Isolation and characterization of microsatellites from Scaptotrigona xanthotricha (Apidae, Meliponini): a stingless bee in the Brazilian Atlantic rainforest Olívia Duarte, Fernanda Gaiotto, Anete Souza, Gustavo Mori, Marco Costa
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Olívia Duarte, Fernanda Gaiotto, Anete Souza, Gustavo Mori, Marco Costa. Isolation and character- ization of microsatellites from Scaptotrigona xanthotricha (Apidae, Meliponini): a stingless bee in the Brazilian Atlantic rainforest. Apidologie, Springer Verlag, 2012, 43 (4), pp.432-435. 10.1007/s13592- 011-0109-6. hal-01003532
HAL Id: hal-01003532 https://hal.archives-ouvertes.fr/hal-01003532 Submitted on 1 Jan 2012
HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2012) 43:432–435 Scientific note * INRA, DIB and Springer-Verlag, France, 2011 DOI: 10.1007/s13592-011-0109-6
Isolation and characterization of microsatellites from Scaptotrigona xanthotricha (Apidae, Meliponini): a stingless bee in the Brazilian Atlantic rainforest
1 1 2 2 Olívia Maria Pereira DUARTE , Fernanda Amato GAIOTTO , Anete Pereira SOUZA , Gustavo Maruyama MORI , 1 Marco Antônio COSTA
1Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Rodavia Ilhéus-Itabuna, Km 16, Ilhéus, Bahia CEP 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 they (Lopes et al. 2010), Melipona bicolor (Peters et al. use odors in marking trails to distant food sources. 1998), and Scaptotrigona postica (Paxton et al. They build their nests in cavities of large living trees 1999), among others. However, these studies are making them susceptible to anthropic actions, such as still scarce when compared to the high diversity deforestation. Due to the reduction in population size within this group. The present study aimed at the as a result of fragmentation and destruction of natural identification and characterization of microsatellite habitat, many species are threatened with extinction loci for S. xanthotricha. (Freitas et al. 2009). Scaptotrigona comprises 30 An enriched genomic library was constructed from a Neotropical species, out of which 18 have been pool of DNA from 30 individuals of the same colony recorded in Brazil (Moure et al. 2007). This genus following Billotte et al. (1999). DNA extraction still harbors many undescribed species and cryptic followed Han and McPheron (1997). Five micrograms species complexes (Silveira et al. 2002). of DNA (300 ng/μl) was digested with RsaI and linked Scaptotrigona xanthotricha shows a wide distribu- to RsaI adapters. The library was enriched for (CT) tion in the Atlantic rainforest ranging from southern to 8 the northeastern Brazil (Silveira et al. 2002). Duarte et and (GT)8 repeats using biotinylated probes attached to al. (2009) reported a remarkable geographical variation streptavidin-labeled magnetic beads as described by in the number and location of the Nucleolus Organizer the manufacturer (MagneSphere Paramagnetic Par- Regions in this species. This finding lent support to the ticles, Promega). Selected fragments amplified by existence of interpopulational divergence or even polymerase chain reaction (PCR) using primers with additional taxa, thus reinforcing the need for more sequences complementary to the adapters and inserted detailed assessment of this group using molecular into the plasmid pGEM-T (Promega) were transformed markers. into competent XL-1 Blue Escherichia coli cells. Once Studies on the genetic diversity and population most of the colonies were transformed, 96 were structure of stingless bees have become frequent selected for long-term storage at −80°Cintoamicro- due to the development of microsatellite markers for several species such as Melipona mondury plate. Forty-eight positive clones were sequenced on an automated sequencer ABI PRISM 377 (Applied Biosystems) using SP6 and T7 primers and the Big Corresponding author: M.A. Costa, [email protected] Dye terminator kit v3.1 (Applied Biosystems). A total Manuscript editor: James Nieh of 32 sequences contained microsatellite regions. Table I. Microsatellite loci developed for S. xanthotricha.
Locus Sequence Repeat motif Ta(°C) Range NA PIC HE HO FIS PNExc I
Sxant 01 F 5′-GTTGCGTATAGCCGGTCGTG-3′ CT (11) 56.7 322 ––––––– R5′-CGCTCCGTGAATGACAGTCC-3′ Sxant 05 F 5′-GGAGAACCGACGACGAAGTG-3′ CT (22) 62 220–242 7 0.745 0.787 0.806 −0.023 0.603 0.082 R5′-TAGTGCCGGCTGGATCATTA-3′ Sxant06 F 5′-GGACGAACGAGAGAGAGA-3′ AG (16) 61.8 151–173 10 0.834 0.860 0.944 −0.098 0.459 0.038 R5′-GGCAAGGATCGTCAGTAT-3′ irstlie for Microsatellites Sxant10 F 5′-GATCGATGCACGAAGAGTTG-3′ AG(20) 61.8 250–300 9 0.814 0.850 0.636 0.252 0.497 0.048 R5′-TTGCGTCGAGGTTACCTTG-3′ Sxant13 F 5′-ATCATGGCGACCGAGAAGAA-3′ GT (15) 61.8 172–186 9 0.748 0.789 0.806 −0.021 0.593 0.079 R5′-ACGAGCATCAGGATGGTTGG-3′ Sxant14 F 5′-CGTAATAGGACGATGTCGTG-3′ GA (12) 65 162–186 14 0.862 0.886 0.970 −0.095 0.398 0.027 R5′-TCATCTGGACGCGGACACA-3′ Sxant15 F 5′-GCGCACGACATCGTCCTATT-3′ GT(12) 54 440–480 2 0.375 0.510 0.333 0.346 0.875 0.375 xanthotricha Scaptotrigona R5′-CGTCAGCCAATCTTGAACTC-3′ Sxant18 F 5′-CCAAGATCTGTCATGCGTG-3′ GA (23) 58 142–186 19 0.927 0.946 0.912 0.036 0.244 0.009 R5′-CAGACGTTACCTGGACGAA-3′ Sxant 19 F5′-TGTCGGTAACGCTATCAGTC-3′ CT (25) 51 510–556 11 0.858 0.887 0.636 0.283 0.408 0.029 R5′-TCGATTCTGTCGGTCTGTC-3′ Sxant20 F 5′-AAGAGAGCAAGAGCGAGAG-3′ CT (26) 56.7 328–376 10 0.854 0.884 0.639 0.277 0.423 0.032 R5′-ACGTGTGTTACAGGCTACAG-3′ Sxant21 F 5′-AGCGGTGGTGATCCTTACGA-3′ AC (14) 64.6 243–260 6 0.575 0.646 0.778 −0.205 0.774 0.195 R5′-CTGCCACCGTGTTAGCCTCT-3′ Sxant23 F 5′-GAACATACGTGTGCATATAC-3′ CT (25) 48.2 370–426 8 0.820 0.857 0.543 0.366 0.492 0.046 R5′-GTACTTGAGCCTAACTGATT-3′ Sxant24 F 5′-TGAAGACGAGAACAATCATC-3′ AG (22) 50.5 150–178 10 0.800 0.836 0.706 0.156 0.520 0.054 R5′-TTACAGCAGCCTTACCAATA-3′ Mean 9.58 0.768 0.81 0.726 –––
Ta annealing temperature, NA allele number, PIC polymorphism information content, HE expected heterozygosity, HO observed heterozygosity, FIS fixation index, PNExc first-parent non-exclusion probability, I non-exclusion probability of identity 433 434 O.M.P. Duarte et al.
Primers for suitable flanking regions of 23 micro- loci were polymorphic, except for Sxant01. The satellites were designed using PRIMER version 2.0 present results suggest that these new microsatellites (Lincoln et al. 1991). Amplifications were performed will be very useful for analyzing the genetic diversity in 25 μl reaction containing 2.5 ng of genomic DNA, and population structure of stingless bees, especially those within the genus Scaptotrigona. 1× Buffer KCl, 7 mM of each primer, 2 mM of each dNTP, 0.03 mM MgCl2, and 1 U of Taq DNA polymerase. The PCR conditions were: 94°C for ACKNOWLEDGMENTS 1 min, followed by 35 cycles of 94°C for 1 min, specific annealing temperature (Table I) for 1 min, 72°C We thank Lucio A. O. Campos for providing for 1 min, and a final extension at 72°C for 5 min. samples of S. xanthotricha and Gabriel A. R. Melo Thirteen loci (GenBank accession numbers JN119815– for the species identification. This research was JN119826) that had the correct size were analyzed for supported by Coordenação de Aperfeiçoamento de — polymorphism in a sample of 36 workers from 18 Pessoal de Nível Superior (CAPES PROCAD NF 2567/2008). O. M. P. Duarte also received a doctoral colonies of S. xanthotricha collected along its distri- scholarship grant from CAPES. bution using denaturing 6% polyacrylamide gels stained with silver nitrate (Creste et al. 2001). We Isolation et caractérisation de microsatellites de selected 12 polymorphic loci (Table I). Scaptotrigona xanthotricha (Apidae, Meliponini): une Allelic frequencies, observed (HO) and expected abeille sans aiguillon de la forêt pluviale atlantique heterozygosities (HE), polymorphic information con- brésilienne. tent (PIC), first-parent non-exclusion probability of each locus (PNExc), non-exclusion probability of Isolierung und Charakterisierung von Mikrosatel- identity (the probability of two randomly selected liten der Stachellosen Biene Scaptotrigona xanthotri- individuals having the same genotype) of each locus cha (Apidae, Meliponini): einer Biene des (I), and combined probabilities were estimated using Atlantischen Regenswalds Brasiliens. CERVUS (Marshall et al. 1998). Number of alleles
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