

first-parent

non-exclusionprobability,Inon-exclusionprobability

ofidentity

Microsatellites for Scaptotrigona xanthotricha 433

Primers for suitable flanking regions of 23 micro-satellites were designed using PRIMER version 2.0(Lincoln et al. 1991). Amplifications were performedin 25 μl reaction containing 2.5 ng of genomic DNA,1× Buffer KCl, 7 mM of each primer, 2 mM of eachdNTP, 0.03 mM MgCl2, and 1 U of Taq DNApolymerase. The PCR conditions were: 94°C for1 min, followed by 35 cycles of 94°C for 1 min,specific annealing temperature (Table I) for 1 min, 72°Cfor 1 min, and a final extension at 72°C for 5 min.Thirteen loci (GenBank accession numbers JN119815–JN119826) that had the correct size were analyzed forpolymorphism in a sample of 36 workers from 18colonies of S. xanthotricha collected along its distri-bution using denaturing 6% polyacrylamide gelsstained with silver nitrate (Creste et al. 2001). Weselected 12 polymorphic loci (Table I).

Allelic frequencies, observed (HO) and expectedheterozygosities (HE), polymorphic information con-tent (PIC), first-parent non-exclusion probability ofeach locus (PNExc), non-exclusion probability ofidentity (the probability of two randomly selectedindividuals having the same genotype) of each locus(I), and combined probabilities were estimated usingCERVUS (Marshall et al. 1998). Number of alleles(NA), fixation index (FIS), and tests for linkagedisequilibrium between loci were calculated usingFSTAT (Goudet 2002). A significance test for FIS wasalso performed using FSTAT based on 240 random-izations with the p value corrected by the Bonferronimethod, p<0.05. Obtained values are depicted inTable I.

The PIC varied from 0.375 to 0.927 with anaverage of 0.768, showing that the described locihave a high polymorphism.

Estimated FIS values for each locus ranged from−0.098, indicating an excess of heterozygotes, to 0.366indicating an excess of homozygotes. However, nosignificant deviations from random mating werefound. Twelve loci were tested for linkagedisequilibrium at a confidence interval of 95%,and only Sxant 14 and Sxant15 showed linkagedisequilibrium.

Allele number, HE, HO, and PIC in S. xanthotrichawere considerably higher than those found in M.mondury (Lopes et al. 2010), M. bicolor (Peters et al.1998), and S. postica (Paxton et al. 1999).

These 13 primer pairs were succesfully transferredfor three other species of Scaptotrigona, Scaptotrigonadepilis, Scaptotrigona bipunctata, and S. postica, usingthe same amplification conditions (Table I). Twelve

loci were polymorphic, except for Sxant01. Thepresent results suggest that these new microsatelliteswill be very useful for analyzing the genetic diversityand population structure of stingless bees, especiallythose within the genus Scaptotrigona.

ACKNOWLEDGMENTS

We thank Lucio A. O. Campos for providingsamples of S. xanthotricha and Gabriel A. R. Melofor the species identification. This research wassupported by Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES—PROCAD NF2567/2008). O. M. P. Duarte also received a doctoralscholarship grant from CAPES.

Isolation et caractérisation de microsatellites deScaptotrigona xanthotricha (Apidae, Meliponini): uneabeille sans aiguillon de la forêt pluviale atlantiquebrésilienne.

Isolierung und Charakterisierung von Mikrosatel-liten der Stachellosen Biene Scaptotrigona xanthotri-cha (Apidae, Meliponini): einer Biene desAtlantischen Regenswalds Brasiliens.

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