characterization of microsatellite loci of tetragonisca angustula

8/7/2019 Characterization of microsatellite loci of Tetragonisca angustula

1/5

T E CH N I CA L N O T E

Characterization of microsatellite loci of Tetragonisca angustula(Hymenoptera, Apidae, Meliponini)

R. M. Brito F. O. Francisco A. M. T. Domingues-Yamada P. H. P. Goncalves F. C. Pioker A. E. E. Soares M. C. Arias

Received: 15 June 2009 / Accepted: 17 June 2009 / Published online: 28 June 2009

Springer Science+Business Media B.V. 2009

Abstract An enriched genomic library was constructed

from Tetragonisca angustula, a stingless bee specieswidely distributed in Brazil. The library was screened using

two simple-repeat oligonucleotide probes and 21 micro-

satellite primer pairs were designed flanking a selection of

repeat sequences within positive clones. The polymor-

phism of the microsatellite loci was analyzed by screening

a sample of 19 unrelated T. angustula workers. Fifteen out

of 21 loci were shown to be polymorphic, with observed

heterozygosity estimates ranging from 0.00 to 0.89. The

primers were also successfully used to amplify microsat-

ellite loci from other stingless bee species, Tetragonisca

fiebrigi, Tetragonisca weyrauchi, Lestrimelitta maracaia

and Schwarziana quadripunctata. The results from vari-

ability analyses suggest that the microsatellite loci isolated

from T. angustula will be useful in further population

studies for the species and also for other Meliponini.

Keywords Microsatellites Tetragonisca angustula

Population genetics Meliponini

The decline of bee species has been observed worldwide

and has usually been associated with degradation of theirnatural habitats, abusive use of pesticides, and direct

human action by mismanagement of apiaries (Klein et al.

2007). This issue has been of major concern (see Ghazoul

2005; Steffan-Dewenter et al. 2005) since in a short time it

may negatively affect the genetic variability of both plants

and bees, and also the economy since several crops depend

on bee pollination (Klein et al. 2007).

Deforestation in the Neotropical Region has been mas-

sive over the last 500 years. Some environments have been

dramatically reduced, such as the Atlantic forest in Brazil

which currently occupies less than 8% of its original area.

For bees in particular, damage to the environmental is a

threat not only by reducing nesting sites but also by iso-

lating populations in forest fragments which may lead to

inbreeding depression. In honeybees low genetic variability

is related to the production of diploid males due to csd

(complementary sex determination) gene homozygosis

(Beye et al. 2003). This locus determines whether a fer-

tilized egg will become a female (heterozygous) or a dip-

loid male (homozygous) (Cook 1993; Beye et al. 2003).

The diploid males are sterile and killed by workers, hence

reducing the effective number (Ne) of the population

(Zayed and Packer 2005). According to Kerr and Ven-

covsky (1982), a similar genetic system is observed in

stingless bees. However, recent data showed no evidence

for the existence of a csd locus in other Hymenopteran

species (Hasselmann et al. 2008). Therefore it is unclear if

low heterozygosis may represent a real risk for Meliponini

bees, the major pollinator group of Angiosperm according

to Michener (2000).

Analysis of population genetics, structuring and migra-

tion dynamics are frequently surveyed through codomi-

nant, generally neutral, nuclear markers such as

R. M. Brito (&) F. O. Francisco A. M.

T. Domingues-Yamada P. H. P. Goncalves M. C. Arias

Departamento de Genetica e Biologia Evolutiva, Instituto de

Biociencias, Universidade de Sao Paulo, Rua do Matao,277, Sao Paulo, SP 05508-090, Brazil

e-mail: [email protected]

F. C. Pioker

Departamento de Ecologia, Instituto de Biociencias,

Universidade de Sao Paulo, Rua do Matao, travessa 14,

n. 321, Sao Paulo, SP 05508-900, Brazil

A. E. E. Soares

Departamento de Genetica, Faculdade de Medicina de Ribeirao

Preto, Universidade de Sao Paulo, Av. Bandeirantes,

3900, Ribeirao Preto, SP 14049-900, Brazil

123

Conservation Genet Resour (2009) 1:183187

DOI 10.1007/s12686-009-9045-4


2/5

microsatellites (Sunnucks 2000). Few microsatellite prim-

ers are available for stingless bees (tribe Meliponini). To

date only three species have had their genomes screened to

develop microsatellite primers: Melipona bicolor (Peters

et al. 1998), Scaptotrigona postica (Paxton et al. 1999) and

Trigona carbonaria (Green et al. 2001). Those loci have

been successfully employed to access the polymorphism at

intra-specific level. However, despite these primers havingsucceeded in some cross-species amplification experiments

(Peters et al. 1998; Paxton et al. 1999), we have found a

very low polymorphism level when they were employed in

a heterologous basis in population surveys of Plebeia

remota, Partamona helleri and Partamona mulata (Fran-

cisco et al. 2006). This low genetic variability may be a

natural consequence of habitat fragmentation, but also, can

be due to technical artifacts by the non-amplification of

some allele (null alleles) caused by primer mispairing at

the heterologous template during the PCR annealing step.

We are particularly interested in measuring the genetic

variability of Tetragonisca angustula, one of the mostpopular stingless bees in the Neotropical region. This

species, widely distributed from southern Mexico to

southern Brazil (Camargo and Pedro 2008), is very com-

mon in urban and natural areas and is easily handled by

amateur and professional beekeepers. In the present work

we introduce a set of primers designed from the genome of

Tetragonisca angustula which will enable us in future

population surveys to accurately detect their genetic vari-

ability avoiding then misinterpretations of data due to null

alleles when using heterologous primers.

Total DNA was extracted from a pool of 15 individuals

using a phenol:chlorophorm protocol. The DNA was ana-

lyzed in 0.8% agarose gel and a sharp band (*500 ng/ll) of

high molecular weight was observed under a UV light. The

enriched microsatellite genomic library was constructed

according to Billotte et al. (1999) with some modifications.

Genomic DNA (5 lg) was digested with 50 U of Rsa I and

then linked to 10 lM of Rsa21 (50CTCTTGCTTACGCGT

GGACTA30) and Rsa25 (50TAGTCCACGCGTAAGCAA

GAGCACA30) adaptors. Fragments were selected by (GA)8and (AGA)5 probes and then cloned into pGem

-T (Pro-

mega) vector and transformed into E. coli DH5a lineage.

From a total of 96 selected colonies, 80 were sequenced. The

sequences were aligned using the online software Multi-

Align (Corpet 1988) to verify a possible correspondence of

clones to the same locus. After the analysis of sequence

content for the presence of direct repeats, 21 primer pairs

were designed flanking the repeats using the Primer 3 tool,

available online (http://frodo.wi.mit.edu/primer3/input.htm )

(Table 1).

Nineteen unrelated workers from different Brazilian

regionswerescreened for the microsatellite loci. Alsoworkers

of Tetragonisca fiebrigi, Tetragonisca weyrauchi,

Lestrimelitta maracaia and Schwarziana quadripunctata

were tested for cross-species amplifications. T. angustula

specimens and the other Meliponini species wereidentified by

Dr Joao M. F. Camargo, FFCLRP/USP, Ribeirao Preto, Bra-

zil. Template DNA was extracted from the thorax of a single

individual per colony using the Chelex method (Walsh et al.

1991). The optimal annealing temperature of each primer pair

was optimized using a Robocycler Gradient 96 (Stratagene).Amplifications were carried out in 10 ll reaction volumes

containing 3.6 ll of deionized Milli-Q water; 19 PCR buffer;

0.3 llofMgCl2 25 mM; 0.2 llofeach primer10 lM; 2 ll of

betaine 5 M; 2 ll of template DNA and 1 U Taq DNA

polymerase (Fermentas). PCR reactions were performed in an

AmpliGene thermocycler (Applied Biosystems) following

the conditions: 4 min at 94C, then 35 cycles of 30 s at 94C,

30 s at specific annealing temperature (Table 1), and 30 s at

72C, followed by a final elongation step of 5 min at 72C.

Amplified fragments were electrophoresed in 9% polyacryl-

amide gels and silver stained. Genic diversity, observed het-

erozygosity (HO), expected heterozygosity (HE), number ofalleles and allelic frequencies were estimated using formulas

inserted in Microsoft Excel by the second author (Francisco

2009). Tests for HardyWeinberg Equilibrium and Linkage

Disequilibrium were calculated by GENEPOP v4.0 (Rousset

2008).

Fifteen out 21 loci tested were polymorphic (P[ 0.05) in

a population survey comprising individuals from assorted

collection sites covering the geographic distribution of Te-

tragonisca angustula. The average value for allelic diversity

(A = 8.94) was higher than the observed for other Melipo-

nini species such as Melipona bicolor(3.88), Scaptotrigona

postica (5.67) and Trigona carbonaria (3.60) (Peters et al.

1998; Paxton et al. 1999; Green et al. 2001). The levels of

observed heterozygosity were lower than the expected

(Table 1) and significant deviations from the HardyWein-

berg expectations were found in all loci except at Tang48,

Tang60, Tang61, and Tang70. Such results were expected

since we analyzed workers collected from colonies

2,000 km apart in some cases which surely do not represent a

panmictic population. Linkage disequilibrium was detected

(P\ 0.05) between loci Tang03/Tang17, Tang11/Tang57,

Tang11/Tang60, Tang57/Tang68, Tang65/Tang79, and

Tang68/Tang77. The PCR products from cross-species

amplification tests produced fragments of expected sizes for

all species analyzed, nonetheless non PCR product for

Tang57 locus was detected for L. maracaia (Table 2).

These loci can be assessed in studies of natural popu-

lations of T. angustula in order to detect how habitat

degradation is affecting their genetic variability. Also this

primer set will be useful for detection of endogamy,

especially in highly managed small populations from me-

liponaries and will allow us to test Kerr and Vencovskys

hypothesis. The present work also will contribute to

184 Conservation Genet Resour (2009) 1:183187

123
http://frodo.wi.mit.edu/primer3/input.htmhttp://frodo.wi.mit.edu/primer3/input.htm


3/5

Table1

Characteristicsof16microsatellitelocifromTetragoniscaangustula

Locus

Repeatmotif

GenBank

accessionnumber

Primersequences(5

0

3

0

)

Ta(C)

k

Allelesize

range(bp)

HO

HE

Tang03

(AG)11

GQ184627

F:GGAACATTTGTTGAAGGAATT

TG

60.0

6

196210

0.32

0.75*

R:GCCGCATTGGTTTTCTTAAT

Tang11

(GA)22

GQ184612

F:TATTCCTATTCACGCGATGC

53.0

13

158190

0.58

0.86*

R:AGACGATATGGTGGCATTCA

Tang12

(GA)24

GQ184613

F:CCAGATGCAACCCTTTGACT

53.0

14

176218

0.68

0.88*

R:AGGCCCATCGAAGACCAT

Tang17

(AG)23

GQ184614

F:GTAATGTGGAACGTCTACG

52.0

10

138172

0.53

0.84*

R:GATAATCGCGCGAGTGGAG

Tang29

(GA)26

GQ184615

F:CGGTCTTGAAGTGCGGAATA

55.0

11

171207

0.68

0.90*

R:CAGGAACGCGTAACCAACTT

Tang40

(TCAC)7TCAT(TC)14TGT(TCTTC)3

GQ184616

F:TACGTGACAACTTCCGAATG

52.5

11

110188

0.42

0.78*

R:CGCCGCTAGTTCCCATATC

Tang48

(CT)13

GQ184617

F:TGACGGATAAAGAGAGGTCGA

G

55.0

6

233243

0.58

0.53

R:CTCTCGGATTCCTTGAGCTT

Tang57

(TC)5TT(TC)2TGTT

(TC)18

GQ184618

F:GCCGATTTATGGCAACGATA

60.0

11

138188

0.58

0.84*

R:TCGAATTTATAGTCTTCCGATTC

Tang60

(AG)27

GQ184619

F:GAGAAAACGATGAATGCCG

60.0

8

110132

0.63

0.74

R:TGAGAGAAGGCAAGTTGTTGA

Tang61

(TA)5

GQ184620

F:GCTGTCGAATGTCTCTAAACC

55.0

2

110112

0.05

0.05

R:TAGTCACATGGGCAAGATGC

Tang65

(AG)14

GQ184621

F:TGCTCGTTATAATTGCACCA

55.0

7

171195

0.68

0.76*

R:CAGCTCAAGCCGTAAAGATG

Tang68

(TC)10

GQ184622

F:TAACGGAGCCGAGGATACAG

60.0

2

220224

0.00

0.43*

R:CGATGAAATCGTGGATGAAG

Tang70

(AG)10

GQ184623

F:GGTTAGGGCGGTCGACTTAT

55.0

5

200208

0.63

0.72

R:TGGTTCTCTCCGTTTTCGAC

Tang77

(CT)16CC(CT)3

GQ184624

F:CGTTTGAACGATGAACTGGA

55.0

10

175225

0.89

0.79*

R:CCTATTTCCGACGCTCTGTC

Tang78

(CT)23

GQ184625

F:CGAATACGATCTGCACTCCTC

55.0

16

208260

0.74

0.88*

R:ATTCACGACGATACGCCACT

Tang79

(TC)21

GQ184626

F:CTAGGCCGGACGACAGATTC

48.0

11

118140

0.47

0.85*

R:TGAACTGTCTTCCTATCGTCTG

Flankingprimers,optimalannealingtemperatures(Ta),numberofalleles(k),a

llelesizerange,observed(HO)andexpected(HE)heterozygosityestimatedfrom19unrelatedworkers.GenBank

accessionnumbersforclonedsequ

encesarealsogiven

*DenotessignificantHWEdeparture(P\

0.05)

Conservation Genet Resour (2009) 1:183187 185

123


4/5

molecular population surveys on other Meliponini species

providing a set of new microsatellite primers successfully

tested for Tetragonisca fiebrigi, Tetragonisca weyrauchi,

Lestrimelitta maracaia and Schwarziana quadripunctata.

Acknowledgements We would like to express our gratitude to Susy

Coelho Oliveira for the technical support; Dr Joao M. F. Camargo for

the identification of specimens; Dr Anete P. de Souza for protocols

adaptation; Dr. Timothy Schaerf for the English revision; Conselho

Nacional de Desenvolvimento Cientfico e Tecnologico for a PhD

scholarship to AMTDY, Coordenacao de Aperfeicoamento de Pessoalde Nvel Superior for a master degree scholarship to PHPG, and

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo for PhD

scholarships to FOF and FCP and for financial support (BIOTA 2004/

15801-0).

References

Beye M, Hasselmann M, Fondrk MK, Page RE Jr, Omholt SW (2003)

The gene csdis the primary signal for sexual development in the

honeybee and encodes an sr-type protein. Cell 114:419429. doi:

10.1016/S0092-8674(03)00606-8

Billotte N, Lagoda PJR, Risterucci AM, Baurens FC (1999) Microsat-

ellite-enriched libraries: applied methodology for the develop-ment of SSR markers in tropical crops. Fruits 54:277288

Camargo JMF, Pedro SRM (2008) Meliponini Lepeletier, 1836. In:

Moure JS, Urban D, Melo GAR (eds) Catalogue of bees

(Hymenoptera, Apoidea) in the Neotropical Regiononline

version. Available at http://www.moure.cria.org.br/catalogue.

Accessed 10 May 2009

Cook JM (1993) Sex determination in the Hymenoptera: a review of

models and evidence. Heredity 71:421435. doi:10.1038/hdy.

1993.157

Corpet F (1988) Multiple sequence alignment with hierarchical

clustering. Nucl Acids Res 16:1088110890

Francisco FO (2009) FOFpop v.2.0, a pool of Microsoft Excel sheets

to analyze genotypic data. Available at: http://www.ib.usp.br/

*lgea/fofpop. Accessed 15 May 2009

Francisco FO, Brito RM, Arias MC (2006) Alelle number and

heterozygosity for microsatellite loci in different stingless bee

species (Hymenoptera: Apidae, Meliponini). Neotrop Entomol

35:638643. doi:10.1590/S1519-566X2006000500011

Ghazoul J (2005) Buzziness as usual? Questioning the global

pollination crisis. Trends Ecol Evol 20:367373. doi:10.1016/

j.tree.2005.04.026

Green CL, Franck P, Oldroyd BP (2001) Characterization of

microsatellite loci for Trigona carbonaria, a stingless beeendemic to Australia. Mol Ecol Notes 1:8992. doi:0.1046/

j.1471-8278.2001.00041.x

Hasselmann M, Gempe T, Schitt M, Nunes-Silva CG, Otte M, Beye

M (2008) Evidence for the evolutionary nascence of a novel sex

determination pathway in honeybees. Nature 454:519522. doi:

10.1038/nature07052

Kerr WE, Vencovsky R (1982) Melhoramento genetico em abelhas. I.

Efeito do numero de colonias sobre o melhoramento. Rev Bras

Genet 5:279285

Klein A-M, Vaissie BE, Cane JH, Steffan-Dewenter I, Cunningham

SA, Kremen C, Tscharntke T (2007) Importance of pollinators in

changing landscapes for world crops. Proc R Soc B 274:303

313. doi:10.1098/rspb.2006.3721

Michener CD (2000) The bees of the world. The John Hopkins

University Press, BaltimorePaxton RJ, WeibSchun N, Quezada-Euan JJG (1999) Characteriza-

tion of dinucleotide microsatellite loci for stingless bees. Mol

Ecol 8:690692

Peters JM, Queller DC, Imperatriz-Fonseca VL, Strassmann JE

(1998) Microsatellite loci for stingless bees. Mol Ecol 7:783787

Rousset F (2008) GENEPOP007: a complete re-implementation of

the GENEPOP software for Windows and Linux. Mol Ecol Res

8:103106. doi:10.1111/j.1471-8286.2007.01931.x

Steffan-Dewenter I, Potts SG, Packer L (2005) Pollinator diversity

and crop pollination services are at risk. TREE 20:651652. doi:

10.1016/j.tree.2005.09.004

Table 2 Cross-species

amplification tests: (?)

successful amplification and (-)

no amplification

Loci Tetragonisca

fiebrigi

Tetragonisca

weyrauchi

Lestrimelitta

maracaia

Schwarziana

quadripunctata

Tang03 ? ? ? ?

Tang11 ? ? ? ?

Tang12 ? ? ? ?

Tang17 ? ? ? ?

Tang29 ? ? ? ?Tang40 ? ? ? ?

Tang48 ? ? ? ?

Tang57 ? ? - ?

Tang60 ? ? ? ?

Tang61 ? ? ? ?

Tang65 ? ? ? ?

Tang68 ? ? ? ?

Tang70 ? ? ? ?

Tang77 ? ? ? ?

Tang78 ? ? ? ?

Tang79 ? ? ? ?

186 Conservation Genet Resour (2009) 1:183187

123
http://dx.doi.org/10.1016/S0092-8674(03)00606-8http://www.moure.cria.org.br/cataloguehttp://dx.doi.org/10.1038/hdy.1993.157http://dx.doi.org/10.1038/hdy.1993.157http://www.ib.usp.br/~lgea/fofpophttp://www.ib.usp.br/~lgea/fofpophttp://www.ib.usp.br/~lgea/fofpophttp://dx.doi.org/10.1590/S1519-566X2006000500011http://dx.doi.org/10.1016/j.tree.2005.04.026http://dx.doi.org/10.1016/j.tree.2005.04.026http://dx.doi.org/0.1046/j.1471-8278.2001.00041.xhttp://dx.doi.org/0.1046/j.1471-8278.2001.00041.xhttp://dx.doi.org/10.1038/nature07052http://dx.doi.org/10.1098/rspb.2006.3721http://dx.doi.org/10.1111/j.1471-8286.2007.01931.xhttp://dx.doi.org/10.1016/j.tree.2005.09.004http://dx.doi.org/10.1016/j.tree.2005.09.004http://dx.doi.org/10.1111/j.1471-8286.2007.01931.xhttp://dx.doi.org/10.1098/rspb.2006.3721http://dx.doi.org/10.1038/nature07052http://dx.doi.org/0.1046/j.1471-8278.2001.00041.xhttp://dx.doi.org/0.1046/j.1471-8278.2001.00041.xhttp://dx.doi.org/10.1016/j.tree.2005.04.026http://dx.doi.org/10.1016/j.tree.2005.04.026http://dx.doi.org/10.1590/S1519-566X2006000500011http://www.ib.usp.br/~lgea/fofpophttp://www.ib.usp.br/~lgea/fofpophttp://dx.doi.org/10.1038/hdy.1993.157http://dx.doi.org/10.1038/hdy.1993.157http://www.moure.cria.org.br/cataloguehttp://dx.doi.org/10.1016/S0092-8674(03)00606-8


5/5

characterization of microsatellite loci of tetragonisca angustula

Documents