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An optimized method for the generation of AFLPmarkers in a stingless bee (Melipona quadrifasciata)

reveals a high degree of intracolonial geneticpolymorphism

Gustavo R. Makert, Robert J. Paxton, Klaus Hartfelder

To cite this version:Gustavo R. Makert, Robert J. Paxton, Klaus Hartfelder. An optimized method for the generationof AFLP markers in a stingless bee (Melipona quadrifasciata) reveals a high degree of intracolonialgenetic polymorphism. Apidologie, Springer Verlag, 2006, 37 (6), pp.687-698. <hal-00892224>

Apidologie 37 (2006) 687–698 687c© INRA/DIB-AGIB/ EDP Sciences, 2006DOI: 10.1051/apido:2006043

Original article

An optimized method for the generation of AFLP markersin a stingless bee (Melipona quadrifasciata) reveals a high

degree of intracolonial genetic polymorphism*

Gustavo R. Ma, Robert J. Pb, Klaus Hc

a Depto. Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazilb School of Biological Sciences, Queen’s University Belfast, Belfast, UK

c Depto. Biologia Celular e Molecular e Bioagents Patogênicos, Faculdade de Medicina de Ribeirão Preto,Universidade de São Paulo, Ribeirão Preto, Brazil

Received 22 June 2005 – Revised 26 February 2006 – Accepted 28 February 2006

Abstract – This study describes an optimized protocol for the generation of Amplified Fragment LengthPolymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a re-striction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown programin the selective PCR amplification step and product labelling by incorporation of α[33P]dATP. In an analysisof 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analy-sis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony.Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), theobserved rates of within-colony polymorphism are remarkably high, considering that the workers of eachcolony were all offspring of a singly mated queen.

amplified fragment length polymorphism / genotyping / stingless bee / genetic polymorphism /Melipona / Apidae

1. INTRODUCTION

Since its introduction over ten years ago(Vos et al., 1995), the methodology for gener-ating amplified fragment length polymorphism(AFLP) markers has become increasingly pop-ular for genotyping and genome mapping. Abrief PubMed survey in 2005 returned over1400 entries, most representing AFLP stud-ies on plants and microorganisms, yet only48 entries on the use AFLP strategies in in-sects. This is surprising considering the ad-vantages of AFLP analysis over traditionalDNA fingerprinting protocols such as RFLP(higher resolution) and RAPD (higher repro-ducibility) (Mueller and Wolfenbarger, 1999;

Corresponding author: G.R. Makert,gustavo@rge.fmrp.usp.br* Manuscript editor: Walter S. Sheppard

Garcia et al., 2004; Nybom, 2004; Benschand Åkesson, 2005). Even though insects aregenerally rather small, it is not the quantityof DNA required for individual analyses thatshould hamper the generation of AFLP mark-ers. Rather, when initiating a study on the stin-gless bee, Melipona quadrifasciata, we noteda series of problems with standard protocolsthat required optimization before reliable andconsistent results could be obtained on geneticvariation within and between colonies.

Stingless bees of the genus Melipona havelong attracted research interest because of theirpeculiar mode of caste determination. Distinctfrom all other highly eusocial bees, the broodcombs of Melipona species lack specific queencells; rather, all male and female brood isreared in cells of similar size, provided withsimilar amounts of larval food (Ihering, 1903).The proportion of queens in the female brood

Article published by EDP Sciences and available at http://www.edpsciences.org/apido or http://dx.doi.org/10.1051/apido:2006043

688 G.R. Makert et al.

is exceptionally high in most Melipona speciesand shows relatively little variation when com-pared to the frequency of male brood (Velthuiset al., 2005). These findings have spurred theformulation of two hypotheses. The first is aproximate explanation for caste determination,which proposes a genetic mechanism of castedetermination. It is based on a two locus-twoallele system where only females that are het-erozygous at both loci can become queens.It was postulated over fifty years ago (Kerr,1946, 1950), but despite a series of attemptsit has never been conclusively confirmed ex-perimentally or genetically. The second is anultimate explanation for caste ratios in thisgenus which proposes that caste determinationmay be modelled on the basis of a potentialconflict of interest over caste fate (Ratnieks,2001). This model, developed using a single-locus inclusive fitness approach, attributes acertain degree of choice over caste fate (selfdetermination) to the developing larvae.

Testing these hypotheses requires screen-ing large numbers of polymorphic loci. Yet,genetic variability within species or evenwithin colonies of stingless bees is still poorlystudied, and different approaches have re-sulted in quite discrepant estimates. While lev-els of polymorphisms were observed to below in isozyme studies (Falcão and Contel,1990; Machado and Contel, 1991), polymor-phism levels of 25–45% were detected forRAPD markers when comparing Meliponaquadrifasciata colonies (Tavares et al., 2001;Waldschmidt et al., 2002). Since RAPD mark-ers are frequently criticized for their notori-ously low levels of reproducibility, we decidedto adopt an AFLP approach to test whetherthis methodology can generate markers of ap-propriate quantity and reproducibility. SinceAFLP combines the advantages of RAPD withthe reliability of RFLP, this seemed like apromising strategy because RFLP mapping ofthe mitochondrial genome of different sting-less bees already indicated high levels of ge-netic diversity among species (Francisco et al.,2001; Weinlich et al., 2004).

We initiated this study with a commer-cially available and frequently employed sys-tem (AFLP Analysis system I, Invitrogen)which has been successfully used in a num-

ber of insect species, such as the gall midgeOrseolia oryzae (Behura et al., 2000; Katiyaret al., 2000), the fruit fly Ceratitis capitata(Corsini et al., 1999), the damselfly Nehalen-nia irene (Wong et al., 2001), the sweet potatowhitefly, Bemisia tabaci (Göçmen and Devran,2002), the dung beetle Onthophagus taurus(Simmons et al., 2004) and also the honeybee, Apis mellifera (Suazo and Hall, 1999;Rueppell et al., 2004). Since we could not ob-tain reproducible results (consistent bands us-ing the same DNA extracts as templates) withthis system in the stingless bee M. quadrifas-ciata, even after testing different strategies ofDNA extraction to obtain DNA of adequatepurity for an insect AFLP study (Reinekeet al., 1998), we decided to go through a com-plete optimization process of the AFLP reac-tions with this species. The detailed descrip-tion of critical steps given here may be of useto overcome similar problems in other prob-lematic species and promote the use of thispowerful methodology in a wider range of beespecies.

2. MATERIALS AND METHODS

2.1. Bee samples

We collected a total of 580 adults of the stin-gless bee Melipona quadrifasciata Lepeletier fromthree colonies kept in the apiary of the Departmentof Genetics on the campus of the University of SãoPaulo at Ribeirão Preto over a period of one year.Brood combs containing brood in the late pupalstages were removed from the colonies and werekept in Petri dishes in a temperature controlled en-vironment (28 ◦C). Bees emerging from the combswere sorted according to sex and caste and weresnap-frozen individually in liquid nitrogen. Subse-quently they were stored in 99% ethanol at –20 ◦C.

2.2. DNA extraction

DNA was extracted from the thoraces of individ-uals using a high-salt extraction protocol (Paxtonet al., 1996). Per bee we used 350 µL SET buffer(0.15 M NaCl, 0.02 M Tris, 1.0 mM EDTA pH 8.0),supplemented with 6.5 µL proteinase K (20 mg/mL)and 15.5 µL SDS (25%) for digestion at 55 ◦Cfor 2 h. Subsequently, 300 µL of 6 M NaCl were

Stingless bee AFLP markers 689

added and the samples were vortexed and cen-trifuged at 15 000 g for 10 min. The supernatantcontaining DNA (550 µL) was transferred to a newtube with 150 µL 10 mM Tris-HCl (pH 8.0) towhich 1.4 mL ice-cold absolute ethanol was added.This was stored overnight at –20 ◦C to precipitatethe DNA, then centrifuged at 12 000 g for 15 minto pellet the DNA. The pellet was rinsed in 70%ethanol, vacuum dried and dissolved in 100 µL dis-tilled water. This extraction yielded approximately5 µg DNA per bee, and 1 µL of this DNA (approx-imately 50 ng) was sufficient for each AFLP reac-tion.

2.3. Two-step restriction enzymedigestion, adapter primer ligationand amplification

Instead of the frequently used single-step diges-tion with restriction enzymes, we performed a two-step digestion. In the first reaction, the EcoRI di-gestion, 1 µL DNA was added to 20 µL Y+/Tangobuffer (Fermentas) with 0.5 U EcoRI enzyme (Fer-mentas). This mixture was incubated for 30 min at37 ◦C. For the second digestion step, 0.5 U Tru1Ienzyme (Fermentas) in 1 µL Y+/Tango buffer wasadded to the reaction mixture of the first digestionstep and rapidly heated to 65 ◦C for 30 min. Atthis temperature, EcoRI is inactivated and Tru1I hasits optimal activity. For ligation of adaptor primers(all primers from Sigma-Genosys) to the restric-tion enzyme cleavage sites, we added to digestionproducts (21 µL) a mixture (4 µL) of 2.5 pmolEcoRI adapter and 25 pmol Tru1I-adapter (Tab. I)with 0.5 U T4 DNA-ligase (Fermentas) in T4 lig-ation buffer. These core primers are complemen-tary to the EcoRI and Tru1I (MseI) adaptor oligonu-cleotides and have one additional 3’ nucleotide (Aor C respectively). The ligation reaction mix was in-cubated at 22 ◦C for 3 h. After ligation, the reactionmixture was diluted with 5 volumes of ddH2O andstored at –20 ◦C.

The amplification protocol followed the originaltwo-step amplification strategy (Vos et al., 1995).For the preamplification reaction, 60 µM dNTPs,25 ng of each preamplification primer (Tab. I),200 µM spermidine and 0.5 U Taq DNA polymerasein 10× reaction buffer (Finnzymes) were brought toa volume of 11.5 µL before adding 1 µL of the lig-ation reaction as template. The preamplification re-action commenced with a 10 min denaturation step(95 ◦C), followed by a 30 cycle program (denatura-

tion 30 s at 95 ◦C, annealing 60 s at 56 ◦C and ex-tension 60 s at 72 ◦C) and terminating with a 20 minextension step at 72 ◦C. For the second (selective)amplification, the products of the preamplificationreaction were diluted with 20 volumes of ddH2O.

The second (selective) amplification reactionswere carried out in a total volume of 10 µL byadding 1 µL of the diluted preamplifiction reactionto a mix (9 µL) of 1 µL 10× amplification buffer(same as preamplification), 6 µM dATP and 200 µMeach of dCTP, dGTP and dTTP, 25 ng of one ofthe EcoRI and 25 ng of one of the Tru1I amplifi-cation primers (Tab. I), 200 µM spermidine, 0.5 UTaq DNA polymerase and 1.25 µCi α[33P]dATP.The amplification reaction protocol started with a10 min denaturation step (95 ◦C), followed by atouchdown cycle profile (45 cycles total), and ter-minated with a 20 min extension step at 72 ◦C. Thetouchdown profile (denaturation 30 s at 95 ◦C, an-nealing 60 s and extension 60 s at 72 ◦C) startedwith an annealing temperature of 65 ◦C which wasgradually reduced by 0.7 ◦C in each of the follow-ing 12 cycles. In the final 33 cycles, the annealingtemperature was kept constant at 56 ◦C. All amplifi-cation reactions were performed in an MJ ResearchPTC-100 thermal cycler.

2.4. Electrophoresis, autoradiographyand analysis of AFLP bands

The amplification products of each reaction(10 µL) were mixed with 7 µL stop solution (20 mMEDTA; 0.1% xylene cyanol; 0.1% bromophenolblue; 96% formamide) and denatured for 5 min at96 ◦C before they were electrophoretically sepa-rated under denaturing conditions (ca. 50 ◦C) on a43 cm long sequencing gel (8 M urea; 6% polyacry-lamide). As marker for comparison between gelsand as internal standard to monitor the amplificationreactions, we used a single DNA sample amplifiedby the primer combination EcoRI-aac/Tru1I-cta.The gels were dried for 2 h at 80 ◦C before expo-sure to X-ray film (Kodak Biomax) for 3 days.

In the 25 samples per colony of Melipona workerDNA that were run in parallel on each film, wecounted all clearly visible bands and scored themas total bands per primer combination. In a sec-ond round we scored the percentage of polymorphicmarkers for each primer set and colony. By pairwisecomparisons between the samples from the threecolonies we next ascertained in a cumulative man-ner the total number of bands and the number of

690 G.R. Makert et al.

Table I. Oligonucleotide adapters and primers.

Oligonucleotide SequenceEcoRI adapter forward 5’-CTCGTAGACTGCGTACC-3’EcoRI adapter reverse 5’-AATTGGTACGCAGTCTAC-3’Tru1I adapter forward 5’-GACGATGAGTCCTGAG-3’Tru1I adapter reverse 5’-TACTCAGGACTCAT-3’preamplification EcoRI-a 5’-GACTGCGTACCAATTCA-3’preamplification Tru1I-c 5’-GATGAGTCCTGAGTAAC-3’amplification EcoRI-aag 5’-GACTGCGTACCAATTCAAG-3’amplification EcoRI-aac 5’-GACTGCGTACCAATTCAAC-3’amplification Tru1I-cta 5’-GATGAGTCCTGAGTAACTA-3’amplification Tru1I-cat 5’-GATGAGTCCTGAGTAACAT-3’

polymorphic bands that could be obtained by eachprimer combination.

2.5. Microsatellite analysis

For the analysis of microsatellite loci in the threecolonies of Melipona quadrifasciata, we used 50DNA samples of workers that were also used in theAFLP analyses. Of the microsatellite primers de-veloped for Melipona bicolor (Peters et al., 1999),Mbi105 and Mbi233 were the ones that gave thebest results with the M. quadrifasciata samples, interms of polymorphism and band resolution. Theamplification reactions were carried out in a totalvolume of 25 µL, with PCR-Master Mix (Promega)and 400 nmol of Mbi105 or Mbi233 primer and1 µL of template DNA under the following condi-tions: 94 ◦C for 3 min, 40 cycles of 94 ◦C for 30 s,52 ◦C for 30 s and 72 ◦C for 60 s, followed by a fi-nal extension at 72 ◦C for 10 min. The amplificationproducts were separated by electrophoresis in 6%polyacrylamide gels and revealed by silver staining(Sanguinetti et al., 1994).

3. RESULTS

3.1. AFLP Protocol optimization

Since our initial attempts with a commer-cial AFLP kit did not give satisfactory resultswith DNA extracted from workers of the sting-less bee M. quadrifasciata because bands gen-erated by the same DNA template were notreproducible across AFLP trials,we system-atically tested different DNA extraction pro-cedures, restriction enzyme digestion proto-cols, and amplification and product labelling

protocols. The final working protocol for thisspecies is detailed in Section 2: Materials andMethods. Typical results of the series of adap-tations and optimization steps are presented inFigure 1.

The first step in the optimization pro-cess was the choice of the DNA extractionmethod. We initially tested a modified phe-nol/chloroform extraction that includes a pro-teinase K digestion step and addition of SDS.This method has been tested for genomic DNAanalyses of stingless bees (Waldschmidt et al.,1997). Next, we tested an extraction protocolthat uses a high salt (6 M NaCl)/ethanol pre-cipitation of DNA in combination with SDSand a proteinase K digestion step. However,when used in conjunction with the commercialAFLP kit, we did not obtain satisfactory re-sults with any these protocols (Fig. 1A), eventhough all these methods resulted in high qual-ity DNA. With the high-salt extraction method,which we finally adopted because of its ease ofuse, we obtained approximately 5 µg DNA perbee thorax.

Consequently, we decided to redesign theAFLP protocol itself, and this involved a se-ries of sequential modifications. We had no-ticed that AFLP protocols with insect genomicDNA frequently substituted MseI, the enzymeused in the original protocol in conjunctionwith EcoRI (Vos et al., 1995), for another re-striction enzyme. Since we did not want tosubstitute the AFLP adaptor and primer com-binations, we decided to test the efficiency ofthe restriction enzyme Tru1I in combinationwith EcoRI. Tru1I cuts at the same recognitionsite as MseI, but has a much higher optimum

Stingless bee AFLP markers 691

Figure 1. Results of optimization stepsin the AFLP protocol for the stinglessbee, Melipona quadrifasciata. All DNA ex-tractions on worker bees were performedwith a high-salt buffer, SDS and pro-teinase K digestion. Fragments of diges-tion reactions were labelled by incorpora-tion of α[33P]dATP and resolved on se-quencing gels under denaturing conditions.(A) single-step digestion with EcoRI andMseI and two-step amplification with AFLPprimers (Invitrogen) performed with differ-ent mixes of dNTP and varying concen-trations of primers (0.3–1.0 ng/µL), shownin lanes 1–9; (B) two-step digestion withEcoRI and Tru1I followed by a two-step am-plification with two sets of AFLP primers(lanes 1–8 EcoRI-aag/Tru1I-cat, lanes 9–16 EcoRI-aag/Tru1I-cta; these primers hadthe same sequence as those shown in Fig-ure 1A); (C) digestion and the first roundof amplification was performed as in Fig-ure 1B, but the second, selective amplifica-tion followed a touchdown protocol. The 19samples were analyzed with primer combi-nation EcoRI-aag/Tru1I-cat.

temperature than MseI and EcoRI, thus re-quiring a two-step digestion protocol. Whileneeding more pipetting steps, our two-stepprotocol could be performed in two 30 minincubations, whereas the usual double digestperformed in a single-step in most AFLP pro-tocols is much more time consuming, requir-ing an incubation for 2–3 h (Vos et al., 1995;Behura et al., 2000; Simmons et al., 2004). Aninitial typical result of the two-step digestionprotocol performed on DNA extracted with thehigh-salt protocol is illustrated in Figure 1B. Itshows a clear improvement when compared toFigure 1A, in terms of the number of score-able bands. Yet, the markers were still too in-consistent, appearing in some reactions but notothers using the same DNA template.

The refinement of the banding pattern intoa high resolution and consistent AFLP profilefor individual M. quadrifasciata workers wasfinally achieved by optimization of the am-plification conditions into a touchdown proto-

col (Fig. 1C). The preamplification step is aPCR with core primers that are complemen-tary to the adapter sequence, plus one addi-tional nucleotide at the 3’ end (Forward PrimerEcoRI-a; Reverse Primer Tru1I-c). The sec-ond amplification round uses longer selectiveadapter primers that have three overhangingnucleotides at their 3’ end. Starting this sec-ond PCR round with a Tm of 65 ◦C and gradu-ally dropping the Tm to 56 ◦C in a touchdownprotocol allowed for higher stringency in theamplification reactions. As detection method,we opted for radioactive labelling of amplifica-tion products by incorporation of α[33P]dATPduring elongation. This direct labelling is fre-quently used in microsatellite analyses (Paxtonet al., 1996) and has also been employed inAFLP fingerprinting (Katiyar et al., 2000). Iteliminates the primer labelling step required inmost radioactive PCR-based genotyping pro-tocols. This direct labelling protocol may sub-tly affect band intensity in the autoradiographs

692 G.R. Makert et al.

since the degree of α[33P]dATP incorpora-tion will depend on the AT-content of thesynthesised fragments, though the effect islikely to be minor.

3.2. AFLP fingerprinting in Meliponabees

On the basis of the optimized protocol wecould consistently amplify DNA samples ofM. quadrifasciata workers that had emergedfrom brood combs of three colonies. Samplingbees as they emerged from the brood combscontrolled for genetic origin, as drifting ofadult workers among colonies can be a fre-quent phenomenon in these bees (Palmer et al.,2002). The results for the primer pair EcoRI-aag and Tru1I-cat are shown in Figure 2 for aset of 25 workers from each colony, illustrat-ing the large number of scorable markers ob-tained with this protocol. In addition, it illus-trates that most polymorphic markers fall intothe median gel region (fragments from 100to 1000 bp), which has excellent resolution.The larger markers (fragments > 1000 bp) areoften represented as relatively weak bands,whereas the lower region of the gel (frag-ments < 100 bp) contains mainly monomor-phic markers. Table II lists the total number ofAFLP bands and the percentage that are poly-morphic, detected by the four combinations ofthe two specific amplification primers. Fromthese we calculated the percentage of poly-morphic markers within each colony and thepercentage overall polymorphism. A notableresult is that with only four primer combina-tions we could score a mean of 630 markersfor each M. quadrifasciata colony. Across allprimer combinations for all colonies, 44% ofmarkers were polymorphic. This figure repre-sents the overall polymorphism for this set ofcolonies, whereas intracolony polymorphismvaried between 27 and 38%. Intracolony poly-morphism thus accounts for most of the ge-netic variation in this dataset. Nevertheless,there is still additional variation that can be de-tected when comparing rates of genetic poly-morphism within colonies (27–38%) to the ob-served overall polymorphism (χ2, 22.85/2, P <0.01). This genetic variation at the population

level is not unexpected and is probably due tothe mating of virgin queens with geneticallyunrelated males.

In contrast, the high degree of geneticvariation among females collected from thesame M. quadrifasciata colony was a surpris-ing result if we consider that these coloniesshould have originated from a single queenthat had mated with a single male. To con-firm monandry and monogyny, we genotyped50 workers by analysis of two microsatelliteloci (Mbi105 and Mbi233). These showed al-lele patterns that were consistent with monog-yny and monandry (data not shown), indicat-ing that workers were full sisters.

4. DISCUSSION

4.1. Optimization steps in the AFLPprotocol for Meliponaquadrifasciata

High quality genomic DNA is a general de-mand in AFLP protocols and has proven crit-ical in AFLP reactions with different insectspecies (Reineke et al., 1998). Generally, thesemethods use CTAB and a phenol/chloroformextraction protocol. We modified this becauseprevious studies describing DNA extractionprocedures for stingless bees have shown theimportance of a step incorporating proteinaseK digestion and the substitution of CTAB bySDS (Waldschmidt et al., 1997) before thephenol/chloroform extraction. In addition, wetested a high-salt DNA extraction procedurethat is frequently employed in microsatellitestudies on bees (Paxton et al., 1996). Since wedid not obtain satisfactory results when usingany of these extracts as DNA templates in con-junction with a commercial AFLP kit we con-cluded that the problems were more likely inthe digestion and/or amplification steps, andnot because of poor quality of extracted DNA.We decided to opt for the high-salt DNA ex-traction protocol because it is well-suited fora high-throughput protocol due to its relativesimplicity and the low toxicity of chemicalsemployed.

The combination of restriction enzymesthat has been explored and successfully stan-dardized for AFLP analyses in plants and also

Stingless bee AFLP markers 693

Figure 2. AFLP results for workers sampledfrom three colonies of Melipona quadrifas-ciata. Amplification reactions shown in thispanel were performed with primer combina-tion EcoRI-aag/Tru1I-cat. The marker (M)shown in the panel for colony 48 was al-ways generated as an internal marker by am-plification of digested DNA for the samebee with the primer combination EcoRI-aac/Tru1I-cta. In the electrophoresis runs itpermitted consistent assignment of bandsacross gels.

Table II. AFLP markers scored for three Melipona quadrifasciata colonies with four sets of primers. Foreach colony, 25 workers were sampled and analyzed. The total number of polymorphic markers for eachcolony (last column) represents estimates for intracolony genetic polymorphism, whereas the total of poly-morphic markers for all colonies, obtained from a cumulative marker analysis of the three colonies (lastline) gives an estimate of overall genetic polymorphism for this species.

primer set 1 primer set 2 primer set 3 primer set 4 all primerscolony 21

n workers 25 25 25 25 100total # bands 157 149 156 123 585polymorphic 48 36 43 31 158% polymorphic 27.0

colony 48n workers 25 25 25 25 100total # bands 160 148 175 171 654polymorphic 71 56 72 51 250% polymorphic 38.2

colony 3n workers 25 25 25 25 100total # bands 158 151 168 190 667polymorphic 54 52 54 47 207% polymorphic 31.0

all coloniesn workers 75 75 75 75 300total # bands 168 171 185 195 719polymorphic 88 72 87 69 316% polymorphic 44.0

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most animals is EcoRI with MseI. Insects seemto be an exception in this respect, since evenwithin the relatively few technical reports oninsect species we found descriptions of twonew enzyme combinations, EcoRI and PstI inthe potato beetle (Hawthorne, 2001) and PstIand TaqI in the silkworm (Tan et al., 2001).This suggests that the choice of restriction en-zymes is an important step that demands at-tention in the establishment of AFLP proto-cols with insect species. In our experimentswe substituted Tru1I for MseI in a two-stepdigestion and amplified the fragments in atouchdown-PCR protocol. With these modi-fications we obtained reproducible AFLP re-sults with amounts of DNA that were at least10 times lower than those used in most otherstudies on insects (Corsini et al., 1999; Behuraet al., 2000; Hawthorne, 2001).

An interesting question to be asked in thiscontext, is why AFLP fingerprinting and QTLmapping performed in a single digestion stepwith EcoRI and MseI worked well for Apiscerana, A. nigrocincta (Smith et al., 2003)and A. mellifera (Arechavaleta-Velasco andHunt, 2004; Rueppell et al., 2004), but not forthe closely related stingless bees that we in-vestigated. The frequent substitution of MseIin restriction enzyme combinations for AFLPfingerprinting in insect species (Hawthorne,2001; Tan et al., 2001) may be related to dif-fering base composition of their genomes, es-pecially AT-richness, and thus the probabilityof recognition site frequency. For the presentcase, however, this argument should not applysince MseI and Tru1I differ in temperature op-tima but not in restriction site. An interestingalternative to the protocol we present in thisstudy could be a three-enzyme digestion sys-tem (Smith et al., 2003).

4.2. Genetic polymorphism in Meliponaquadrifasciata

The degree of overall genetic polymor-phism detected by AFLP analysis in ourdataset is similar to that observed in previ-ous studies that employed RAPD approacheson either drones of M. quadrifasciata (Tavareset al., 2001), or a much larger number of

colonies collected from a wide geographicalrange (Waldschmidt et al., 2002). Not surpris-ingly, all these DNA-based dominant markersystems revealed higher degrees of geneticpolymorphism than isozyme studies that onlyexamined one or few loci (Falcão and Contel,1990; Machado and Contel, 1991). Our resultsbased on an AFLP methodology confirm theRAPD results at the population level. In addi-tion, they show that a large part of this geneticvariability is in fact due to an unexpectedlyhigh level of polymorphism within colonies.

The high genetic variability that we ob-served within colonies of M. quadrifasciataposes an interesting problem that needs to bediscussed at two levels. The first one is re-lated to the mechanism(s) that may generatesuch genetic variation, and the second oneconcerns its population genetic implications.In relation to the first aspect, the high de-gree of polymorphism suggests that each lo-cus may have several allelic states (maximum3 per colony) with differing dominance rela-tions among them and/or that some loci exhibitcodominant rather than dominant inheritance.AFLP bands are generally treated as domi-nant markers, but careful quantitative analysishas also revealed evidence for codominance(Piepho and Koch, 2000).

An alternative (or additional) mechanismthat could account for the observed within-colony genetic variation would be a high re-combination frequency. A much elevated re-combination frequency was found in the honeybee, Apis mellifera (Hunt and Page, 1995;Beye et al., 1999), exceeding by a factor offive the recombination frequencies in otherHymenoptera, including a bumble bee, Bom-bus terrestris (Gadau et al., 2001). Such highrecombination frequencies are possibly re-lated to levels of social organization. Particu-larly at the transition from primitively eusocial(bumble bees) to highly eusocial organization(honey bees) an increase in recombination fre-quencies could produce a genetically more di-verse worker force and might reduce suscepti-bility to pathogen attacks (Gadau et al., 2001).

Maintaining a genetically diverse workerpopulation may be a much more severeproblem for stingless bees than for the honeybee with its multiple mating system and a

Stingless bee AFLP markers 695

swarming process that disperses genotypesover a relatively large area. With the excep-tion of the facultatively polygynous speciesMelipona bicolor (Nogueira-Neto, 1997;Velthuis et al., 2006), all stingless beescolonies are headed by a single queen and inmost cases, these queens had mated with asingle male only (Paxton et al., 1999; Peterset al., 1999). Furthermore, the swarmingprocess in stingless bees is not an abruptfission between the mother and daughtercolonies, but rather occurs over a prolongedperiod. It involves intense traffic of workersand frequent transport of materials betweenthe old nest and the new site (Engels andImperatiz-Fonseca, 1990; Nogueira-Neto,1997), and therefore dispersal by swarmingis slow and spatially restricted. The obviousconsequences for small populations would bea decrease in genetic diversity if there wereno counteracting mechanisms to maintainit. The significant contribution of workersto male production in many stingless beespecies (Tóth et al., 2002, 2004; Paxtonet al., 2003; Velthuis, 2005) in conjunctionwith a high recombination frequency couldrepresent mechanims that allow maintenanceof sufficiently high genetic diversity in evenrelatively small populations of stingless bees.Even though high recombination rates haveso far only been demonstrated for honey bees(Hunt and Page, 1995; Gadau et al., 2001),studies on karyotype (Rocha et al., 2002) andmitochondrial genome evolution (Weinlichet al., 2004; Arias et al., 2006) suggest thatstingless bees could harbor rather flexiblegenomes, as well.

In conclusion, we describe an optimizedand efficient protocol for AFLP fingerprintingof a highly eusocial bee. This protocol primar-ily differs from the original one (Vos et al.,1995) in using a two-step restriction enzymedigestion with EcoRI and Tru1I. The largenumber of markers obtained in an analysis ofworker bees from three colonies of the sting-less bee M. quadrifasciata makes plain the po-tential of this optimized AFLP protocol in ad-vanced genetic studies on stingless bees, espe-cially for high resolution analyses as requiredfor linkage analyses. Such linkage analysis onlarge numbers of queens, workers and males

collected from single colonies should revealwhether or not and how many loci segregatewith caste phenotype, thus allowing one to ad-dress the long-standing question of a geneticpredisposition to caste in the genus Melipona.

In addition, AFLP-based linkage maps mayprovide a means to investigate the proposedassociation between levels of eusociality andrecombination rates. Stingless bees are aninteresting group for exploring this associa-tion. They are closely related to honey bees(Michener, 2000; Engel, 2001) and have at-tained a similar level of social organization.But they differ from honey bees in their so-ciogenetic colony structure since the workerscomprising a stingless bee colony are gener-ally all full sisters, produced by a singly matedqueen (Paxton et al., 1999; Peters et al., 1999;Paxton, 2000; Palmer et al., 2002).

ACKNOWLEDGEMENTS

We would like to thank Wolf Engels and ThomasHaug for hosting parts of this project at the Univer-sity of Tübingen, Germany, Paulo Nogueira Netofor providing colonies used in this study, and toWeyder Santana for assistance in the collection ofbees. Valuable comments on a previous versionof the manuscript were made by Deborah Smith.This research was supported by a DAAD/CAPESdoctoral fellowship to GRM and a grant from theDeutsche Forschungsgemeinschaft to RJP.

Résumé – Une méthode optimisée pour générerdes marqueurs AFLP chez une abeille sans ai-guillon (Melipona quadrifasciata) révèle un fortdegré de polymorphisme génétique intra co-lonie. Les études de génétique des populationset de cartographie des liaisons génétiques néces-sitent la production d’une grande quantité de mar-queurs polymorphiques exploitables avec fiabilité.La méthode de l’AFLP (polymorphisme de lon-gueur du fragment de restriction) peut générer ra-pidement des centaines de marqueurs reproduc-tibles, permettant ainsi de caractériser le génotypeavec une résolution élevée à des coûts relative-ment bas et une efficacité en temps élevée. Ellea été largement appliquée à la caractérisation dessouches bactériennes et des cultivars de plantes ettrouve maintenant son application dans le génoty-page animal. Les espèces d’insectes représententsouvent un défi pour cette technique, car elles re-quièrent fréquemment des étapes d’optimisationdans le protocole AFLP. Notre étude présente un tel

696 G.R. Makert et al.

protocole optimisé pour une abeille sans aiguillon,Melipona quadrifasciata Lepeletier. Les modifica-tions les plus importantes apportées aux protocolesstandards comportent une digestion de l’enzyme derestriction (EcoRI et Tru1I) dans une procédure endeux temps, combinée avec un programme « touch-down » (diminution progressive de la températured’hybridation des amorces) à l’étape d’amplifica-tion sélective par PCR et le marquage du produitpar l’incorporation d’α[33P]dATP (Fig. 1).Lors d’une analyse de 75 ouvrières prélevées dans3 colonies de M. quadrifasciata, nous avons ob-tenu un total de 719 marqueurs (Fig. 2). L’ana-lyse de la variabilité génétique a révélé un poly-morphisme moyen des marqueurs intra-colonie de32 % (Tab. II). Comparé au pourcentage global desmarqueurs polymorphiques (44 %) détecté dans noséchantillons d’abeilles, les taux observés de poly-morphisme intra-colonie sont remarquablement éle-vés, étant donné que les ouvrières d’une coloniedescendent toutes d’une même reine fécondée unefois. Nous considérons que cela peut être dû à untaux élevé de recombinaisons, semblable à celui ob-servé chez l’Abeille domestique (Apis mellifera L.).En raison de sa grande efficacité le protocole opti-misé peut fournir un outil intéressant pour générerdes cartes de liaisons génétiques pour les abeillessans aiguillon, par exemple pour des études compa-ratives d’organisation du génome. En outre l’ana-lyse de la ségrégation des marqueurs AFLP par lephénotype de caste peut fournir un outil pour abor-der de manière empirique l’hypothèse de longuedate et fort débattue de la prédisposition génétiquedes castes chez ce genre d’abeilles sans aiguillon.

Melipona / abeille sans aiguillon / génotypage /polymorphisme génétique / AFLP / Apidae

Zusammenfassung – Die Optimierung einerAFLP-Marker-Methode für Stachellose Bienedeckt einen hohen genetischen Polymorphis-mus in Völkern von Melipona quadrifasciataauf. Populationsgenetische Studien und Untersu-chungen zur genetischen Kopplung von Loci erfor-dern die Erstellung einer grossen Anzahl zuverläs-sig auswertbarer Marker. Die Amplifizierte Frag-mentlängen Polymorphismus (AFLP)-Methode istin der Lage, innerhalb kurzer Zeit hunderte repro-duzierbarer Marker zu produzieren und somit ei-ne hochauflösende Genotypierung bei verhältnis-mässig geringen Kosten und hoher Zeiteffizienzzu leisten. Diese Methode wird inzwischen häufigzur Charakterisierung von Bakterienstämmen undpflanzlichen Kultivaren eingesetzt und findet nunauch häufiger Anwendung in der Genotypierungbei Tieren. Insekten stellen in dieser Hinsicht of-fensichtlich eine Herausforderung dar, da bei ihnendiese Methode oft umfassende Optimierungen inden Versuchsprotokollen erfordert. Die vorliegendeUntersuchung stellt ein optimiertes Protokoll für ei-

ne Stachellose Biene, Melipona quadrifasciata, vor.Die wichtigsten Veränderungen gegenüber Stan-dardprotokollen sind der Restriktionsenzymverdau(EcoRI und Tru1I) in zwei Schritten, kombiniert miteinem Touchdown-Programm im selektiven PCR-Amplifizierungsschritt und der Produktmarkierungdurch den Einbau von α[33P]dATP (Abb. 1).In einer Untersuchung, die mit 75 Arbeiterinnenaus drei M. quadrifasciata Völkern durchgeführtwurde erzielten wir insgesamt 719 Markerbanden(Abb. 2), wobei durchschnittlich 32 % dieser Mar-ker einen genetischen Polymorphismus innerhalbder jeweiligen Kolonie zeigten (Tab. II). Vergli-chen mit dem Gesamtpolymorphismus von 44 %zwischen den drei Völken war der Anteil poly-morpher Marker innerhalb der einzelnen Völkervergleichsweise hoch, insbesondere wenn man be-trachtet, dass die Königinnen, die diese Arbeiterin-nen produzierten, mit jeweils nur einem Drohn ver-paart waren. Eine Erklärung hierfür könnte eine mitder Honigbiene vergleichbare hohe Rekombinati-onsrate darstellen.Durch seine hohe Effizienz bietet sich dieses op-timierte Protokoll auch für vergleichende Studienzur Genomorganisation an, z.B. zur Erstellung vonKopplungskarten des Genoms Stachelloser Bienen.Ausserdem kann die Analyse der Segregation ein-zelner Marker mit dem Kastenphänotyp eventuellein Werkzeug darstellen, die kontrovers diskutiertegenetische Basis der Kastendetermination bei die-ser Gattung Stachelloser Bienen aufzuklären.

Amplifizierter Fragmentlängen Polymorphis-mus / Genotypierung / Stachellose Biene / gene-tischer Polymorphismus /Melipona / Apidae

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